Activation of disulfide bond cleavage triggered by hydrophobization and lipophilization of functionalized dihydroasparagusic acid

Article abstract of DOI:10.1039/c8ob01055b

Concisely synthesized and functionalized dihydroasparagusic acid (DHAA) derivatives were used to show that the introduction of a hydrophobic functional group dramatically reduced air oxidation activity at the dithiol moieties and dominantly activated the cleavage of S-S bonds in proteins, presumably due to the hydrophobization and lipophilization. Notably, the reaction sites of water-reactive dithiol moieties behaved similarly to hydrophobic and lipophilic functional groups, which suggests impersonation of the reaction site.

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Full paper
Activation of Disulfide Bond Cleavage Triggered by
Hydrophobization and Lipophilization of Functionalized
Dihydroasparagusic Acid
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
a
a
DOI: 10.1039/x0xx00000x
Fuyuhiko Inagaki,* Miyuki Momose, Naoya Maruyama, Kenkyo Matsuura, Tsukasa Matsunaga, and Chisato Mukai
www.rsc.org/
Concisely synthesized and functionalized dihydroasparagusic acid (DHAA) derivatives were used to show that the
introduction of a hydrophobic functional group dramatically reduced air oxidation activity at the dithiol moieties and
dominantly activated the cleavage of S-S bonds in proteins, presumably due to the hydrophobization and lipophilization.
Notably, the reaction sites of water-reactive dithiol moieties behaved similarly to hydrophobic and lipophilic functional
groups,
which
suggests
impersonation
of
the
reaction
site.
-
2
The disulfide (S-S) unit between two cysteine amino acids avoiding contact between H O (or OH ) and -SH might protect
in proteins is one of the most significant bonds for the against oxidation of the thiol. In addition, an increase in
1
formation of a tertiary structure. Casadio and coworkers lipophilicity should activate S-S bond cleavage in proteins
predicted that approximately 20% of human proteins contain (lipophilization) because proteins represent lipophilic areas in
2
5
bonded cysteines.
β-Mercaptoethanol (β-ME) and racemic water (Figure 1c). Here, we report the activation of S-S bond
2S,3S)-1,4-dimercaptobutane-2,3-diol (dithiothreitol (DTT), cleavage caused by the hydrophobization and lipophilization of
(
6
Cleland′s reagent) are widely used as reducing agents for the thiol moieties in functionalized dihydroasparagusic acid
,7
9
3
cleavage of S-S bonds in proteins during sodium dodecyl (DHAA) derivatives.
4
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure
1
a,b). Compared with DTT,
β
-ME is relatively stable and
-ME has less reducing ability
Although DTT exhibits greater reducing ability
8
storable in solution. However,
β
9
,10
than DTT.
due to its intramolecular cyclization, it also undergoes air
oxidation more quickly and is therefore difficult to handle.
Recent efforts in our laboratory have focused on
controlling reactivity at the reaction site via the introduction of
11
functional groups. In general, an amine absorbent reacts
with not only CO but also H O (moisture) because of the
2
2
hydrophilicity of amine functionality. However, in a previous
study, we found that the introduction of a hydrophobic
arylalkyl group into hydrophilic amines conferred CO
1
2
1c
selectivity.
absorbed CO
In particular, m-xylylenediamine (MXDA)
2
only, without water contamination. Therefore,
we hypothesized that the water-reactive functional group
located near the lipophilic group might masquerade as a
hydrophobic group (hydrophobization). It is well known that
air oxidation of thiols is highly dependent on pH in the
aqueous solution; this fact encouraged us to hypothesize that
Figure 1. Reactivity concerning thiol-disufide interchange
reaction and air oxidation of thiol unit. a) The reaction
equations show both S-S bond cleavage and air oxidation of
common dithiols. b) General thiols for the SDS-PAGE and
developed dithiols based on DHAA are drawn. c) Our
conceptual design for the selective S-S bond cleavage of
proteins using DHAA derivative is depicted.
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extremely stable solid and can be stored for several months at
room temperature. Thus, short-step syntheses of various types
of DHAA derivatives were achieved.
