Bioconjugation with Thiols by Benzylic Substitution

Article abstract of DOI:10.1002/chem.201706149

A benzylic substitution of 3-indolyl(hydroxyl)acetate derivatives with thiols proceeded specifically in the presence of amino, carboxy, and phosphate groups in weakly acidic aqueous solutions under nearly physiological condition, while no reaction occurred at pH over 7. Kinetic studies revealed that the reaction followed second-order kinetics (first-order in the reactant and first-order in thiol) in contrast with the S_N1 mechanism of common benzylic substitution of alcohols. The utility of the present method for functionalization of biomacromolecules was demonstrated using several model proteins, such as lysozyme, insulin, trypsin, and serum albumin. The catalytic bioactivity of lysozyme in lysis of Micrococcus lysodeikticus cells was completely retained after the modification.

Full text of DOI:10.1002/chem.201706149

DOI: 10.1002/chem.201706149
Communication
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Proteins |Hot Paper|
Bioconjugation with Thiols by Benzylic Substitution
Kenji Watanabe* and Takashi Ohshima*[a]
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Abstract: A benzylic substitution of 3-indolyl(hydroxyl)-
acetate derivatives with thiols proceeded specifically in
the presence of amino, carboxy, and phosphate groups in
weakly acidic aqueous solutions under nearly physiologi-
cal condition, while no reaction occurred at pH over 7. Ki-
netic studies revealed that the reaction followed second-
order kinetics (first-order in the reactant and first-order in
thiol) in contrast with the SN1 mechanism of common
benzylic substitution of alcohols. The utility of the present
method for functionalization of biomacromolecules was
demonstrated using several model proteins, such as lyso-
zyme, insulin, trypsin, and serum albumin. The catalytic
bioactivity of lysozyme in lysis of Micrococcus lysodeikti-
cus cells was completely retained after the modification.
Conjugation between natural functional groups in biomole-
cules and synthetic compounds has various applications, such
as the synthesis of biologic tools for elucidating biofunctions
of living organisms in situ,[1] preparing antibody-drug conju-
gates,[2] and constructing tailor-made libraries of bioactive mol-
ecules.[3] Therefore, many scientists are interested in these reac-
tions, but the development of suitable reactions for biocon-
junction remains difficult. Criteria for bioconjugation are (i) suf-
ficient reactivity in aqueous medium at mild temperature, (ii)
functional group selectivity/tolerance, (iii) high regioselectivity,
and (iv) safety of reagents and co-products formed during the
reaction.[4,5] These requirements limit the reaction types appli-
cable for bioconjugation.
Figure 1. Preceding reactions used in bioconjugation (a), present study (b)
and 3-indolyl(hydroxyl)acetates having reactive benzylic CÀO bonds (c).
groups at the benzylic position can be activated by an action
of Lewis and Brønsted acids,[18] and substitution reactions by
nucleophiles proceeded via carbocation intermediates[19] Thus,
we hypothesized that benzylic substitutions of appropriately
designed reactants could be used for a selective bioconju-
gation with thiol at a weakly acidic pH under nearly physiologi-
cal conditions (Figure 1b). Since this reaction, involving the ac-
tivation of benzylic hydroxyl group, proceeds under weakly
acidic conditions, the applicable pH range differs from those of
the preceding reactions (Figure 1a). Moreover, since this reac-
tion gives water as the only co-product, and does not use
heavy metal, light irradiation, or radical initiator, it is highly bio-
compatible.
Cysteine is often used as a target for selective bioconju-
gation because it has a reactive thiol group, except when
forming a disulfide bond.[5] Michael addition of thiol to a,b-un-
saturated carbonyl[6]/sulfone,[7] substitution of thiol with haloal-
kane[8]/haloarene[9]/organopalladium[10]
compounds,
thiol-
ene,[11] thiol-yne[12] and disulfide exchange[13] are representative
reaction types used in this field. The Michael addition and sub-
stitution reactions are often carried out at pH 6.5–7.5[14] and
pH 7.2–9.0,[4] respectively (Figure 1a). Although, the reactions
are well established and have already been applied to the
functionalization of bioactive peptides and protein conju-
gates,[5,15] concerns regarding the exchange of thiol in the mal-
eimide adduct, oxidation of thiol to disulfide, and competition
with amine nucleophile at high pH still remain.
