Synthesis of water-soluble hypervalent iodine reagents for fluoroalkylation of biological thiols

Article abstract of DOI:10.1039/c9ob02115a

New open-chain and water-soluble hypervalent iodine reagents were synthesized and used for the transfer of fluoroalkyl groups to sulfur atoms of cysteine and cysteine-containing peptides under biocompatible conditions. Some of the reagents displayed excellent reactivity despite their limited stability in aqueous media. In reactions with a short cysteine-containing peptide, in addition to the expected S-fluoroalkylated product, a range of side-products were obtained. The amount of side-products depended on the conditions used (type of reagent, concentration, and pH). With highly activated hypervalent iodine reagents, a new reactive mode was observed-reaction with disulfides to form fluoroalkyl thiols.

Full text of DOI:10.1039/c9ob02115a

Organic &
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Synthesis of water-soluble hypervalent iodine
reagents for fluoroalkylation of biological thiols†
Cite this: Org. Biomol. Chem., 2019,
17, 10097
Iveta Klimánková,a Martin Hubálek, a Václav Matoušekb and Petr Beier
*
a
New open-chain and water-soluble hypervalent iodine reagents were synthesized and used for the trans-
fer of fluoroalkyl groups to sulfur atoms of cysteine and cysteine-containing peptides under bio-
compatible conditions. Some of the reagents displayed excellent reactivity despite their limited stability
in aqueous media. In reactions with a short cysteine-containing peptide, in addition to the expected
S-fluoroalkylated product, a range of side-products were obtained. The amount of side-products
depended on the conditions used (type of reagent, concentration, and pH). With highly activated hyperva-
lent iodine reagents, a new reactive mode was observed – reaction with disulfides to form fluoroalkyl thiols.
Received 30th September 2019,
Accepted 11th November 2019
DOI: 10.1039/c9ob02115a
rsc.li/obc
sulfide formation and reaction on the Trp residue.16
Acetyl coenzyme A (AcCoSH) was trifluoromethylated with 2 to
Introduction
Protein modification is a widely used tool to investigate their AcCoSCF3 in a good yield and used as a probe for protein–
biological function and to facilitate drug design.1–6 High protein interaction using 19F NMR.17 All these reactions were
selectivity, reactivity under biocompatible conditions and conducted in organic solvents (DCM, MeOH, and MeCN)
stability of the conjugates are crucial requirements for a without or with only a small amount of water and at low temp-
successful bioconjugation method. Cysteine bioconjugation eratures (−78 °C to −40 °C), which was crucial according to
utilizes sulfur’s high nucleophilicity, and common strategies one mechanical study with reagent 1.18 Later, we and the
include substitution reactions with haloacetamides, Michael Togni group reported the extension of reagents 1 and 2 to their
additions to maleimides or vinyl sulfones and disulfide tetrafluoroethylene19 analogues of general structures 3 and 4
exchange.4 However, these reactions suffer from limitations, (R = ArO, ArS, nitrogen heterocycle such as benzimidazole,
such as low selectivity7–9 and stability of adducts.1 Therefore,
new reagents and reactive modes are being developed.1,6
A recently introduced reactive mode for thiol bioconjugation
employs cyclic hypervalent iodine compounds (λ3-iodanes) for
the transfer of alkynyl10 or fluoroalkyl groups.7 Moreover,
introduction of fluroalkyl groups gives the possibility to use
19F NMR for reaction monitoring and determination of the
protein structure and dynamics.11–13
In 2006, the group of A. Togni introduced iodanes 1 and 2
for electrophilic trifluoromethylation of various substrates
including thiols, N-oxides, phosphines and carbon nucleo-
philes (Fig. 1).14 Their excellent reactivity with thiols was soon
harnessed in trifluoromethylation of thiol-containing bio-
molecules. A uridine 2-SH derivative was trifluoromethylated
on sulfur with 1.15 A heptapeptide octreotide was trifluoro-
methylated with 1 or 2 to afford a mixture of Cys trifluoro-
methylation accompanied by side products consisting of di-
aInstitute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences,
Flemingovo náměstí 2, 166 10 Prague 6, Czech Republic. E-mail: beier@uochb.cas.cz
bCF Plus Chemicals, s.r.o., Karásek 1767/1, 621 00 Brno-Řečkovice, Czech Republic
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9ob02115a
Fig. 1 Fluoroalkylated λ3-iodanes.
