Arsinous acid as a thiol binding group: Potential cysteine peptide tagging functionality that binds a single thiol

Article abstract of DOI:10.1039/c3nj01462b

A simple aryl arsinous acid (ArAs(CH₃)OH) was prepared by ortho mercuration of p-cresol followed by Pd-catalyzed reaction with methylarsenic dibromide, purification as the mercaptoethanol adduct, and deprotection using a silver salt. Isothermal titration calorimetry experiments showed reaction with mercaptoethanol with an association constant of 6 × 10⁴ M ^-1. Rapid exchange of thiols at the arsenic atom was demonstrated by ¹H-NMR.

Full text of DOI:10.1039/c3nj01462b

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Arsinous acid as a thiol binding group: potential
cysteine peptide tagging functionality that
binds a single thiol†
Cite this: New J. Chem., 2014,
38, 1368
Received (in Montpellier, France)
22nd November 2013,
Xiaofei Liang and Dale G. Drueckhammer*
Accepted 7th January 2014
DOI: 10.1039/c3nj01462b
www.rsc.org/njc
A simple aryl arsinous acid (ArAs(CH3)OH) was prepared by ortho
mercuration of p-cresol followed by Pd-catalyzed reaction with
methylarsenic dibromide, purification as the mercaptoethanol
adduct, and deprotection using a silver salt. Isothermal titration
calorimetry experiments showed reaction with mercaptoethanol
with an association constant of 6 Â 104 MÀ1. Rapid exchange of thiols
at the arsenic atom was demonstrated by 1H-NMR.
The fluorescent labeling of proteins, especially within living
cells, has been a valuable tool for studying protein function.1–4
Of particular value has been fusion of the green fluorescent
protein and its variants for visualization and tracking of the
protein of interest.5 The substantial size of the fluorescent protein
domain and the time delay from translation to formation of the
fluorophore limit many applications. A very useful development has
been the fluorescent biarsenical probe FlAsH, commonly used as the
bis-ethanedithiol adduct 1 and related analogues 2 pioneered by
Tsien’s group.6 FlAsH usually labels a simple tetracysteine motif
CCXXCC (commonly CCPGCC), though binding to other sequences
and ‘‘bipartite’’ dicysteine pairs has also been demonstrated.7,8 The
need for orthogonal probes that bind alternate cysteine arrange-
ments has been recognized, and demonstrated with some success in
the probe AsCy3 3, which targets a sequence of two cysteine pairs
separated by five amino acids (Fig. 1).9
Fig. 1 FlAsH and other fluorescent bisarsenical probes as their ethane-
dithiol adducts.
bind cysteine pairs within a-helical peptides but show highest
A limitation in design of arsonous acid-based tags is that the affinity for a structure designed for hairpin formation.6,10,11
binding of two cysteine thiols to a single arsenic atom requires Cline et al. have shown that CH3As(OH)2 binds to i,i + 1;
that the peptide or protein adopt a structure which allows i,i + 2; i,i + 3; and i,i + 4 dicysteine pairs with almost equal
formation of a cyclic complex. While providing high affinity affinity, while CD measurements indicate the a-helical popula-
per arsenic functionality, this may require major conformation tion is increased in binding to the i,i + 4 peptide but decreased in
change of the dithiol structure, as illustrated in Fig. 2, and may other dicysteine arrangements.10 An alternate and perhaps more
not permit binding to a regular repeating peptide structure. flexible approach would be to utilize arsenic functionality in
Indeed, FlAsH and other arsenic(III) compounds do not generally which each arsenic atom binds to only a single thiol group,
displayed on a backbone structure such that they are positioned
for binding to a specifically spaced cysteine pair within an a-helical
peptide or other regular structure. Such an approach might be
achieved using arsinous acids (RR’AsOH), having only one arsenic–
oxygen bond for displacement by a thiol. Such compounds might
Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400,
USA. E-mail: dale.drueckhammer@stonybrook.edu; Tel: +1 631-632-7923
† Electronic supplementary information (ESI) available: 1H-NMR spectra of 8
and 9. See DOI: 10.1039/c3nj01462b
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Fig. 2 Major conformational change required for formation of a cyclic
adduct 6 between a dithiol 4 and an arsonous acid or arsine oxide 5.
Scheme 1 Completed synthesis of an arsinous acid model compound 9.
make and use the dibromide in part because of its lower volatility
and resulting greater ease of handling relative to the dichloride.
