Rapid, stable, chemoselective labeling of thiols with Julia- Kocienski-like reagents: A serum-stable alternative to maleimide-based protein conjugation

Article abstract of DOI:10.1002/anie.201306241

Exquisite chemoselectivity for cysteine has been found for methylsulfonylphenyloxadiazole compounds under various buffer conditions. Furthermore, the resulting protein conjugates have superior stability to cysteine-maleimide conjugates in human plasma (HS

Full text of DOI:10.1002/anie.201306241

Angewandte
Chemie
DOI: 10.1002/anie.201306241
Bioconjugation
Hot Paper
Rapid, Stable, Chemoselective Labeling of Thiols with Julia–Kocien´ski-
like Reagents: A Serum-Stable Alternative to Maleimide-Based
Protein Conjugation**
Narihiro Toda, Shigehiro Asano, and Carlos F. Barbas III*
Proteins modified with fluorescent or biologically active
agents are powerful reagents in chemistry, biology, and
medicine.[1] Selectivity for certain of the natural 20 amino
acids in the presence of many unprotected amino acid
Figure 1. Methylsulfonylbenzothiazole (1), a selective protein-thiol
blocking reagent.
residues is necessary for the selective modification of
proteins.[2] The development of bioconjugation reactions has
largely focused on the modification of lysine and cysteine side
chains. Conjugation to cysteine, which is a rare amino acid
and often exists as a disulfide pair in native proteins, can be
readily achieved as a consequence of the relatively low
pKa value and potent nucleophilicity of the thiolate anion.[3]
Significant research efforts have been made to identify
reagents that enable blocking or labeling of protein thiols
with high selectivity and conversion yields.[4] Among those,
alkylation reagents for thiols (such as a-halocarbonyl deriv-
atives) or Michael acceptors (such as maleimide derivatives)
are the most commonly used, and their reactivity profiles
have been studied extensively. Indeed, antibody–drug con-
jugates made through the maleimide–cysteine conjugation
method are approved drugs.[5] Additionally, maleimide–
cysteine conjugation is used to make albumin-binding pro-
drugs, which are rapidly and selectively bound to the cysteine-
34 position of endogenous albumin in the blood.[6] Although
maleimide chemistry is a powerful tool for the selective
modification of proteins, limitations have been reported.[7]
The succinimide linkage of the maleimide addition product is
susceptible to hydrolysis, and the thioether moiety undergoes
exchange reactions with reactive thiols (such as those of
albumin) and with free cysteine and glutathione residues
through the retro-Michael reaction. As a result, heteroge-
neous mixtures of the conjugates can be produced in the
blood that have different pharmacokinetics, in vivo efficacy,
and toxicity.[8] Therefore, a cysteine-selective conjugation
method that results in a stable linkage is desired. Recently,
methylsulfonylbenzothiazole (1, MSBT; Figure 1) was
reported as a thiol-blocking reagent.[9] The stability of
MSBT-blocked thiols, however, and the broad versatility of
methylsulfonyl-functionalized heteroaromatic compounds in
thiol conjugation chemistry remains unexplored. Herein, we
designed and optimized methylsulfonyl-functionalized
heteroaromatic derivatives for rapid protein/peptide conju-
gation and demonstrate that these conjugates can be signifi-
cantly more stable than maleimide–cysteine conjugates in
human plasma, thereby providing a promising and versatile
new approach to protein and peptide conjugates for chemis-
try, biology, and medicine.
The recent disclosure of MSBT (1, Figure 1) as a selective
protein thiol blocking reagent[9] stimulated our thinking
concerning the reactivity of molecules in this structural
class. The compound 2-(alkylsulfonyl)benzothiazole is
known as a substrate for a modified one-pot Julia–Lythgoe
olefination.[10] Furthermore, Kocienski and co-workers
´
reported the use of related phenyltetrazole derivatives in
a
stereoselective synthesis of trans-1,2-disubstituted
alkenes.[11] One-pot Julia–Lythgoe olefination by these sub-
strates is believed to proceed through a Smiles rearrangement
on the heteroaromatic ring (see Figure S1A in the Supporting
Information). Furthermore, benzimidazole-derived proton-
pump inhibitors are also subject to the Smiles rearrangement
under acidic conditions (see Figure S1B in the Supporting
Information).[12] The reactivity of MSBT and this class of
molecules led us to hypothesize that a broader class of
heteroaromatic methylsulfones might be exploited to develop
a new class of thiol-reactive molecules for thiol-selective
conjugation of peptides and proteins.