Screening of several substrates with a reactive CÀO bond at
the benzylic position (see Table S1 in the Supporting Informa-
tion) revealed that the class of water-soluble indole derivatives
shown in Figure 1c was the most reactive against thiol nucleo-
philes. These indole derivatives were synthesized from com-
mercially available starting materials in 1–7 steps (see the Sup-
porting Information).
Our research group has been studying the direct catalytic
activation of allylic alcohol through p-allyl metal formation.[16]
During the course of our studies,[17] we realized that hydroxyl
1
Figure 2 shows the H NMR spectra of the reaction mixtures
of 3-indolyl(hydroxyl)acetate 2 (0.010m) and glutathione in
sodium phosphate (0.20m) D2O solutions at the indicated pD
for 378C (after 4 h). At pD 7.4 with 1.2 equivalents of gluta-
thione, almost no changes in the aromatic and benzylic signals
of 2 were observed (Figure 2a) compared with the signals of 2
in D2O without glutathione (Figure 2d), suggesting that 2 did
not react with glutathione at pD 7.4. Full conversion of 2 and
new signals attributed to the thiol-adduct 8 were observed in
the pD 5.4 solution (Figure 2b; see the Supporting Information
[a] Dr. K. Watanabe, Prof. Dr. T. Ohshima
Graduate School of Pharmaceutical Sciences
Kyushu University
Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)
E-mail: kwatanabe@kyudai.jp
ohshima@phar.kyushu-u.ac.jp
Homepage: http://green.phar.kyushu-u.ac.jp/index_e.html
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under https://doi.org/10.1002/
chem.201706149.
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for characterization of 8). When 2 was reacted with 0.6 equiva-
lents of glutathione, a mixture of 8 and unreacted 2 was ob-
served (Figure 2c), suggesting that 2 selectively reacted with
thiol and was intact against the amino and carboxylate nucleo-
philes of the peptide. The reaction at pD 4.0 proceeded
smoothly, similar to that at pD 5.4 (see Figure S1 in the Sup-
porting Information). 3-indolyl(hydroxyl)acetate 2 remained
intact against both amine and thiol nucleophiles even at
pD 9.5 (Figure S1), suggesting that the activation of benzylic
hydroxyl group was essential in the reaction. Exchange of thiol
was not observed by the treatment of 8 with 10 equivalents of
2-mercaptoethanol at pD 5.4 and 378C, indicting a high stabili-
ty of the thioether linkage under physiological conditions (see
FigureS2 in the Supporting Information). In addition, 2 itself
was stable at pD 5.4 (no decomposition).
Next, the kinetics of the reaction between 2 (0.00010m) and
glutathione (0.0050m) were studied in a UV/Vis experiment
(Figure 3a). Systematic spectral changes with the existence of
isosbestic points indicated clean consumption of 2, even in di-
luted conditions. When excess amounts of glutathione were
present (50 equiv), the concentration of glutathione during the
reaction can be considered as steady-state, and the reaction
was assumed to be a pseudo first-order reaction [Eqs. (S1), (S2)
in the Supporting Information].
Figure 2. NMR spectra of reaction mixtures of 2 (0.010m) and glutathione in
sodium phosphate (0.20m) D2O solutions at 378C for 4 h. a) 2 + glutathione
(1.2 equiv) at pD 7.4. b) 2 + glutathione (1.2 equiv) at pD 5.4. c) 2 + gluta-
thione (0.6 equiv) at pD 5.4. d) 2 only in D2O. The aromatic and benzylic
peaks were shown for clarity. The reaction mixtures were neutralized with
NaDCO3 before the NMR measurements.
Non-linear fitting using Eq. (S2) to the absorbance changes
showed excellent agreement (R2 =0.9998) and supported a
second-order reaction (Figure 3b). The reaction orders with re-
Figure 3. a) UV/Vis spectral changes of 2 (0.00010m) upon reaction with glutathione (0.0050m) in a pH 5.4 sodium phosphate (0.20m) aqueous solution at
378C. The spectra were recorded at 2 h interval. b) Non-linear fitting of absorbance changes of 2 at 316 nm to Eq. (2). c) Correlation between k values and
^
^
number of methoxy on indole: *=1, =2, ~=3, *=4, =5. d) Steric effect of thiol nucleophiles. e) Eyling plot of the reaction between 2 and gluta-
thione at varied temperatures (27, 37, 47 and 578C). f) Plots of k values at varied pH (pH 3.5, 4.5, 5.4, 6.4 and 7.4).