With syntheses of the desired DHAA derivatives in hand,
we next investigated the relative oxidation rates in air. The air
1
4
oxidation of the dithiol to the disulfide in Tris-HCl buffer (pH
) at room temperature was monitored by observing the
7
degree of disappearance of dithiol (Figure 2a). When DHAA
was exposed to air, the half-life was 15 h. The oxidation rate
was greater for 4a-OEG (t1/2 = 9 h) than for DHAA. In contrast,
the oxidation rates of 4b-Bu (t1/2 = 23 h) and 4c-Bn (t1/2 = 25 h)
were slower than that of DHAA. Thus, increasing the
hydrophobicity of the reducing agent provided resistance to
air oxidation. Although the reduction agents were fully
dissolved in solution, reactivity appeared to be strongly
effected by higher hydrophobicity (cf. ClogP: DHAA 0.29, 4a-
OEG 0.40, 4b-Bu 1.72, 4c-Bn 1.92).
Scheme 1. Concise routes for syntheses of DHAA and its
derivatives.
1
2
DHAA , which has anti-inflammatory effects, antioxidant
activity, and tyrosinase inhibitory activity, is the reduced form
of asparagusic acid, which can be isolated from several species
1
3
of asparagus. Oxidation of the propane-1,3-dithiol unit forms
1
the 5-membered ring of 1,2-dithiolanes. Whitesides et al
4
15
reported that the rate constants of ring closing/opening
reactions with propane-1,3-dithiol/5-membered 1,2-dithiolane
are greater than those of reactions with butane-1,4-dithiol/6-
membered 1,2-dithiane. In addition, the carboxylic acid moiety
of achiral DHAA has additional potential for concise
transformation. Although several types of synthetic methods
Figure 2. Comparable experiments of DHAA derivatives for the
reactivity of air oxidation a) The half-life of disappearance of
1
6
for DHAA have been reported, most pathways require basic
conditions for deprotecting the S-functionalities, which would
cause the undesired oxidation of dithiols to form asparagusic
dithiols (DHAA, 4a-c) in Tris buffer (pH 7)/CH
3
CN (4:1) due to
the air oxidation were calculated from H NMR. b) The H/D
interchange examinations of DHAA and 4c-Bn in CDCl and
O. The relative integrals of SH were monitored by H NMR
with time.
1
3
1
7
acid.
Initially, our effort was directed toward the
1
D
2
development of concise syntheses of DHAA and its derivatives
via the use of acidic conditions during the last deprotection
step. For this purpose, a trityl group was chosen for S-
To our knowledge, the oxidation mechanisms of dithiol in
20
air remain unclear, presumably due to their complexity.
1
8
protection.
bromomethyl)propanoic acid (
protected thiol under basic conditions to produce S-protected
DHAA ( ) in 74% yield (Scheme 1). After deprotection of the
trityl group by the addition of TFA and (i-Pr)
Commercially
available
3-bromo-2-
-
O (or OH ) must be crucial for air
However, the effect of H
2
(
1) was reacted with trityl-
oxidation because the oxidation rate is highly dependent on
solution pH. To further investigate oxidation resistance, we
examined an H/D interchange reaction (Figure 2b). After the
2
1
9
3
SiH , DHAA was
addition of D
1
.5 mL), the change from -SH to -SD was monitored using H
2 3
O (25 ꢀL) to a solution of dithiol in CDCl (20 mM,
observed in 91% yield. On the other hand, atmospheric
oxidation of the dithiol easily proceeded in Tris-HCl buffer (pH
0
NMR. In the case of DHAA, the thiol proton was almost entirely
converted to deuterium after only 30 min. In contrast, >3 h
was required for the H/D interchange of 4c-Bn. This result also
suggests that the hydrophobicity of the benzyl group imparts
pseudo-hydrophobic properties to thiol moieties.
