Mechanistic study on the facilitation of enzymatic hydrolysis by α-glucosidase in the presence of betaine-type metabolite analogs

Article abstract of DOI:10.1016/j.tet.2014.06.028

Recently we reported that betaine-type metabolite analogs structure-dependently facilitate enzymatic hydrolysis reaction for α-glucosidases, β-glucosidases, and alkaliphosphatases. To understand the facilitation mechanism for enzymes, in this study we exp

Full text of DOI:10.1016/j.tet.2014.06.028

Tetrahedron 70 (2014) 5895e5903
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Tetrahedron
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Mechanistic study on the facilitation of enzymatic hydrolysis by
a-glucosidase in the presence of betaine-type metabolite analogs
Yuichi Nakagawa a, Shiro Sehata a, Satoshi Fujii a, Hiroaki Yamamoto b, Akihiko Tsuda b,
,c,
Kazuya Koumoto a
*
a Department of Nanobiochemistry, FIRST (Frontiers of Innovative Research in Science and Technology), Konan University,
7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
b Department of Chemistry, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
c FIBER (Frontier Institute for Biomolecular Engineering Research), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently we reported that betaine-type metabolite analogs structure-dependently facilitate enzymatic
Received 30 January 2014
Received in revised form 7 June 2014
Accepted 7 June 2014
hydrolysis reaction for
itation mechanism for enzymes, in this study we expanded the analog library and measured the prop-
erties of analog solutions. The structural investigation on -glucosidase-mediated hydrolysis reaction
a-glucosidases, b-glucosidases, and alkaliphosphatases. To understand the facil-
a
Available online 12 June 2014
indicated that suitable structures to facilitate the enzyme reaction efficiently should have the ammonium
cation in the betaine structure possess triplicate aliphatic chains from C1 to C7 without any polar
functional groups. Analyses of the solution properties revealed that such analogs possess a large hy-
dration layer with low water density. Such a specific hydration environment is generated by the char-
acteristic structure of the betaine-type metabolite analogs. The characteristic hydration indirectly
regulates enzyme activity and stability. These findings not only increase our understanding enzyme
activation by betaine-type metabolite analogs, but also will contribute to the molecular design of enzyme
regulators.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
several mol/L yet they do not significantly inhibit cellular func-
tions.18,19 This ability may be exploited to improve enzymatic ac-
Enzymes are one of the most ecological and useful catalysts
under ambient conditions and have extensively been investigated
for use in biological, diagnostic, and industrial fields. However, the
low activity and stability of enzymes is sometimes a problem.
Therefore, the enhancement of the enzymatic activity and stability
is of great significance. In order to obtain active and stable enzymes,
several strategies have been reported, such as screening new en-
zymes from natural sources,1 site-specific mutations of enzymes
utilizing genetic engineering,1,2 the addition of organic molecules
into the reaction buffer,3e16 and so on. Above all, the addition of
organic molecules in the reaction buffer is particularly useful be-
cause it is easy and inexpensive. As organic additives, cellular small
molecules, such as amino acids, polyols, and various metabolites
have been used.8e16 Some metabolites are specifically accumulated
in cells when they face extremely harsh conditions.17 Under such
conditions, the concentration of metabolites sometimes reaches
tivation and stabilization.
To investigate the molecular function of metabolites, we re-
cently synthesized an analog library20 (Fig. 1) derived from the
naturally-occurring metabolite glycine betaine (2-N,N,N-trimethy-
lammonium acetate), which is a common metabolite found in
microorganisms, animals, and plants.17 We modified the chemical
structure in ways analogous to natural changes in cellular metab-
olite structures; that is, changed the linker length in compound 1
(glycine betaine) between the ammonium cation and carboxylate
anion (analogs 2, 3), changed the bulkiness of the ammonium
group (analogs 4e8), or changed the number of intramolecular
zwitterions (analog 9) (Fig. 1). We found that these analogs
structure-dependently regulate the thermal stability of DNA
duplexes.20
A subsequent study revealed that these analogs increase enzy-
matic activity just by dissolving them into the reaction buffer.21
A
kinetic study using a-glucosidase revealed that the activating effect
of the analogs worked not only by enhancing the rate constant but
also by improving thermostability, salt tolerance, and substrate
specificity. Interestingly, enzyme activation was commonly
* Corresponding author. Tel.: þ81 78 303 1465; fax: þ81 78 303 1495; e-mail
address: koumoto@center.konan-u.ac.jp (K. Koumoto).
http://dx.doi.org/10.1016/j.tet.2014.06.028
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