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imidazole, and pyrazole), which allowed the attachment of
structurally diverse R groups in thiol conjugation.7,20,21 Their
reactivity was found to be similar to reagents 1 and 2 20 and
their conjugation to biologically relevant thiols such as the
protected cysteine or 1-thio-β-D-glucose was demonstrated.20,21
Unfortunately other biological thiols such as glutathione or
penicillamine afforded only low yields of the products.21
Again, low solubilities of reagents 3 and 4 in water required
the use of mostly organic reaction media and low temperature
for better control of the reaction. Starting from
N-methyltyramine, reagent 3b·TFA was prepared in five steps
and used subsequently for the attachment of functional
groups such as fluorescent groups, reactive groups for azide–
alkyne cycloaddition or cross-coupling reactions, oligo(ethyle-
neglycol) chain, biotin, etc. via amide or sulfonamide for-
mation or alkylation on nitrogen. One such reagent, a rhoda-
mine-containing sulfonamide 5, showed good water solubility
and high Cys-selectivity in protein labelling, which outper-
formed commercial maleimide or iodoacetamide reagents.7
In this work, previously reported 3b·TFA and newly syn-
thesized reagents (Fig. 1) are evaluated in fluoroalkylation of
biological thiols in aqueous media at laboratory temperature.
A detailed reaction monitoring allowed the identification of
optimal reaction conditions and limitations of thiol fluoroalky-
lation. Additional advantage of tetrafluoroethylene-containing
reagents 3 and 4 over reagents 1 and 2 is in the possibility of
monitoring the fate of the fluoroalkyl group by 19F NMR since
the CF3 group of 1 and 2 can be partially converted into
intractable CF3H or carbon dioxide (vide infra).
Scheme 2 Synthesis of reagent 1·BetCl and polymeric reagents 6 and 7.
Scheme 3 Synthesis of reagents 4 and salt 4b·HCl.
Scheme 4 Decomposition of 1·BetCl and 3a·AcCl in water.
Results and discussion
Synthesis and stability of the reagents
Using this knowledge, reagent 1 was modified with charge-
carrying betaine chloride to 1·BetCl. For the introduction of
negatively charged groups, a modification of 1 and 3a with
2-sulfobenzoic acid anhydride was successfully performed.
However, the formed reagents 6 and 7 had a polymeric struc-
ture and were insoluble in water or aqueous buffer media
(Scheme 2).
We developed a new protocol for the synthesis of reagents
4a and 4b·Boc starting from 1-acetoxy-1,2-benziodoxol-3-(1H)-
one, where the use of DMF as the solvent was crucial for
obtaining good yields. The reaction of 4b·Boc with HCl
afforded salt 4b·HCl analogous to 3b·TFA (Scheme 3).7
Stability of the prepared reagents in aqueous media was
investigated. Reagent 1·BetCl decomposed in water within a
few minutes; however, the reaction with thiols was found to be
much faster (vide infra). Similarly, 3a·AcCl was also unstable in
the presence of water and afforded acid 8 (Scheme 4). On the
other hand, cyclic reagents (3a and 4a) were stable in water.
Reagent 3a was synthesized in analogy to literature-reported
synthesis (Scheme 1).20 An improved isolation protocol was
based on the formation of hydrochloride salt 3a·HCl, which
precipitated from diethyl ether solution. Basification with satu-
rated bicarbonate solution restored the cyclic structure of 3a
which is soluble in organic solvents. The search for water
soluble λ3-iodanes led us to the application of charged struc-
tures. We found that 3a does not react only with HCl to yield
3a·HCl (as described for 1 by Toste)22, but also with acetyl
chloride to provide 3a·AcCl (Scheme 1).
Reactivity studies
Efficiency of the prepared cyclic aryloxytetrafluoroethylene-con-
taining λ3-iodanes in fluoroalkylation of cysteine was evaluated
in aqueous buffers of pH 4–8 with a small amount of a co-
Scheme 1 Synthesis of reagent 3a and salts 3a·HCl and 3a·AcCl.