To explore the reactivity of this class of molecules and the
relative stability of the thiol conjugates they might form, we
synthesized a family of these molecules, guided by the known
[*] Dr. N. Toda,[+] Dr. S. Asano,[+] Prof. Dr. C. F. Barbas III
The Skaggs Institute for Chemical Biology and the Departments of
Chemistry and Molecular and Cell Biology
´
reactivity of Julia–Kocienski reagents. A generalized syn-
thesis is shown in Scheme 1. Methylation of the thiol group of
heteroaromatic derivatives 2 gave the corresponding methyl
thioether compounds. Methyl thioether derivatives 3 were
converted into methylsulfone compounds 4 by oxidation with
hydrogen peroxide in the presence of ammonium molybdate
as a catalyst.[13]
Next, in order to study protein conjugation, fluorescein
derivatives of the sulfone and maleimide compounds 7 and 10
were synthesized (Scheme 2). The methyl thioethers 5 (see
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail: carlos@scripps.edu
[+] These authors contributed equally to this work.
[**] This study was supported by National Institutes of Health Pioneer
Award DP1 CA174426.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201306241.
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eimide 10 as a single isomer at the 5-position. PEGylated
derivative 11 was synthesized with commercially available
NHS-PEG derivative SUNBRIGHT ME-050AS from azide
6c in two steps (Scheme 3).
Scheme 1. Synthesis of methylsulfone derivatives. Reagents and con-
ditions: a) CH3I, TEA, THF, RT, 1–2 h, 3a 78%, 3b 94%, 3c 84%;
b) (NH4)6[Mo7O24]·4H2O, 30% H2O2, C2H5OH, RT, 2-6 h, 4a 76%, 4b
54%, 4c 84%, 4d 89%. TEA=triethylamine, THF=tetrahydrofuran.
Scheme S2 in the Supporting Information) were oxidized with
mCPBA to afford the corresponding methylsulfone com-
pounds 6. The azide groups of compounds 6 were reduced by
hydrogenation or through the Staudinger reaction, and these
primary amines were coupled with commercially available
NHS-5(6)-fluorescein to yield the desired fluorescein com-
pounds 7. The maleimide derivative was prepared by coupling
Boc-protected 1,3-propanediamine (8) with NHS-5(6)-fluo-
rescein. Deprotection gave the primary amines, and a subse-
quent coupling with Sulfo-SMCC yielded the desired mal-
Scheme 3. Synthesis of PEGylated derivative. Reagents and conditions:
a) 1. PPh3, H2O, THF; 2. SUNBRIGHT ME-050AS, CH3CN, 14%.
We first examined the relative reactivity of sulfones (1,
4a–d) and commercially available maleimide (12) with (R)-2-
acetamido-N-benzyl-3-mercaptopropanamide (13; Table 1
and see Figure S2a–d in the Supporting Information). Com-
pounds 1, 4a–d, or 12 were added to a solution of 1.2 equiv-
alents of protected cysteine 13 in THF/200 mm PBS, and the
reactions monitored by HPLC (220 nm). The conversion of
phenyltetrazole compound 4a, which is known as a key
Table 1: Reaction of methylsulfonyl or maleimide derivatives 1, 4a–d,
and 12 with protected cysteine 13.[a]
Entry
1
Substrate
R
Conversion [%] [5 min][b]
1
45[c] (14a)
2
3
4
4a
4b
4c
93 (14b)
>99 (14c)
no reaction
5
6
4d
12
no reaction
Scheme 2. Synthesis of fluorescein derivatives. Reagents and condi-
tions: a) mCPBA, CH2Cl2, 6a 51%, 6b 63%, 6c 37%; b) 1. 10% Pd/C,
H2, THF or PPh3, H2O, THF; 2. NHS-5(6)-fluorescein, TEA, DMSO, 7a
12%, 7b 28%, 7c 14%; c) NHS-5(6)-fluorescein, TEA, DMSO, 9a
38%, 9b 54%; d) 1. TFA, CH2Cl2; 2. Sulfo-SMCC, TEA, DMF, 51%.
Boc=tert-butoxycarbonyl, mCPBA=m-chloroperbenzoic acid, NHS-
5(6)-fluorescein=[fluorescein-5(6)-carboxamido]hexanoic acid N-
hydroxysuccinimide ester, DMSO=dimethylsulfoxide, TFA=trifluoro-
acetic acid, DMF=N,N-dimethylformamide.