Reaction of the commercially available cacodylic acid with con-
centrated HBr formed the product in a retro-Arbusov type reaction
(Scheme 1),18 and the product was isolated by extraction into
dichloromethane. The Pd-catalyzed mercury to arsenic substitu-
tion followed procedures for the reaction with arsenic trichloride
Fig. 3 Synthetic approaches to arsinous acid and aryl arsonous acid
derivatives.
also be less toxic than the arsine oxides (RAsQO) or the hydrated but to our knowledge had not been demonstrated with an arsenic
arsonous acid form (RAs(OH)2), which apparently exhibit their bromide or with any alkyl dihaloarsenic derivative.19 The reaction
toxicity by forming cyclic adducts with essential dithiols.12–14
proceeded and the product was isolated as the mercaptoethanol
We sought to develop synthetic methodology and explore thiol adduct 8, which was purified by chromatography on silica gel.
binding properties of an arsinous acid. The limited literature on Mercaptoethanol was chosen to improve the aqueous solubility of
arsinous acid synthesis rely on either reaction of a Grignard reagent the thiol adduct, though that feature was not utilized in the work
with an alkyl arsenic dihalide or similar reagent (Fig. 3a) or reported here. Efforts to cleave the arsenic–sulfur bond with a
nucleophilic substitution by an arsonous acid dianion on an alkyl mercury salt by published procedures gave a mixture of products.6
halide followed by reduction of the initial product (Fig. 3b).15 We developed an alternate deprotection procedure using silver
Synthesis of FlAsH and related arsonous acids is accomplished nitrate in methanol. The silver–thiolate complex was removed by
by mercuration ortho to a phenolic hydroxyl group followed by a filtration after addition of a small amount of lithium chloride to
Pd-catalyzed substitution of the resulting aryl-mercurial with precipitate excess silver ion.
arsenic trichloride (Fig. 3c).6 The resulting aryl arsenic dichloride
product is isolated as a dithiol adduct and subsequently hydro- was converted to the arsinous acid form 9 when dissolved in water
lyzed to the arsenic acid. and was used directly without further purification. The products 8
The methanol adduct isolated upon evaporation of methanol
We chose p-cresol 7 as the precursor for a simple model and 9 appear quite stable and show no decomposition upon
compound, with the methyl group serving to block substitution at standing in pure form or in DMSO or aq. solution.
the para-position. The ortho-mercuration of 7 followed standard
Formation of the complex 8 between 9 and mercaptoethanol was
procedures for the mercuration of phenols also used in the studied by isothermal titration calorimetry (Fig. 4). An association
synthesis of FlAsH and derivatives (Scheme 1). Formation of the constant of 6 Â 104 MÀ1 was determined. This represents approxi-
methyl-substituted arsinous acid derivative required reaction with a mately 4-fold and 30-fold greater affinity than the values of 1.6 Â
methylarsenic dihalide rather than the trihalide. While methyl- 104 MÀ1 and 1.9 Â 103 MÀ1 for the first and second association
arsenic dibromide and the corresponding dichloride are both constants for binding of methylarsonous acid [CH3As(OH)2]
known compounds, they are not available from common sources to two equivalents of thiol.12 The high affinity of 9 for thiols
and a convenient synthesis is not described.16,17 We chose to further supports this approach to the development of selective
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70 1C bath for 24 h, then allowed to cool to room temp. The
suspension was filtered to remove the insoluble solid which by
1H-NMR appears to be the 2,6-bis-mercurated product (aromatic
singlet at d 6.94 in DMSO-d6). The filtrate was evaporated to about
10 mL, 40 mL water was added, and the mixture was cooled to 4 1C
for 40 minutes. The resulting suspension was vacuum filtered to
recover the solid product. 1H-NMR indicated about 20% unreacted
1
7 along with the desired monomercurated product. H-NMR dH
(300 MHz: DMSO-d6) 2.02 (3H, s, OAc), 2.20 (3H, s, Ar-CH3), 6.73
(1H, d, J = 8 Hz), 6.93 (1H, dd, J = 2, 8 Hz), 7.01 (1H, d, J = 2 Hz). This
crude product was stable indefinitely at room temp., and was used
in the next step without purification.
2-(((2-Hydroxyethyl)thio)(methyl)arsino)-4-methylphenol 8
Fig. 4 Isothermal titration calorimetry profile for titration of 0.5 mM 9
in pH 7 phosphate buffer (1.3 mL) with 10 mL injections of 6 mM
2-mercaptoethanol.
To the crude mercuric acetate derivative of p-cresol (0.54 g,
1.47 mmol) and palladium(II) acetate trimer (8 mg, 0.036 mmol
Pd) under N2 was added a solution of methylarsenic dibromide
(1.30 g, 5.0 mmol) in dry THF (17 mL). Diisopropylethylamine
(1.20 mL, 7.5 mmol) was added and the solution was stirred at
room temp. for 18 h. The reaction mixture was poured into a
solution of 2-mercaptoethanol (0.80 mL, 0.89 g, 11.4 mmol) in aq.
sodium phosphate (70 mL, 0.25 M, pH 7.0) and acetone (70 mL).