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Table 1 Fluoroalkylation of cysteine using λ3-iodanesa
Table 2 Trifluoromethylation of cysteine using 1·BetCla
Yield (%)
Entry
Reagent
pH
9
10
8
1
2
3
4
5
6
7
8
3a
3a
4
7
4
7
8
4
7
8
4
7
4
7
8
9
5
2
—
—
—
—
6
2
2
6
—
24
—
—
8
3
3
2
2
9
4
Entry
Amino acid
pH
Time
Yield (%)
3a·AcCl
3a·AcCl
3a·AcCl
3b·TFA
3b·TFA
3b·TFA
4a
18
13
28
98
91
65
6
11
13
94
82
1b
2b
3b
4c
5c
6c
7c
8c
9c
Cys
Cys
Cys
Trp
4
7
8
4
7
4
7
4
7
10 min/24 h
10 min/24 h
10 min/24 h
10 min
10 min/3 h
10 min
10 min
10 min
10 min
11 (94/94)
11 (99/99)
11 (97/57)
12 (0)
12 (<1/9), 13 (0/3)
11 (30), 12 (24), 13 (8)
11 (46), 12 (17), 13 (5)
11 (79), 14 (2)
11 (73), 14 (<1)
—
6
Trp
9
Cys + Trp
Cys + Trp
Cys + Tyr
Cys + Tyr
10
11
12
13
4a
3
4b·HCl
4b·HCl
4b·HCl
8b
24b
14b
a Acetate (pH 4) or phosphate (pH 7 and 8) buffers were used. b 1·BetCl
(1.5 equiv.) was used. c 1·BetCl (1.0 equiv.) was used. 19F NMR yields
using potassium trifluoroacetate as the internal standard are shown.
a In the case of 3b·TFA and 4b·HCl, MeCN was used as the co-solvent,
and in other cases, DMSO was used as the co-solvent. Acetate (pH 4) or
phosphate (pH 7 and 8) buffers were used. 19F NMR yields using 2,2,2-
trifluoroethanol or potassium trifluoroacetate as internal standards
are shown. b Trace amounts of ArOCF2CF2X (X = Cl, I) were observed by
19F NMR in the product mixture.
solvent (Table 1). The low yield of the fluoroalkylation with
reagents 3a, 3a·AcCl and 4a was caused by their low solubility
in the reaction media. The activated salt reagent 3a·AcCl out-
performed the cyclic reagents 3a and 4a. The highest conver-
sion was achieved with the reagent 3b·TFA in acidic or neutral
pH (entries 6 and 7). In basic buffer, solubility of 3b·TFA was
not complete, which led to the lower yield of fluoroalkylated
cysteine 9 (entry 8). On the other hand, reagent 4b·HCl
afforded the best yield of 9 in neutral or basic media (entries
12 and 13), which was consistent with the report of Capone
and co-workers16 showing that 2 reacted cleanly only with thio-
lates. This was supported by the observation, that at pH 4,
most of the reagent 4b·HCl remained unreacted.
Decomposition side-products 8 and 10 (accompanied by in-
organic fluoride formation) formed from excess of reagents
were detected in all reaction mixtures. Moreover, the formation
of trace amounts of halides ArOCF2CF2X (X = Cl, I) was
observed in some cases.
Next, the reagent 1·BetCl was evaluated in trifluoromethyl-
ation of cysteine, tryptophan and tyrosine (Table 2). It showed
excellent reactivity with cysteine in buffers of different pH
values as judged by the observed 19F NMR yields of trifluoro-
methylated cysteine 11. Stability of product 11 under acidic or
neutral conditions was very good; however, at pH 8, transform-
ation of 11 to dipeptide 15 was observed by 19F NMR and
HPLC-MS. Its presence can be explained by the formation of
Scheme 5 Formation of dipeptide 15.
fluoromethyl radicals in the presence of sulfur nucleophiles.
Competition experiment between cysteine and tyrosine revealed
a much higher reactivity of cysteine.
Product stability studies
Good stability of conjugates is essential for a successful bio-
conjugation method. Therefore, stability of conjugates 17a,
17b and 17c was tested under various conditions. The conju-
gates showed good stability in excess of a strong acid
(Scheme 6), and according to the literature20, also towards a
large excess of an external thiol. On the other hand, the litera-
ture described the β-elimination of “CF3SH” and the formation
of dehydroalanine in the presence of a strong base (cf. NaOH
or t-BuOK).16,24 Conjugates 17b and 17c were found to be
stable at pH 8 and also in 1 M NH4HCO3 buffer (Scheme 6). In
the presence of a strong oxidant (TCICA), oxidation on sulfur
was observed, which decreased the stability of conjugates, due
to the subsequent elimination of fluoroalkyl sulfenic acid
(Scheme 6).