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spect to 2 and glutathione were 1.09 and 0.93 (sum=2) as de-
termined from the relationship between the initial rate and the
substrate concentrations (see Figure S3 and S4 in the Support-
ing Information). Interestingly, although benzylic substitution
of alcohols generally proceeded through an SN1 mechanism,
where formation of the carbocation intermediates is the rate-
determining step,[19e] the present benzylic substitution reaction
had a second-order dependency on the concentration of both
substrates, indicating that the reaction between the iminium
cations and thiols are the rate-determining step (Figure 1b). To
the best of our knowledge, this is the first example of a
second-order reaction of benzylic substitution of alcohols in
homogeneous aqueous media.
large excess amounts of phosphate or acetate anion existed
against 2 (2000 equiv), counteranions in the buffer solution
seemed to have minor effects on the reaction. Substrate 2 re-
mained reactive at pH 6.4 (entry 9; 4.1-fold reduced rate),
while the reactivity completely diminished at pH 7.4 (entry 10),
consistent with the results of NMR studies.
Figure 3e shows the Eyring plot obtained from the k values
of 2 at various temperatures (27 to 578C). The activation pa-
rameters Ea, DG°, DH°, and ÀTDS° at 378C were determined
to be 17.1, 21.1, 16.5, and 4.6 kcalmolÀ1, respectively, and the
DH° term was the predominant requirement for the activation.
Interestingly, the steep increase in the k values was observed
at the pH below 4.5 (Figure 3 f). In addition, ethyl 2-hydroxy-2-
(1H-indol-3-yl)acetate (7), which has an ester instead of a car-
boxylate, exhibited no reactivity at pH 5.4 (Table 1, entry 7);
the carboxy moiety was essential for the reaction.[18c]
Similar experiments were performed with various 3-indolyl-
(hydroxyl)acetates 1–7, and the rate constants (k) of those sub-
strates are summarized in Table 1. The k values increased de-
The utility of the present reaction for site-selective bioconju-
gation was studied using lysozyme (chicken egg-white) as a
model protein. Lysozyme is a 14.3 kDa enzyme that catalyzes
the hydrolysis of peptidoglycans on bacterial cell walls. The
enzyme contains four disulfide bridges, and the C6ÀC127
bridge is exposed on the protein surface, while the other three
bonds are buried inside.[20] Although cysteine can be mutage-
netically introduced to proteins,[21] thiols derived from reduc-
tion of native disulfide are also often use for bioconjugation.[22]
Reduction of native enzymes with 1.0 equivalent of tris(2-car-
boxyethyl)phosphine hydrochloride (TCEP)[22] at pH 4.5, and
the following one-pot addition of 2 afforded bioconjunction
compound 9 (Figure 4). ESI-TOF MS of 9 showed m/z=1461.2,
Table 1. Rate constants (k) of substrates 1–7 and calculated electron den-
sity of the aromatic systems.
[a]
Entry
Substrate
k [mÀ1 sÀ1
]
Electron density
1
2
3
4
5
6
7
8[b]
9[c]
10[d]
1
2
3
4
5
6
7
2
2
2
3.9Æ0.5”10À3
8.0Æ1.5”10À3
15.0Æ1.6”10À3
3.3Æ0.6”10À3
7.3Æ1.0”10À3
8.8Æ0.9”10À3
no reaction
7.4Æ1.3”10À3
1.5Æ0.1”10À3
no reaction
À1.507
À0.976
À0.437
À1.537
À1.006
À1.006
À1.588
À0.976
À0.976
À0.976
[a] Average of three experiments (Æ standard error). [b] pH 5.4 sodium
acetate. [c] pH 6.4 sodium phosphate. [d] pH 7.4 sodium phosphate.
pending on the number of methoxy groups on the indole (Fig-
ure 3c), indicating that the electron density of the indole aro-
matic systems influenced the reactivity (Table 1, entries 1–5).
When the number of methoxy groups was the same, N-alkylat-
ed and NH-free indole-substrates had similar k values (entry 1
vs. 4, entry 2 vs. 5), indicating that the N-alkylation of indole
had a minor influence on the reaction rate. When 18, 28 and 38
thiols were used as the nucleophiles, the k values decreased
with increasing steric congestion, but very importantly, even 38
thiols had high reactivity (Figure 3d).