1
) to form asparagusic acid in quantitative yield. When acidic
3
8
conditions were used for deprotection of the trityl group,
oxidized asparagusic acid was not isolated. Thus, the described
pathway is useful for avoiding undesired oxidation. In addition,
the carboxylic acid group of the S-protected intermediate
was readily transformed into the corresponding amides. In
fact, the condensation reaction between and 3,6-
2
Our next endeavor focused on the cleavage of peptide S-S
bonds using DHAA and its derivatives. For this purpose, we
initially applied an excess amount (10 equiv.) of the protected
2
dioxaheptylamine (3a) with EDC and HOBt followed by
deprotection of the trityl group under acidic conditions
afforded the dithiol amide 4a-OEG in 50% yield in 2 steps. The
cystine derivative
5
as a simple mimic of the target molecule
Figure 3a). When 1 equiv. of DHAA was treated with in Tris
CN (2:5) at room temperature for 1.5 h, the
(
5
buffer (pH 7) /CH
3
other amides 4b-Bu
from amines 3b-d and
, DHAA, and ), we detected no unpleasant odors; this lack of
,
4c-Bn, and 4d-Arg were also produced
desired thiol
Tris buffer from 7 to 8, 54% yield (calculated from dithiol) of
the thiol product was obtained (entry 1). Thus, same solution
Tris buffer (pH 8) /CH CN (2:5)) was applied for this
6 was hardly observed. After changing the pH of
2
in high yield. Using this route (TrtSH,
2
4
6
odor is a significant advantage of the use of the thiol
derivatives as a reducing agent. In addition, DHAA is an
(
3
preliminary examination. Although the yield was slightly lower
2
| J. Name., 2012, 00, 1-3
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for 4a-OEG (48%) than for DHAA, 4b-Bu (60%) and 4c-Bn (78%)
HS
HS
O
R
a)
ꢀ
afforded better yields than DHAA (entries 2-4). In all cases, 80-
NHBoc
S
O
NHBoc
Dithiol
1 equiv.)
Tris buffer (pH 8) /
CH CN (2:5)
MeO
(
2
•
MeO
SH
8
5% yields of unreactive disulfide
material ) were recovered, which would indicate the
5
(calculated from starting
S
OMe
NHBoc
O
O
5
3
5
(10 equiv.)
6
rt, 1.5 h
a
(% yield)
stoichiometric reduction by dithiol. Thus, the introduction of a
hydrophobic group into DHAA tended to activate S-S bond
cleavage.
Entry
Dithiol
6
1
2
3
4
DHAA
4a-OEG
4b-Bu
4c-Bn
54%
48%
60%
72%
We continued our investigation by conducting additional
comparative experiments to examine S-S bond cleavage in
a
Calculated from dithiol.
2
1
b)
c)
antibodies.
The disulfides in the immunoglobulin (Ig)
monomer, which is a basic functional unit of antibodies, are
essential for connecting the two heavy chains and two light
chains. After adding the reducing agents (1.0 mM) to mouse
1
00
90
OEG
8
7
6
5
4
3
0
0
0
0
0
0
a 3
IgG2 κ isotype ctrl antibody solution in Tris buffer/CH CN (4:1)
and incubating for 15 min at room temperature, we quenched
the reactions by adding iodoacetamide and processed the
2
samples for SDS-PAGE to analyze disulfide bonds cleavage.
2
20
1
0
Images obtained after staining with Coomassie Brilliant Blue
dye are shown in Figure 3b. In this experiment, intact antibody
0
H
2
L
2
H
L
uncleaved
cleaved
2 2
(H L ) was detected near 250 kDa as seen in the sample
without thiol. After the treatment with lipophilic thiols 4b-Bu
and 4c-Bn, the fully cleaved heavy chain (H) and light chain (L)
were observed around 50 kDa and 25 kDa, respectively, as
main bands. In contrast, hydrophilic thiols such as DTT, DHAA,
d)
0
0
.5
.4
0.3
4
a-OEG, and 4d-Arg (cf. ClogP 0.30) produced no or only faint
0
0
.2
.1
0
H and L bands, indicating much less efficient S-S bond cleavage
of antibody. The quantitative data from three independent
experiments revealed that the appearance of the heavy chain
and the light chain was 9.3- and 2.8-fold higher, respectively,
without DTT
dithiol
OEG
DHA A 4a-PEG 4b-Bu 4c-Bn 4d-Arg TCEP
by the most efficient lipophilic dithiol 4c-Bn, compared with Figure 3. Activity for S-S bond cleavage of reduction agents. a)
DHAA (Figure 3c).