Application in peptide derivatization
A
cysteine-containing heptapeptide with the amino acid
cyclic intermediate 16, which is known to form dipeptides sequence DAVACAK (21) was used for the investigation of Cys
(Scheme 5).23 Tryptophan alone reacted sluggishly, but in an labelling using reagents 1·BetCl, 3a, 4a, 3a·AcCl, 3b·TFA and
equimolar mixture with cysteine, competing reactions were 4b·HCl (Scheme 7). First, the treatment with the reductant
observed with better selectivity to 11 over 12 or 13 at neutral TCEP was performed to ensure that the Cys residue is in the
pH (Table 2). These results indicate the formation of tri- reduced thiol form.25 Next, a ten-fold excess of the reagent was
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Elimination of 23 gives rise to alkene 25, which was not
directly observed but underwent a Michael reaction with the
starting peptide to yield traces of 26. Acylation of the nitrogen
of 21, 22 and 24 with ArOCF2COF (formed by the decompo-
sition of excess of the iodine reagent) afforded side-products
27–29 at pH 7. In the acidic media (pH 4), no formation of
27–29 was observed due to the protonation of the reactive
amino group, and also elimination of 23 was suppressed. The
presence of the starting peptide 21 in the product mixture
after the final treatment with TCEP can be explained by an
incomplete reaction or formation of the disulfide of 21 and
the subsequent reduction. We propose that most of unreacted
21 in the product mixture originated from the disulfide
formed by oxidation with hypervalent iodine reagents and the
subsequent reduction with TCEP. When N-acetyl cysteine was
used instead of TCEP in the last step, the disulfide of 21 and
the mixed disulfide of 21 and N-acetyl cysteine were obtained
(see ESI, Table S2†). Peptide concentration influenced the
Scheme 6 Stability of conjugates 17a–c.
added. After 1 hour26 at ambient temperature, excess of TCEP degree of fluoroalkylation. A comparison of reaction of 21 with
was added to quench the unreacted hypervalent iodine 4b·HCl (10 equiv.) at 0.15 mM and 1.5 mM peptide concen-
reagent. From the data shown in Scheme 7 and Table 3, it is trations revealed a much better yield of product 22b and less
evident that strong signals of the product 22 were observed in side products at a higher peptide concentration (see ESI,
all cases, but the highest conversion of 21 was achieved using Table S3†). In summary, the reaction was very fast and led to
reagent 3a·AcCl in acidic buffer for the formation of 22a, and the desired fluoroalkylation products 22, and depending on
using reagent 4b·HCl in neutral buffer for the formation of the reagent and conditions, yielded other side-products as
22b. A detailed MS analysis revealed the formation of some well. Side reactions were caused by the presence of excess of
side-products. Sulfur oxidation products 23 or 24 were formed iodine reagents, especially at neutral or basic pH.
in limited amounts. The formation of sulfoxide 23 was caused
by the presence of oxidizing hypervalent iodine reagents and only a minimum amount of unreacted peptide 21 is not its low
water (for
control experiment in H2O18 vide infra). oxidation power but rather reactivity with the peptide in both
The reason why 3a·AcCl provided high conversion to 22 and
a
Scheme 7 Fluoroalkylation of peptide DAVACAK (1.2 mM) using reagents 1·BetCl, 3a, 3a·AcCl, 4a, 3b·TFA and 4b·HCl.
Table 3 Area under the curve (AUC × 102) determined by the integration of extracted ion chromatogram (XIC) for the selected precursor ion of
LC-MS measurement of the crude reaction mixture of fluoroalkylation of DAVACAK (21) with reagents 1·BetCl, 3, 4, 3b·TFA and 4b·HCla
Reagent
Product
3a
pH 4
3a
pH 7
3a·AcCl
pH 4
3a·AcCl
pH 7
4a
pH 7
Reagent
Product
3b·TFA
pH 4
3b·TFA
pH 7
4b·HCl
pH 7
Reagent
Product
1·BetCl
pH 4
21
22a
23
24
26
27a
28a
29a
137
101
4
210
56
—
9
—
3
11
243
13
20
1
—
—
—
13
94
2
17
6
0.5
—
—
47
79
7
8
3
0.5
0.5
4
21
22b
23
24
26
27b
28b
29b
91
46
7
56
—
—
—
—
59
54
4
28
—
2
59
85
2
25
0.5
1
21
22c
23
24
26
27c
28c
29c
73
490
9
14
—
—
—
—
8
—
—
—
—
—
0.5
25
10
0.5
5
a See Scheme 7 for conditions and product structures.
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Table 4 Reaction of 3a, 3a·AcCl and 4a with dimethyldisulfide
was formed already during the fluoroalkylation step. Therefore,
reaction media degassing or preferentially oxidized additives
will not prevent the formation of traces of sulfoxide 23.