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Y. Nakagawa et al. / Tetrahedron 70 (2014) 5895e5903
linker length between the ammonium cation and carboxylate an-
ion. Taking structural differences into consideration, we designed
analogs 2 and 3. Similarly, to assess the bulkiness of the ammonium
cation, analogs 4e6 were designed. Also, to assess functional
groups introduced into the ammonium cation, analogs 4, 7, and 8
were designed, which have similar bulkiness but different func-
tional groups attached to the ammonium group. However, the
changes in these compounds do not have major structural
consequences.
We therefore newly designed and synthesized analogs 10e21
(Fig. 2) Our previous study revealed that analogs 4e6, which possess
aliphatic chains attached to the ammonium cation, strongly accel-
erated enzymatic hydrolysis, and the acceleration was intensified as
the aliphatic chains become longer. Therefore, long aliphatic chains
introduced into the ammonium cation may be important in the fa-
cilitation of enzymatic hydrolysis. Eight analogs (10e17) were syn-
thesized to test this. Analogs 10e13 possess triplicate n-pentyl, n-
hexyl, n-heptyl, and n-octyl chains in the ammonium cation, re-
spectively. They have more bulky ammonium cation moieties than
analogs 4e6. In addition, in order to investigate, which of the in-
troduced aliphatic chains affects enzymatic activity, analogs 14e17
were synthesized. Analogs 14 and 15 have duplicate n-hexyl chains
and either one n-butyl or one n-octyl chain attached to the ammo-
nium cation, and analogs 16 and 17 have duplicate n-butyl chains
and either one methyl or one n-octyl chain.
Fig. 1. Chemical structure of metabolite analogs derived from glycine betaine.
observed for different types of hydrolases, such as a-glucosidase, b-
glucosidase, and alkaline phosphatase. Furthermore, the facilitation
of enzymatic hydrolysis strongly depended on the chemical struc-
ture and the concentration of the analogs. In particular, efficient
Fig. 2. Chemical structure of newly synthesized metabolite analogs.
facilitation was achieved by analogs possessing relatively long ali-
phatic chains containing the ammonium cation. However, we did
not obtain any information as to why the analogs structure-
dependently facilitated enzymatic reactions. In the current study,
we expanded the library of analogs to clarify the structural con-
tribution to the facilitation of enzymatic hydrolysis and in-
vestigated the mechanism.
Our previous study also suggested that having a polar group as
part of the ammonium cation, such as in analog 7, diminishes the
enzymatic activation compared with aliphatic analogs that possess
similar bulkiness (analogs 4 and 8).21 However, it was difficult to
conclude whether structural effects caused by the introduced
functional groups were responsible. Therefore, we added analogs
that possess other functional groups (18e21). Analogs 18 and 19,
which have one or two hydroxyl groups at the end of the hydro-
carbon, were synthesized to test the influence of polar functional
groups in the ammonium cation. Analogs 20 and 21 were synthe-
sized to test the influence of aromaticity in the ammonium cation.
2. Results and discussion
2.1. Metabolite analog library expansion
The molecular design of the metabolite analogs derived from
glycine betaine (analog 1) was based on the structural diversity of
naturally occurring cellular metabolites. For example, glycine and
2.2. Synthesis and water-solubility of analogs
All of the analogs (10e19) were synthesized according to the
previously reported method.20 All compounds were confirmed
b-alanine, which are both naturally-occurring metabolites, differ in

