Metallothionein-inspired prototype of molecular pincer

Article abstract of DOI:10.1039/c1cc11463h

Study of Zn and Pb release from complexes with natural and synthetic amidothiol motifs inspired the design of a "molecular pincer" that scavenges quantitatively metals from liquid environment and releases them, on-demand, under very mild oxidative conditi

Full text of DOI:10.1039/c1cc11463h

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 8067–8069
www.rsc.org/chemcomm
COMMUNICATION
Metallothionein-inspired prototype of molecular pincerw
Svatava Voltrova,a Denisa Hidasova,a Jan Genzerb and Jiri Srogl*a
Received 14th March 2011, Accepted 25th May 2011
DOI: 10.1039/c1cc11463h
Study of Zn and Pb release from complexes with natural and
synthetic amidothiol motifs inspired the design of a ‘‘molecular
pincer’’ that scavenges quantitatively metals from liquid
environment and releases them, on-demand, under very mild
oxidative conditions.
been proposed to transform oxidatively to non-methallophilic
disulfides, forming a highly beneficial thermodynamic sink
that closes effectively the process gate (cf. Fig. 1). The role
of metal thiolates, including their proposed oxidative trans-
formation,24 may thus be of great importance in elucidating
biological function mechanisms involving metallothioneins.
Given these important observations on living systems, it comes
as no surprise that related chemistries are of great scientific
interest.28
Understanding transport, effective scavenging and on-demand
release of metals and metal ions has been an outstanding issue
straddling various areas of chemistry, technology, medical and
environmental sciences.1–4 An excellent illustration, and one of
several possible solutions to the related problems, can be
found in the area of biological chemistry where researchers
have long recognized the importance of metals and studied
their function in biosystems.1,5–7 While on the one hand,
metals are beneficial for their various roles in diverse enzymatic
cycles, on the other hand, metals may be detrimental to proper
systemic functions due to their high toxicity.8,9 Existence of a
biochemical vehicle that would facilitate controlled transport
of metals is therefore of immense importance.10 Systems that
deliver metals to target sites, where the ‘‘cargo’’ is unloaded
selectively, frequently involve high cysteine containing peptides
that escort metals, such as copper, zinc, etc., from one place to
another.11–15 A prominent example of this molecular platform
is found in the chemistry of metallothioneins16–21 In such
scenario the ease of metal loading to the cysteine is facilitated
primarily by the well-established affinity of soft metals to
sulfur.19,22 Concurrently, however, the strong metal/sulfur
bonding also obstructs selective metal release, particularly
under mild conditions. While, in vitro, the equilibrium can
be shifted conveniently towards metal release by implementing
vigorous chemical means (i.e., using strong acid, varying the
concentration of metals),23 which lead to metal liberation,
those rather harsh conditions would likely be detrimental
when applied in vivo environments.
The purpose of this work is twofold. First, we intend to
supplement the circumstantial evidence of the oxidative release
of metals from metal–thiolate complexes with direct measurements
quantifying chemical liberation of selected metals (Pb, Zn)
from metallocysteine moieties. Second, we employ robust
synthetic model systems based on mercaptoamide/thioimide
motifs featuring a reaction pattern that is inspired structurally
by cysteine. We demonstrate the efficacy of metal release even
under biologically benign conditions thanks to the advantageous
conformation of the sulfur terminus.
In order to substantiate the concept of the bio-relevant
oxidative metal unloading, we synthesized corresponding
metallocysteine Pb (1a, 2a) and Zn (1b, 2b) based thiolates
and subjected them to aerobic oxidative conditions. The top
portion of Fig. 2 depicts the proposed chemical pathway
facilitating oxidative release of metals from cysteine residues.
Metals are liberated by the formation of disulfides and the
concentration of the resultant disulfide thus represents a direct
measure of the release efficacy. Two classes of compounds
have been synthesized and tested; they contain either amide
(1a, 1b) or ester (2a, 2b) functionalities. Interestingly, the
cursory kinetic evaluation of the oxidative metal release
indicated the superior reactivity of the amide 1a, 1b (native
to cysteinyl peptides and hence the metallothioneins) over
the ester 2a, 2b for both metals studied. The critical role
of the cysteinyl amide can be rationalized by an intermittent
Recent experiments on biological assays have revealed that
oxidative pathways can facilitate more pertinent means of metal
unloading.24–27 In this fashion metallophilic thiolates have
a Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Flemingovo nam. 2,
166 10 Prague, Czech Republic. E-mail: jsrogl@uochb.cas.cz
b Department of Chemical and Biomolecular Engineering,
North Carolina State University, Raleigh, USA.
E-mail: jan_genzer@ncsu.edu
w Electronic supplementary information (ESI) available: All relevant
synthetic details and 1H and 13C spectra for all compounds. See DOI:
10.1039/c1cc11463h
Fig. 1 Metallothionein function in cellular metal transport. Metals
bound to the metallothionein complex (A) are delivered to the target
(B), as suggested in ref. 24.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 8067–8069 8067