Figure 4. Preparation of protein conjugates 9–12.
which corresponds to [M+10H]10+ of 9, in which one unit of 2
was introduced to the enzyme (Table 2, entry 1). The amount
of 2 introduced to the protein was determined to be m=1.03
based on the absorbance of 5-methoxyindole at 316 nm (e=
3180MÀ1 cmÀ1; see the Supporting Information). The amount
of remaining thiol in the product was quantified by Ellman’s
Almost the same k values were obtained when the reaction
was performed in sodium acetate solution instead of a sodium
phosphate solution at pH 5.4 (Table 1, entry 2 vs. 8). Although
Table 2. ESI-TOF MS of native and modified proteins (9–12). Reaction time and amounts of 2 (m) and thiol (n) in the products.
Entry
Protein
Calculated m/z
Found m/z
Time [h]
Product
Calculated m/z
Found m/z
m
n
1[a]
2[b]
3[b]
4[c]
lysozyme
insulin
trypsin
[M+10H]10+ =1432.4
[M+5H]5+ =1160.1
[M+17H]18+ =1295.7
[M+47H]47+ =1414.4
1432.4
1160.1
1295.7
1414.4
15
3
15
15
9
[M+10H]10+ =1461.2
[M+5H]5+ =1217.8
[M+17H]18+ =1311.7
[M+47H]47+ =1420.5
1461.2
1217.8
1311.7
1420.5
1.03
0.95
0.89
0.65
0.94
0.85
0.93
0.40
10
11
12
serum albumin
[a] [protein]=0.0001m, [2]=0.0002m (2 equiv). [b] [protein]=0.0001m, [2]=0.0001m (1 equiv). [c] [protein]=0.001m, [2]=0.002m (2 equiv).
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test (Table 1, n=0.94).[13c] Oxidation of thiol to disulfide was
not observed under the weakly acidic pH. The second-order
rate constant of the reaction between 2 and reduced lysozyme
was determined to be 0.13Æ0.01MÀ1 sÀ1 (see Figure S5 in the
Supporting Information), which was moderate compared to
Acknowledgements
Grant-in-Aid for Scientific Research on Innovative Areas (JSPS
KAKENHI Grant Number JP15H05846 in Middle Molecular Strat-
egy for T.O.) and Grant-in-Aid for Young Scientists (B) (JSPS KA-
KENHI Grant Number 16K17902 for K.W.) from JSPS, Platform
Project for Supporting Drug Discovery and Life Science Re-
search (Basis for Supporting Innovative Drug Discovery and
Life Science Research (BINDS)) from AMED (for T.O. and K.W.).
K.W. acknowledges Ajinomoto Award in Synthetic Organic
Chemistry, Japan for financial support. The authors acknowl-
edge Drs. Hiroyuki Morimoto and Kazutru Usui for discussion
in the reaction mechanism and CD measurement. The authors
acknowledge Ms. Megumi Noshita, Mr. Ryohei Yonesaki and
Mr. Tsukushi Tanaka for help in HRMS measurement.
that of maleimide (730MÀ1 À1). The peptides containing the
s
C6 and C127 residues functionalized with 2 were found from
the tryptic digestion of 9 (see the Supporting Information).
The generality of the present reaction for functionalization
of biomacromolecules was studied with several proteins
having various molecular weights: human insulin (5.8 kDa),[23]
bovine trypsin (23.3 kDa),[24] and bovine serum albumin
(66.4 kDa).[25] The results are summarized in Table 2. Insulin has
low solubility at neutral pH, but it is soluble in acidic aqueous
solutions. The disulfide bridge between C7 (A chain) and C7
(B chain) is known to be the most exposed to solvent.[23b] Reac-
tion of reduced insulin with 2 at pH 4.5 gave product 10
(Figure 4), as confirmed by ESI-TOF MS (Figure 4; Table 1,
entry 2).
Conflict of interest
The authors declare no conflict of interest.
Trypsin is a serine protease that shows catalytic activity at
basic pH conditions (pH 8–9). Functionalization of trypsin at
this pH range is problematic because of autodigestion of the
enzyme. Successful protein modification at pH 4.5 was con-
firmed (entry 3). Bovine serum albumin (BSA) has a reactive
thiol at Cys34,[25b] which is often used as a target for surface
modification of the protein. BSA treated with TCEP (1 equiv) re-
acted moderately with 2 (m=0.65, entry 4). The modification
at Cys34 was confirmed by tryptic digestion of 12 (see the
Supporting Information).
Keywords: bioconjugation · nucleophilic substitution · pH-
responsive · protein modifications · sulfur
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Chem. Eur. J. 2018, 24, 3959 –3964
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