The mixture was stirred at room temp. for 1 h and extracted with
ether (3 Â 50 mL). The combined organic layers were dried over
MgSO4, evaporated, and purified by chromatography on silica gel
(25 to 65% acetone in heptane)21 to give 8 as a white powder (0.23 g,
0.84 mmol, 57% yield). 1H-NMR dH (300 MHz: CDCl3) 1.62 (3H, s,
As-CH3), 2.28 (3H, s, Ar-CH3), 2.92 (2H, t, J = Hz, S-CH2), 3.79 (2H, m,
CH2-OH), 6.75 (1H, d, J = 8.1 Hz), 7.05 (1H, dd, J = 2, 8.1 Hz), 7.20
(1H, d, J = 2 Hz). 13C NMR (75 MHz, CDCl3): d = 11.80 (CH3As),
20.50 (CH2S), 34.96 (Ar-CH3), 62.87 (CH2O), 116.29 (CH), 124.88 (C),
130.30 (CH), 131.78 (C), 132.24 (CH), 156.46 (C); HRMS (ESI): m/z
calcd for C10H15AsO2S [M À H]À: 272.9936; found: 272.9939.
dicysteine peptide receptors. Selectivity of arsenic functionality
for thiol groups over other functional groups present in proteins
is illustrated in the binding of arsonous and arsinous acids
to glutathione, mercaptoalcohols, and related compounds.12,20
Formation of the arsenic–thiol complex is rapid on the time-
scale of the ITC experiment.
To address the rate of thiol exchange, 3 eq. of cysteine
were added to a 9 mM solution of 8 in D2O in an NMR tube.
The 1H-NMR spectrum was taken immediately (about 2 min to
mid-point of acquisition) and again after 30 minutes and the
ratio of the mercaptoethanol and cysteine adducts was deter-
mined by integration. The spectra indicated that complete
equilibration was achieved prior to the first spectrum. While
this does not provide an accurate rate measurement or con-
trolled conditions of pH, etc., it does indicate the kind of rapid
exchange necessary for a peptide binding agent.
2-(Hydroxy(methyl)arsino)-4-methylphenol 9
This work has demonstrated a synthetic approach to an arsinous
acid and the high affinity and reversible binding to a thiol. These
results provide a foundation for a new design approach to arsenic-
based receptors for cysteine peptides in which each arsenic
functionality binds a single cysteine thiol group.
8 (0.0079 g, 28.5 mmol) was dissolved in 8 drops methanol in a
test tube and a solution of silver nitrate in methanol (1.25 mL,
0.025 M, 31.25 mmol) was added, with immediate formation of a
light yellow precipitate. After 30 minutes, a solution of lithium
chloride in methanol (50 mL, 0.2 M, 10 mmol) was added. After
10 minutes the suspension was filtered through a cotton plug,
the filtrate was evaporated, and the residue was dissolved in
DMSO-d6 (0.7 mL). 1H-NMR dH (300 MHz: DMSO-d6) 1.46
(3H, s, As-CH3), 2.20 (3H, s, Ar-CH3), 6.68 (1H, d, J = 8 Hz),
6.99 (1H, dd, J = 2, 8 Hz), 7.26 (1H, d, J = 2 Hz). 0.1 mL of the
solution was mixed with 0.5 mL D2O and 0.1 mL of a 0.021 M
solution of pinacol in D2O and the concentration of 9 in the NMR
sample was determined to be 0.026 M by relative integration of the
1H-NMR signals of 9 and the methyl singlet of pinacol at d 1.05.
This corresponds to a total yield of 9 of 18.5 mmol (59% yield).
Experimental
Methylarsenic dibromide
Cacodylic acid (10.0 g, 0.0725 mol) was dissolved in 48% aq. HBr
(57 mL) and the mixture was heated at 130 1C for 5 h. The mixture
was allowed to warm to room temp. and was extracted with
dichloromethane (3 Â 30 mL). The combined organic layers were
dried over MgSO4 and evaporated to give methylarsenic dibromide
(15.3 g, 0.0612 mol, 84% yield) which was used without further
purification. 1H-NMR (CDCl3) d 2.61 (s) (lit.,17 d 2.61 (CCl4)).