View Article Online
Fig. 2 (top) Proposed mechanism of metal capture and release by
cysteines. (bottom) Metal release as indicated by the formation of S–S
bond for Pb (1a, 2a, left) and Zn (1b, 2b, right) for cysteines bearing
ester and amide functionalities. All attempts to isolate the S–N inter-
mediate have been futile. Cu catalyst was used to speed up liberation of
the metal. Compound 1: X = NH, R0 = CH3, R = naphthalene-2-yl;
compound 2: X = O, R0 = 4-CH3-C6H4, R = CH3.
Fig. 3 (top) Proposed mechanism of metal capture and release by
thioimides. (bottom) Metal release as indicated by the formation of
S–N bond for Pb (left) and Zn (right) for thioimides with X = NH
(a, b) or NO2 (c, d). Compound 3: X = NO2, R = CH(CH3)2;
compound 4: X = 4-CH3C6H4CONH, R = CH(CH3)2.
formation of a putative S–N bond in the intermediate (cf. Fig. 2),
which has been postulated to possess a likely influence on the
reactivity of the cysteinyl residue.29,30 Perhaps not surprisingly
the data reveal that the capture/release efficiency depends on
the metal. While the capture of both Pb and Zn is nearly
instantaneous, the kinetics of Pb release is faster than that
of Zn.31
Successful metal release is associated with the formation of
the S–N bond that closes the five-membered ring in the
thioimide form of the pincer. By monitoring the population
of the S–N bond in solution one can gain a quantitative
understanding of metal liberation from the complex. In
Fig. 3 we plot the time dependence of the concentration of
the S–N bonds for the thioimides studied. From the data it is
apparent that the release of both Pb and Zn is almost
quantitative in 20 h regardless of X. Comparing these results
with those obtained from the cysteine series it is evident that
molecular pincers are more effective in liberating metals from
the carrier metalloorganics. While the ability of the molecule
to form stable five-membered rings is the likely explanation for
the observed behavior, based on the current data it is
impossible to specify the exact role of the chemical environment
(i.e., X and R) on the rate and completion of the metals
release. In order to explain the function of X and R on metals
release one has to carry out additional experiments with other
metals and other combinations or X and R.31
Motivated by the prospect of unraveling the mechanism of
metal release, model mercaptobenzamide compounds were
synthesized that bore a molecular pattern analogous to cysteinyl
amide but were stabilized by the structurally advantageous
arrangement of the functional groups, i.e., amide and thiol,
that govern the capture/release of metals (cf. Fig. 3). These
mercaptoamide/thioimide compounds act effectively as
‘‘molecular pincers’’.32 Molecules comprising different combi-
nations of X were synthesized bearing –NO2 or –NH– groups
representing electron withdrawing or donating elements,
respectively (compounds 3 and 4). The corresponding mercapto-
amides were exposed to the same conditions as used in the
aforementioned experiments with cysteinyl amides. In agreement
with the above cysteinyl amide experiments, the resulting
metallothiolates formed instantaneously under ambient conditions
during which metal ions were attached to the sulfur residue as
depicted in the scheme presented in the top portion of Fig. 3.
The same scheme also indicates the proposed mechanism of
metal release from the thioimide moiety. Mild aerobic oxidation
was selected to satisfy simultaneously the criteria for metal
unloading reactions and systemic biocompatibility. Specifically,
all metal release experiments were carried in DMSO at room
temperature utilizing air as oxidant;33 the reaction rate was
enhanced by adding a catalytic amount of Cu(II) acetate
(for details see the Experimental section, ESIw).
In summary, metallothionein-inspired ‘‘molecular pincer’’
systems for loading and release of metals was developed and
discussed. The devised molecule scavenges quantitatively
selected metals from liquid environment and releases them,
on-demand, under very mild oxidative conditions. Importantly,
the conversion of the closed form of the ‘‘pincer’’ (thioimide)
back to the open form (mercaptoamide) is possible; it can
be accomplished by mild reducing agents, e.g. ascorbic
acid. Fundamental understanding of interaction of metals with
thiols may impact many practical areas, including biotechnology
c
8068 Chem. Commun., 2011, 47, 8067–8069
This journal is The Royal Society of Chemistry 2011