>99[d] (14d)
[a] Reagents and conditions: (R)-2-acetamido-N-benzyl-3-mercaptopro-
panamide 13 (1.2 equiv), THF/pH 7.4, 200 mm PBS (1:1=v/v), room
temperature. [b] Conversion was measured by HPLC (220 nm). [c] 86%
[60 min]. [d] Diastereomeric mixture. PBS=phosphate buffered saline.
2
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´
structure for Julia–Kocienski olefination, was faster than that
a protein. We evaluated reactions with recombinant human
of benzothiazole 1, while triazole compound 4c did not react
with 13. We were pleased to discover that phenyloxadiazole
compound 4b reacted most rapidly and provided the desired
conjugate 14c in quantitative yield after only 5 min (Table 1,
entry 3). In contrast, benzimidazole compound 4d, chosen
based on the core structure of known proton-pump inhibitors,
was unreactive. In the case of 4d, activation of the benzimid-
azole ring by strong acid might be necessary for reactivity.
Given the promising reactivity of phenyloxadiazole com-
pound 4b, we studied its reactivity with other potential
nucleophilic species in proteins. Thus, the reactivity of 4b with
amino acid derivates 13 and 15a–e was evaluated (see
Scheme S4 in the Supporting Information). Only the pro-
tected cysteine–phenyloxadiazole adduct was observed. The
other amino acid derivatives (15a–e) did not react with 4b.
Thus, phenyloxadiazole derivative 4b was selective for the
free thiol of cysteine and substantially more reactive than
1 (Table 1).
serum albumin (HSA), in which Cys34 is reactive, and
a fusion protein of our design based on the maltose-binding
protein, MBP-C-HA, which has a single cysteine residue in
the linker between the protein and the HA peptide tag.
Compound 6c (10 equiv) was added to a solution of
recombinant HSA in PBS at room temperature, and the
reaction was incubated for 2 h at room temperature. ESI-MS
analysis indicated that HSA was modified with a single label
(Figure 2A and see Figure S3 in the Supporting Information),
To further investigate of the reactivity of 4b we studied
the effects of buffer concentration and pH dependence on the
reaction of 4b with 13 (see Table S1 in the Supporting
Information). When this reaction was performed in pure
organic solvents (e.g. THF; Table S1, entry 1), the desired
adduct 14c was not observed. In the case of a 1:1 mixture of
THF and water, 14c was detected but conversion was low
(Table S1, entry 2). For reactions at pH 7.4, reaction con-
version increased with increasing buffer concentration
(Table S1, entries 3–7). The conversion in HEPES (2-[4-(2-
hydroxethyl)-1-piperazinyl]ethanesulfonic acid) or Tris
(tris(hydroxymethyl)aminomethane) buffer at pH 7.4 with
THF (1:1, v/v) was similar to that in PBS (Table S1, entries 6,
11, and 12). Conversion was complete within 30 min at pH 7.0
and pH 8.0, but was moderate at pH 5.8 (Table S1, entries 8–
10).
We also studied the substrate scope of the thiol-modifying
reaction (Table 2). 4b reacted with cysteine derivatives 16b
and 16c, which have free amino and carboxy groups,
respectively (entries 2 and 3). These studies suggest that the
sulfone compound should react with the thiol group of
cysteine residues in peptides or proteins regardless of their
location.
Figure 2. Thiol labeling of recombinant HSA and MBP-C-HA using
1,3,4-oxadiazole derivatives 6c, 7c, and 11. A) Conjugation reaction of
oxadiazole compound (6c) with recombinant HSA, and the ESI-MS
spectra of the conjugate. B) Gel image of the conjugation reaction of
fluorescein-attached compound (7c) in the presence or absence of
iodoadetamide. C) Gel image of the conjugation reaction of PEGylated
oxadiazole compound (11) with reduced MBP-C-HA protein.
Next, we investigated whether our sulfone derivatives
were able to selectively modify cysteine in the context of
which suggest that Cys34 was labeled with compound 6c. To
obtain further evidence for specific cysteine labeling we
reduced the disulfide bond of the homodimer MBP-C-HA by
treatment with 1,4-dithiothreitol (DTT) or tris(2-carboxy-
ethyl)phosphine hydrochloride (TCEP). The reduced MBP-
C-HA was incubated in the presence or absence of iodoacet-
amide (IAA, 50 equiv) at room temperature. Fluorescein-
linked phenyloxadiazole 7c (10 equiv) was added to the
reaction solution after 1 h, and then the solution was
incubated for 1 h. Analysis by SDS-PAGE (sodium dodecyl
sulfate polyacrylamide electrophoresis) showed strong fluo-
rescence of the MBP-C-HA conjugate without IAA blocking
(lanes 2 and 4 in Figure 2B) even though pretreatment of
reduced MBP-C-HA with IAA suppressed the labeling for
compound 7c (lanes 3 and 5 in Figure 2B). Unreduced MBP-
Table 2: Reactions of compound 4b with thiol substrates.