Yielda (%)
Conclusions
Reagent
Co-solvent
Time (h)
30
8
31
In conclusion, new hypervalent iodine reagents for fluoroalky-
lation of thiols were synthesized. They included 1,2-benzio-
doxol-3(1H)-one type reagents 4a and 4b, and open-chain acti-
vated reagents 1·BetCl, 3a·AcCl, 4b·HCl, 6 and 7. Reagents 6
and 7 were insoluble in aqueous media and therefore unsuita-
ble for thiol bioconjugation. Salt 1·BetCl was found to be an
excellent thiol fluoroalkylation reagent despite its limited
stability in aqueous media. N-Methyltyramine-derived reagent
3b·TFA efficiently fluoroalkylated free cysteine in acidic or
neutral pH and a related reagent 4b·HCl in neutral or basic
pH. Reagent 1·BetCl trifluoromethylated cysteine in a short
time over a wide range of pH values. Heptapetide DAVACAK
was used as a model for Cys conjugation in peptides. A
detailed MS analysis revealed that, apart from the expected
fluoroalkylated peptide, a range of side products were formed.
They arose from sulfur oxidation and elimination and acyla-
tion of nitrogen with acid fluoride derived from the decompo-
sition of the hypervalent iodine reagent. O18 isotope labelling
experiment proved that the oxygen of the sulfoxide side-
product originated from water and that the oxidation took
place during or before the fluoroalkylation step. The highest
sulfur fluoroalkylation conversion and minimal amount of
side-products were achieved using 3a·AcCl in acidic pH.
Despite the limitations in the reactivity of fluoroalkylated λ3-
iodanes, they represent the only available option for the intro-
duction of fluoroalkyl groups to the sulfur atom under bio-
compatible conditions. A new reactive mode for activated salt
hypervalent iodine reagents was identified – reaction with dis-
ulfides to yield fluoroalkylated sulfides. This reaction contribu-
ted significantly to the overall product yields. It also points to
the limitation of the reagents in cysteine bioconjugation, as
not only thiols but also disulfides can react.
3a·AcCl
3a·AcCl
3a·AcCl
3a·AcCl
3a·AcCl
3a
3a
4a
4a
4a
—
D2O
D2O
Buffer (pH 4)
Buffer (pH 7)
Buffer (pH 7)
D2O
Buffer (pH 7)
D2O
0.5
0.5
20
1
—
18
59
66
66
—
30
—
15
31
—
13
17
20
22
—
27
—
23
57
0
5
16
8
6
—
—
—
—
—
1
144
18
144
18
72
D2O
a
19F NMR yield using potassium trifluoroacetate as the internal
standard.
the free thiol and the disulfide form. This was verified by the
control reaction of 3a·AcCl with dimethyl disulfide in aceto-
nitrile, where no reaction took place. However, addition of
small amounts of water or buffer triggered the reaction to
afford conjugate 30 and products of decomposition of 3a·AcCl
(Table 4). Interestingly, with less activated 3a and 4a, the same
reaction did not take place in the acetonitrile/buffer (pH 7)
mixture, but in acetonitrile/water a very slow reaction was
observed pointing to a possibility of reagent activation with HF
(formed by the decomposition of PhOCF2CF2OH to 8).
Glutathione disulfide was fluoroalkylated with 3a·AcCl (pH
4
or 7) under the same conditions as DAVACAK.
S-Fluoroalkylated glutathione formed cleanly as the major
product (Scheme 8, Table S8†) with a small amount of the
corresponding sulfoxide. When the pre-reduction with TCEP
(step 1) was omitted, only half the amount of the fluoroalky-
lated product was detected, when the same amount of gluta-
thione was consumed. This supports the hypothesis that only
one-half of the disulfide reacted in the fluoroalkylation reac-
tion. Moreover, even the less reactive reagents 3a and 4a fluor-
oalkylated glutathione disulfide under these conditions.
The source of oxygen in the sulfoxide side-product 23 was
investigated. When fluoroalkylation of DAVACAK was per-
formed in H2O18, O18 is clearly incorporated into 23 (see ESI,
Tables S6 and S7†). The amount of 23 was the same after 1 h
or 24 h of reaction. When the fluoroalkylation was performed
in the presence of methionine (20 equiv.), no incorporation of
O18 into the sulfur of methionine was observed, which
suggests that 23 was not formed from 22 but more probably
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the Czech Science
Foundation (17-00598S) and the Academy of Sciences of the
Czech Republic (RVO: 61388963).
Notes and references
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Scheme 8 Fluoroalkylation of glutathione.
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