Y. Nakagawa et al. / Tetrahedron 70 (2014) 5895e5903
5897
with 1H nuclear magnetic resonance (NMR), 13C NMR, fourier
transform-infrared (FTIR), and high resolution electrospray ioni-
zation mass (HR-ESI-MS) spectroscopies (the spectra are shown in
the electronic Supplementary data (ESD)).
concentrations using analogs 1, 4e6, 10e12, 14e17 (Fig. 3b shows
activity over a low concentration range from 0 to 1.0 mM). As
shown in the figure, the activation behavior is altered by the
chemical structures of the metabolite analogs. For analogs 1 and 4,
the activity linearly increases as the analog concentration increases,
and the maximal activities are 1.33 and 2.21 at 1000 mM, re-
spectively. On the other hand, when the alkyl chains are longer than
triplicate ethyl (C2) chains (analog 4), a concentration-dependent
activation behavior (bell-type plots) is observed. For analog 5, the
maximal activity (3.36) appeared at 500 mM. As the chain length
becomes longer than that of analog 5, the analog concentrations
that induce the maximal activation of enzymatic hydrolysis shift to
lower concentrations: the maximal activation concentrations for
analogs 6, 10, 11, 12, 14, 15, 16, and 17 appeared around 100 mM
(4.27), 5 mM (3.96), 0.5 mM (2.99), 0.05 mM (2.15), 1 mM (3.43),
0.1 mM (2.71), 100 mM (3.23), and 1 mM (3.37), respectively
(maximal activities are noted in parentheses). As being consistent
with the previous study,21 the activation observed for the synthetic
analogs is ascribed to both decrease in Km and increase in Vmax
(ESD, Table S1).
We first tested the water solubility of the analogs. Water solu-
bility was tested by unfavorable turbidity increases in aqueous
analog solutions and is summarized in Table 1. When the aliphatic
chain was short, from C1 to C5, the water solubility did not change
significantly (more than 3000 mM). In contrast, the water solubility
for analogs 11 (C6), 12 (C7), and 13 (C8) dropped to 30 mM, 0.3 mM,
and nearly 0 mM, respectively, suggesting that when the aliphatic
chains become longer than C6, hydrophobicity appears. The water
solubilities of analogs 14, 16, and 17, whose total carbon numbers in
the ammonium cation are smaller than that of analog 11, were more
than 3000 mM, whereas that of analog 15 dropped (1.5 mM) and
was close to that of analog 12, suggesting that the total carbon
number of the ammonium cation determines the water solubility of
the analogs.
Table 1
To understand the relationship between the total carbon num-
ber of triplicate aliphatic chains introduced into the ammonium
cation and either the maximal activation concentration or maximal
activity, we plotted them in Fig. 4a and b, respectively. As shown in
Fig. 4a, the maximal activation concentration linearly decreased as
the total carbon number increased. On the other hand, the maximal
activities describe a bell-shape curvature with analog 6 at the top
(Fig. 4b).
Water-solubility of the synthetic analogs
Analogs
Water-solubility (mM)
Total carbon number in
an ammonium cation
Analog 1
Analog 2
Analog 3
Analog 4
Analog 5
Analog 6
Analog 7
Analog 8
Analog 9
Analog 10
Analog 11
Analog 12
Analog 13a
Analog 14
Analog 15
Analog 16
Analog 17
Analog 18
Analog 19
Analog 20
Analog 21
>3000
>3000
>3000
>3000
>3000
>3000
>3000
>3000
1500
3
3
3
6
9
12
5
6
We also assessed the influence of the identity of the introduced
aliphatic chains using analogs 14, 15e17. With respect to the
maximal activation concentration, the plots do not show any re-
lationship for analogs 5, 6, 10e12 and total carbon number in the
ammonium cation. The maximal activation concentration for ana-
logs 16 and 17 is altered by the contribution of another chain; the
concentration for analog 16 (duplicate n-butyl chains and one
methyl chain) is the equal to that for analog 6 (triplicate n-butyl
chains), suggesting that the duplicate n-butyl chains form the
dominant structure and that the contribution of the methyl chain is
negligible. In contrast, the maximal concentration for analog 17
shifted to 100-fold lower than those of analogs 6 and 16, suggesting
that the longer n-octyl chain intensifies the activation effect. Sim-
ilarly, that for analog 14 possessing duplicate n-hexyl chains and
one n-butyl chain in the ammonium cation was nearly equal to that
for analog 11 (triplicate n-hexyl chains), whereas that for analog 15
possessing duplicate n-hexyl chains and one n-octyl chain shifted
to 10-fold lower than those of analogs 11 and 14. These results
indicate that introduced short chains scarcely affect the maximal
activation concentration, whereas long chains intensify the acti-
vation effect.
3b
15
18
21
24
16
20
9
16
4
5
5
4
3000
30
0.3
z0a
3000
1.5
>3000
>3000
>3000
>3000
>3000
>3000
a
Analog 13 did not dissolve in water even at 0.05 mM.
Six carbons are divided in two ammonium groups.
b
2.3. Enzymatic activity in the presence of analogs
Fig. 3a shows the activity (activity¼initial velocity in the pres-
ence of analog/initial velocity in the absence of analog) of the hy-
drolysis reaction by
a-glucosidase as a function of the analog
Fig. 3. Plots of activity as a function of the analog concentration for (a) 0e1000 mM and (b) 0e1.0 mM. Analogs 1 (filled circle), 4 (filled square), 5 (filled diamond), 6 (filled triangle),
10 (filled inversed triangle), 11 (open circle), 12 (open square), 14 (open diamond), 15 (cross), 16 (plus), and 17 (open triangle).