Entry
R-SH
Yield [%]
99 (17a)
78 (17b)
1
2
3
4
83 (17c)
90 (17d)
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Table 3: Stability evaluation.
C-HA migrated as a homodimer of around 100 kDa and did
not fluoresce. Additionally, the fluorescein-attached MBP-C-
HA conjugate was detected by ESI-MS (Figure S4b in the
Supporting Information). These results suggest that com-
pound 7c modified only the single cysteine residue in MBP-C-
HA and is exquisitely chemoselective since no labeling of the
disulfide linked homodimer was observed. The introduction
of poly(ethylene) glycol chains (PEGylation) is widely used to
modify the pharmacokinetic properties of proteins, typically
through maleimide-labeling reactions.[14] To study the poten-
tial of this sulfone approach in protein PEGylation we studied
the reaction of compound 11, which has a linear 5 kDa PEG
chain, with MBP-C-HA. Compound 11 selectively pegylated
MBP-C-HA at a single site (Figure 2C and Figure S5 in the
Supporting Information), thus suggesting the broad potential
of this approach for the preparation of protein therapeutics.
Finally, we investigated the stability of our conjugates
under several conditions. Significantly, under basic conditions,
all the heteroaryl compounds (14a–c) were more stable than
the maleimide–cysteine conjugate (14d, Table 3). For exam-
ple, no degradation of the cysteine–benzothiazole conjugate
14a was observed under these basic conditions. The succini-
mide ring of the maleimide–cysteine conjugate, however, was
opened under these basic conditions, and hydrolyzed products
were observed (see Figure S6d in the Supporting Informa-
tion). No degradation of the heteroaryl conjugates (14a–c)
was observed after 3 days in acidic conditions. 14a–c were
stable in the presence of glutathione at a neutral pH value for
5 days, but an exchange reaction occurred from N-benzylcys-
teine to glutathione in maleimide–cysteine conjugate 14d (see
Figure S8d in the Supporting Information). These results
indicate that these heteroaromatic–cysteine conjugates have
superior stability relative to the maleimide–cysteine conju-
gate in a variety of conditions.
Conjugate
Conditions[a]
% remaining[b]
A
B
C
D
>99
>99
>99
94
14a
A
B
C
D
88
98
>99
>99
14b
14c
14d
A
B
C
D
82
>99
>99
99
A
B
C
D
2.7
71
69
34
[a] A) K2CO3 (4 equiv), THF/H2O (1:1), room temperature, 20 h; B) THF/
0.1m HClaq (1:1), room temperature, 3 days; C) THF/pH 4.0 200 mm
PBS (1:1), room temperature, 3 days; D) glutathione (3 equiv), THF/
pH 7.4 200 mm PBS (2:3), 378C, 5 days. [b] HPLC (254 nm).
analyzed by HPLC, and the amount of conjugate remaining
was calculated based on an internal standard. Cysteine-
heteroaryl conjugates 14a–c were more stable than cysteine–
maleimide conjugate 14d (green line, t1/2 = 4.3 h; Figure 3A);
benzothiazole derivative 14a had the longest half-life (blue
line, t1/2 = 191 h). After 72 h, the solution of 14c was analyzed
by HPLC with compound 2b, which was expected as a by-
product (see Figure S9d in the Supporting Information) and
then by LC/MS to confirm two unknown peaks (see Fig-
ure S9e in the Supporting Information). The result showed
that the peak at 9.79 min in the HPLC trace was compound
2b, and both the exo-methylene b-eliminated product and
compound 2b were detected by LC/MS. To study the stability
of the labeled MBP-C-HA protein monomers, the disulfides
were reduced with TCEP and the protein incubated with
fluorescein-modified reactants (7a–c and 10). After removal
To evaluate our conjugates under therapeutically relevant
conditions we evaluated the stability of the cysteine con-
jugates (14a–d) and MBP-C-HA conjugates (20a–d) in
human plasma. Solutions of the cysteine conjugates (14a–d,
50 mL) in DMSO were added to human plasma (950 mL), and
the solutions were incubated for 72 h at 378C. After
precipitation of protein with CH3CN, the supernatants were
Figure 3. Stability of the cysteine conjugates (14a–d) and MBP-C-HA conjugates (20a–d) in human plasma, benzothiazole (blue), phenyltetrazole
(black), phenyloxadiazole (red), and meleimide (green). A) Time-dependent stability of the cysteine conjugates (14a–d) in human plasma.