ITC experiments
Mercuric acetate derivative of p-cresol
ITC experiments were performed on a Model CSC 4200 micro-
To a solution of p-cresol 7 (4.32 g, 40 mmol) in methanol calorimeter (Calorimetry Sciences Corp.) at 25 1C with stirring
(180 mL) at room temp. was added acetic acid (2 mL) and at 297 rpm. The cell (1.3 mL) contained a 0.5 mM solution of
mercuric acetate (12.8 g, 40 mmol). The mixture was heated in a 9 (0.65 mmol) in 5 mM potassium phosphate buffer pH 7.0
1370 | New J. Chem., 2014, 38, 1368--1371
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8 N. W. Luedtke, R. J. Dexter, D. B. Fried and A. Schepartz,
Nat. Chem. Biol., 2007, 3, 779–784.
9 H. S. Cao, Y. J. Xiong, T. Wang, B. W. Chen, T. C. Squier and
M. U. Mayer, J. Am. Chem. Soc., 2007, 129, 8672–8673.
10 D. J. Cline, C. Thorpe and J. P. Schneider, J. Am. Chem. Soc.,
2003, 125, 2923–2929.
containing 2% DMSO. A solution of 2-mercaptoethanol (6.0 mM)
in the same buffer with 2% DMSO was added in 23 injections of
10 mL each, with 300 s between injections. Data was processed
using BindWorks 3.0 software.
NMR thiol-exchange experiment
The 1H-NMR spectrum was taken of a solution of 8 (9 mmol) in D2O
(1 mL). A solution of L-cysteine hydrochloride in D2O (27 mL, 1 M,
27 mmol) was added and the spectrum was taken immediately and
again after 30 minutes. Both spectra showed formation of equal
amounts of the free mercaptoethanol S-CH2 triplet at d 2.55.
11 D. Ramadan, D. J. Cline, S. Bai, C. Thorpe and
J. P. Schneider, J. Am. Chem. Soc., 2007, 129, 2981–2988.
12 A. M. Spuches, H. G. Kruszyna, A. M. Rich and D. E. Wilcox,
Inorg. Chem., 2005, 44, 2964–2972.
13 K. T. Kitchin and K. Wallace, J. Biochem. Mol. Toxicol., 2006,
20, 35–38.
14 M. Styblo, L. M. Del Razo, L. Vega, D. R. Germolec,
E. L. LeCluyse, G. A. Hamilton, W. Reed, C. Wang,
W. R. Cullen and D. J. Thomas, Arch. Toxicol., 2000, 74,
289–299.
15 P. V. Ioannou, Phosphorus, Sulfur Silicon Relat. Elem., 2002,
177, 1–14.
Acknowledgements
This work was supported by National Science Foundation grant
CHE-0848334.
16 C. K. Banks, J. F. Morgan, R. L. Clark, E. B. Hatlelid,
F. H. Kahler, H. W. Paxton, E. J. Cragoe, R. J. Andres,
B. Elpern, R. F. Coles, J. Lawhead and C. S. Hamilton,
J. Am. Chem. Soc., 1947, 69, 927–930.
Notes and references
1 B. N. G. Giepmans, S. R. Adams, M. H. Ellisman and
R. Y. Tsien, Science, 2006, 312, 217–224.
2 L. W. Miller and V. W. Cornish, Curr. Opin. Chem. Biol., 17 K. Henrick and S. B. Wild, J. Chem. Soc., Dalton Trans., 1975,
2005, 9, 56–61. 1506–1516.
3 K. M. Marks and G. P. Nolan, Nat. Methods, 2006, 3, 591–596. 18 G. O. Doak and L. D. Freedman, Organometallic compounds
4 T. L. Foley and M. D. Burkart, Curr. Opin. Chem. Biol., 2007,
11, 12–19.
of arsenic, antimony, and bismuth, Wiley-Interscience,
New York, 1970, p. 92.
5 R. Y. Tsien, Annu. Rev. Biochem., 1998, 67, 509–544.
6 S. R. Adams, R. E. Campbell, L. A. Gross, B. R. Martin,
19 C. C. Spagnuolo, R. J. Vermeij and E. A. Jares-Erijman,
J. Am. Chem. Soc., 2006, 128, 12040–12041.
G. K. Walkup, Y. Yao, J. Llopis and R. Y. Tsien, J. Am. Chem. 20 J. S. Edmonds, T. Nakayama, T. Kondo and M. Morita,
Soc., 2002, 124, 6063–6076. Magn. Reson. Chem., 2006, 44, 151–162.
7 B. W. Chen, H. S. Cao, P. Yan, M. U. Mayer and T. C. Squier, 21 D. G. Drueckhammer, S. Q. Gao, X. F. Liang and J. Z. Liao,
Bioconjugate Chem., 2007, 18, 1259–1265. ACS Sustainable Chem. Eng., 2013, 1, 87–90.
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