S-S bond cleavage of cystine derivative using DHAA and its
5
The S-S bonds in polypeptides are crucial structures for derivatives 4a-c was described. b) The gel images of SDS-PAGE
maintaining their essential function. Loss of S-S bonds from followed by Coomassie staining after the S-S bond cleavage of
polypeptides would change their activity and has a high mouse IgG2a
κ isotype ctrl antibody using DTT and DHAA
potential for the next generation of pharmaceutical targets. To derivatives are shown. c) The quantitative data of the bands
check this possibility, we tested for the antigen-binding activity corresponding to H L , H, and L in SDS-PAGE analyses (n = 3) as
2
2
of IgG after the treatment with various dithiol derivatives, by shown in b). d) Inhibitory activities of DTT, DHAA, 4a-d, and
2
3
using a Human CRP ELISA Kit . As shown in Figure 3d, DTT TCEP based on the Human CRP ELISA experiments. The
treatment significantly reduced the antigen-binding ability of immobilized antibody specific for human CRP from a Human
anti-CRP antibody (52% inhibition), indicating that the S-S CRP ELISA Kit was treated with 0.1 M reducing agents (DTT,
bond cleavage leads to the loss of antibody activity. In the case DHAA, 4a-d, or TCEP) in Tris buffer (pH 7)/CH CN (4:1) or
3
of DHAA (68%) and 4a-OEG (72%), slightly higher inhibitory buffer alone for 1 h at 37 °C. After washing, the immobilized
activities were observed compared with DTT. Moreover, the antibody was reacted with human CRP standard. The
dithiols 4b-Bu (87%) and 4c-Bn (90%) containing hydrophobic subsequent reactions were conducted according to a standard
groups exhibited the highest class of inhibition, whereas the protocol of the Human CRP ELISA kit and the absorbance was
2
4,25
amino acid-like 4d-Arg (52%) and the phosphine TCEP
63%), well-known alternative reduction reagent for
measured at 450 nm (n = 3).
(
a
In summary, we developed a concise and effective
disulfide, showed only moderate inhibitory activity. These synthetic method for dihydroasparagusic acid (DHAA) and its
results suggest that the lipophilicities of the reduction agents derivatives in which the final stage can be performed under
were strongly associated with their inhibitory activities, which acidic conditions to avoid the formation of undesired
might be applicable to the functional modulation of antibodies disulfides. Air oxidation tests and H/D interchange analyses
or proteins containing S-S bonds.
using functionalized DHAA derivatives showed that the
introduction of a hydrophobic functional group reduced air
oxidation activity based on the observed decrease in the
reaction rate between the thiol moiety and water (as indicated
by comparing DHAA and 4a-OEG with 4b-Bu and 4c-Bn).
Nevertheless, the hydrophobic dithiol moieties had relatively
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high potential for inducing S-S bond cleavage in proteins, as
indicated by the SDS-PAGE and ELISA assays. These
contradictory reactivities might be concluded that the reaction
sites of water-reactive dithiol moieties behaved similarly to
hydrophobic and lipophilic functional groups, which suggests
impersonation of the reaction site (reaction site masquerade).
Further investigation of this objective is currently ongoing.
9
1
(a) G. M. Whitesides, J. E. Lilburn and R. P. Szaajewski, J. Org.
Chem. 1977, 42, 332-337. (b) R. Singh, G. V. Lamoureux, J.
Lees and G. M. Whitesides, Methods in Enzymology, 1995,
2
51, 167-173.
0 Further developments for the S-S bond cleavage under lower
pH are also reported. (a) J. C. Lukesh, III, M. J. Patle and R. T.
Raines, J. Am. Chem. Soc. 2012, 134, 4057-4059. (b) J. C.
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and R. T. Raines, Chem. Commun, 2014, 50, 9591-9594.
1 (a) F. Inagaki, C. Matsumoto, Y. Okada, N. Maruyama and C.
Mukai, Angew. Chem. Int. Ed. 2015, 54, 818-822. (b) F.
Inagaki, Y. Okada, C. Matsumoto, M. Yamada, K. Nakazawa
and C. Mukai, Chem. Pharm. Bull. 2016, 64, 8-13. (c) F.
Inagaki, C. Matsumoto, T. Iwata and C. Mukai, J. Am. Chem.
Soc. 2017, 139, 4639-4642. (d) F. Inagaki, K. Nakazawa, K.
1
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
Maeda, T. Koseki and C. Mukai, Organometallics 2017, 36
005-3008.
2 (a) S. C. Mitchell and R. H. Waring, Phytochemistry 2014, 97
-10. (b) J. Adamczyk, W. Bal and A. Krezel, Inorg. Chem.
,
3
We are grateful for support from a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science, and Technology (Japan).
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Cammisottoa, M. Ottavianic, A. Biancob and A. Venditti,
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Introduction of hydrophilic group into dihydroasparagusic acid (DHAA) indicated higher
reduction ability of disulfide in protein and lower air oxidation.