View Article Online
and environmental sciences, that demand metal scavenging and
metal delivery.1–4,8,12 One can envision that the thioimide/
mercaptoamide dyads would represent outstanding candidates
for such functions, given their excellent synthetic accessibility
complemented by their inherent structural flexibility, which
allows for convenient chemical modification and incorporation
into various environments.
17 B. Gold, H. Deng, R. Bryk, D. Vargas, D. Eliezer, J. Roberts,
X. Jiang and C. Nathan, Nat. Chem. Biol., 2008, 4, 609–616.
18 G. Henkel and B. Krebs, Chem. Rev., 2004, 104, 801–824.
19 T. T. Ngu and M. J. Stillman, Dalton Trans., 2009, 5425–5433.
20 R. D. Palmiter, Proc. Natl. Acad. Sci. U. S. A., 1998, 95,
8428–8430.
21 N. J. Robinson, Nat. Chem. Biol., 2008, 4, 582–583.
22 E. Nieboer and D. H. S. Richardson, Science, 1980, 1, 3–26.
23 A. Deratani and B. Sebille, Anal. Chem., 1981, 53, 1742–1746.
24 S. G. Bell and B. L. Vallee, ChemBioChem, 2009, 10, 55–62.
25 W. Feng, F. W. Benz, J. Cai, W. M. Pierce and Y. J. Kang, J. Biol.
Chem., 2005, 281, 681–687.
The authors wish to thank to the Czech Academy of
Sciences (Z40550506, M200550908) for financial support.
26 H. J. Hartmann and U. Weser, BioMetals, 2000, 13, 153–156.
27 Y. J. Kang, Exp. Biol. Med., 2006, 231, 1459–1467.
28 Z. Zhang, M. G. Lindale and L. S. Liebeskind, J. Am. Chem. Soc.,
2011, 133, 6403.
Notes and references
1 Cell Biology Of Metals and Nutrients, ed. Rudiger Hell and
¨
R.-R. Mendel, Springer, Heidelberg, Germany, 2010.
2 K. F. Lam, K. L. Yeung and G. McKay, Langmuir, 2006, 22,
9632–9641.
3 M. M. Matlock, B. S. Howerton and D. a. Atwood, Ind. Eng.
Chem. Res., 2002, 41, 1579–1582.
4 M. M. Matlock, B. S. Howerton, M. A. Van Aelstyn, F. L. Nordstrom
and D. A. Atwood, Environ. Sci. Technol., 2002, 36, 1636–1639.
5 P. J. Sadler and Z. Guo, Pure Appl. Chem., 1998, 70, 863–871.
6 S. W. Ragsdale, Chem. Rev., 2006, 106, 3317–3337.
7 H. Sun and Z.-F. Chai, Annu. Rep. Prog. Chem., Sect. A, 2010,
106, 20.
8 O. Andersen, Chem. Rev., 1999, 99, 2683–2710.
9 J. D. Walker, M. Enache and J. C. Dearden, Environ. Toxicol.
Chem., 2003, 22, 1916.
29 For biological relevance of mercaptobenzamides see: K. Bobrowski,
G. L. Hug, D. Pogocki, B. Marciniak and C. Schoneich, J. Am.
Chem. Soc., 2007, 129, 9236–9245.
¨
30 C. E. Paulsen and K. S. Carroll, ACS Chem. Biol., 2010, 5, 47–62.
31 Complete understanding of metal binding to and release from
mercaptoamides and subsequent formation of cyclic thioimides
can only be achieved by varying systematically the chemical
environment around the active center of mercaptoamides and
testing metals bearing various degrees of ‘‘softness’’. Such a study
is beyond the scope of the current communication. A more
comprehensive set of results will be reported in a follow-up longer
format, in which detailed metal release kinetic studies will be
complemented by computational approaches, that will quantify
the metal-specific binding constants.
10 Z. Ma, F. E. Jacobsen and D. P. Giedroc, Chem. Rev., 2009, 109,
4644–4681.
11 L. A. Finney and T. V. O’Halloran, Science, 2003, 300, 931–936.
12 Y.-F. Lin, A. R. Walmsley and B. P. Rosen, Proc. Natl. Acad. Sci.
U. S. A., 2006, 103, 15617–15622.
32 For biological relevance of mercaptobenzamides, see: L. M. M.
Jenkins, S. R. Durell, A. T. Maynard, S. J. Stahl, J. K. Inman,
E. Appella, P. Legault and J. G. Omichinski, J. Am. Chem. Soc.,
2006, 128, 11964–11976.
13 T. V. O’Halloran and V. C. Culotta, J. Biol. Chem., 2000, 275,
25057–25060.
14 A. C. Rosenzweig, Acc. Chem. Res., 2001, 34, 119–128.
15 S. Tottey, D. R. Harvie and N. J. Robinson, Acc. Chem. Res.,
2005, 38, 775–783.
33 In addition to DMSO, water was also evaluated as a reaction
medium. Soluble Pb cysteinate was subjected to the releasing
condition in water; the kinetics of metal release essentially copied
the ones obtained in DMSO. While the aqueous environment is
more relevant to natural biological conditions it was not used in
our study owing to the inherent solubility issues influencing the
generality of our findings.
16 P. Coyle, J. C. Philcox, L. C. Carey and A. M. Rofe, Cell. Mol. Life
Sci., 2002, 59, 627–647.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 8067–8069 8069