B) Relative reaction rate of the conjugation reaction of fluorescein-attached compounds (7a–c and 10) with MBP-C-HA protein in PBS (pH 7.4).
C) Gel image and plot of percentage remaining versus time of fluorescein-attached MBP-C-HA conjugates (20a–d).
4
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of excess small molecules, the MBP-C-HA conjugates (20a–
d) were mixed with human plasma, and the solutions were
incubated for 72 h at 378C. The phenyloxadiazole derivative
7c reacted more rapidly than the other fluorescein-modified
reactants with MBP-C-HA. The efficiency of the reaction of
7b was similar to that of maleimide compound 10, which is
known to react with cysteine residues of proteins (Figure 3B
and see Figure S10 in the Supporting Information). Benzo-
thiazole derivative 7a reacted slowly with MBP-C-HA and
the protein monomer was re-oxidized to the disulfide dimer
(see Figure S10 in the Supporting Information, red arrow).
The stabilities of MBP-C-HA conjugates (20a–d) in human
plasma were analyzed by SDS-PAGE (Figure 3C). No
fluorescent bands were observed at the molecular weight
expected for the sulfone–HSA conjugates of 7a–c. Fluores-
cent bands gradually appeared in the maleimide–MBP-C-HA
conjugate lanes (Figure 3C, red arrow) at the molecular
weight of HSA. This assay was designed to explore the
exchange reaction from labeled MBP-C-HA to Cys34 of
HSA, the most abundant protein in human plasma and
a troublesome side reaction of therapeutic drug conjugates.
We observed in the Coomassie-stained gel of maleimide–
protein conjugate 20d that the free MBP-C-HA monomer,
which was re-generated by an exchange reaction with HSA in
human plasma, formed a heterodimer with another protein,
which was likely HSA (Figure 3C, green arrow). The MBP-C-
HA homodimer was not formed under these conditions
(Figure 3C, blue arrow). The heterodimer was also observed
when the benzothiazole–MBP-C-HA conjugate (20a) was
incubated in plasma. As previously mentioned, the conjuga-
tion reaction with benzothiazole derivative 7a was slow;
therefore, the solution included MBP-C-HA homodimer,
formed by reoxidation during the conjugation reaction, and
an exchange reaction from the MBP-C-HA homodimer to an
S-S exchanged heterodimer occurred (Figure 3C, green
arrow). The half-lives of conjugates 20a–d were 135, 84.4,
117, and 59.9 h, respectively. All the heteroaromatic–protein
conjugates were more stable than the maleimide–protein
conjugate in plasma.
In conclusion, we have developed a class of sulfone
derivatives for applications in protein conjugation chemistry,
and we have compared the newly synthesized conjugates to
maleimide conjugates. Methylsulfonyl-functionalized five-
membered monocyclic compounds, such as phenyltetrazole
or phenyloxadiazole, reacted rapidly and specifically with
thiols in small molecules and proteins with exquisite chemo-
selectivity at biologically relevant pH values (pH 5.8–8.0).
Designer heteroaromatic sulfones allowed for the selective
introduction of a fluorophore and poly(ethylene) glycol
chains (PEGylation), and provided protein conjugates with
superior stability compared to maleimide-conjugated proteins
in human plasma. Given the speed, selectivity, and stability of
the sulfone–cysteine reactions described herein, we anticipate
that this “thiol-click” approach will find broad application in
peptide and protein chemistry and for the development of
antibody drug conjugates.[5,8a,15,16]
Received: July 18, 2013
Published online: && &&, &&&&
Keywords: chemoselectivity · cysteine · drug conjugates ·
.
phenyloxadiazole · protein conjugation
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Communications
Bioconjugation
N. Toda, S. Asano,
C. F. Barbas*
&&&& — &&&&
Rapid, Stable, Chemoselective Labeling of
´
Thiols with Julia–Kocienski-like Reagents:
A Serum-Stable Alternative to Maleimide-
Based Protein Conjugation
Exquisite chemoselectivity for cysteine
has been found for methylsulfonyl-
phenyloxadiazole compounds under var-
ious buffer conditions. Furthermore, the
resulting protein conjugates having
superior stability to cysteine–maleimide
conjugates in human plasma (HSA=
human serum albumin, MBP-C-HA=
maltose-binding protein). This new thiol-
click reaction offers a new approach to
generate stable protein conjugates and
pegylated proteins.
6
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ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 6
These are not the final page numbers!