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Y. Nakagawa et al. / Tetrahedron 70 (2014) 5895e5903
Fig. 4. Relationship between total carbon number of aliphatic chains introduced into ammonium cation and either (a) maximal activation concentration or (b) maximal activity for
analogs 5, 6, 10e12 (filled circle) and analogs 14 (open diamond), 15 (cross), 16 (plus), and 17 (open triangle).
In contrast, with respect to the maximal activity, analogs 14,
15e17 obey the relationship between the maximal activities seen
for analogs 5, 6, 10e12 and the total carbon number in the am-
monium cation. This suggests that the activity is mostly determined
by the total carbon number of the analogs, where the identity of the
introduced aliphatic chain is not so significant.
analog 19 possessing two hydroxyl groups did not increase activity
at all (activity¼1). On the other hand, aromatic analogs (20 and 21)
facilitated enzymatic hydrolysis similar to those of analogs 4 and 8.
In summary, to activate enzymatic hydrolysis, the ammonium
cation should be covered with apolar hydrocarbons without any
polar functional groups. In this regard, the analogs must be rea-
sonably soluble in aqueous media.
2.4. Influence of introduced functional groups in the am-
monium cation
2.5. Comparison of the solution properties of the analogs
To obtain insights as to how these analogs facilitate the enzy-
matic hydrolysis, we compared their solution properties. Betaine-
type metabolite analogs facilitate different hydrolysis reactions
Using analogs 4, 7, 8, 18e21, we then examined the influence of
the functional groups introduced into the ammonium cation. For
these analogs, the activity linearly increased as the concentration
increased from 0 to 1000 mM. Therefore, in order to compare the
additional effects of the analogs, the activities at [analogs]¼
1000 mM were represented as bar graphs as shown in Fig. 5.
catalyzed by a-glucosidases, b-glucosidases, alkaline phosphatases,
and DNA polymerases at almost the same concentrations.20,21 This
feature suggests that the analogs indirectly regulate enzymatic
activity rather than engaging specific interactions between en-
zymes and analogs. We thus compared the solution properties by
measuring dynamic light scattering (DLS), osmolarity, water den-
sity, and dielectric constants of the analog solutions.
First, to analyze the molecular association behavior of the ana-
logs, DLS measurements were carried out. To obtain reliable scat-
tering intensities, the concentrations of the analogs were adjusted
to above 300 mM. DLS measurements therefore were performed for
analogs 1, 4e6, and 10 whose water solubilities were higher than
300 mM (Table 1). The obtained average grain diameters for the
analogs were plotted as functions of the analog concentrations
(Fig. 6). For analogs 1, 4e6, the average grain diameters did not
change over concentration ranges from 300 to 1000 mM, indicating
that these analogs were dissolved in aqueous media as monomers.
Fig. 5. Comparison of the activity in the presence of analogs 4, 7, 8, 18e21 ([analogs]¼
1000 mM).
Based on molecular dynamics calculations,22 the molecular sizes
of analogs 4, 7, 8, 18e21 are similar to each other (the following
values are the longest molecular length of each molecule: 4:
0.69 nm, 7: 0.63 nm, 8: 0.65 nm, 18: 0.77 nm, 19: 0.77 nm, 20:
0.75 nm, 21: 0.82 nm). Therefore, the activity change should be
related to the introduced functional group. Similar to our previous
study,21 the activity in the presence of analog 7 was considerably
smaller than those for analogs 4 and 8. We hypothesized that polar
functional groups introduced in the ammonium cation should di-
minish the activation effect of the analogs. The present results
strongly support our hypothesis, given that the activity of analog 18
possessing one hydroxyl group in the ammonium cation was
smaller than that of analog 7, and furthermore, the activity of
Fig. 6. Plots of average grain diameters estimated from DLS measurements as a func-
tion of the analog concentration for analogs 1 (filled circle), 4 (filled square), 5 (filled
diamond), 6 (filled triangle), 10 (filled inversed triangle).

Y. Nakagawa et al. / Tetrahedron 70 (2014) 5895e5903
5899
On the other hand, the diameter for analog 10 slightly increased
from 1.4 nm to 4.0 nm as its concentration increased, indicating
that its monomers weakly and dynamically associated together in
the aqueous media. This result accounts for the drastic decrease in
the water solubility of analogs 11e13 and 15 due to their molecular
associations. Additionally, although analog 10 weakly associated in
the solution, it dissolved as a monomer at least at the maximal
activation concentration (5 mM). This implies that monomer dis-
persion of the analogs in solution is important to efficiently facili-
tate the enzymatic reaction.
In order to understand the hydration environment surrounding
the analogs, the water density of the analog solutions was mea-
sured. As mentioned above, although the osmolarities for analogs 5
and 6 were almost identical, the enzyme activation behavior was
different (Fig. 3), suggesting that the hydration environments sur-
rounding the analogs were also different. Ionic compounds are
known to hydrate strongly, where water molecules tightly sur-
round the compounds via ionedipole interactions. In contrast, the
charged moiety of the betaine-type analogs is covered with apolar
aliphatic chains. Apolar surfaces dislike tightly packed water mol-
ecules, which induce unfavorable polarity.25,26 Therefore, to solvate
betaine-type analogs possessing relatively long aliphatic chains,
water should be less tightly packed onto the molecular surface, and
furthermore, plenty of water molecules must participate to coun-
teract unfavorable charges via ionedipole interactions.
Fig. 8 shows plots of the water density for analogs 1, 4e6 as
a function of their concentrations. As the aliphatic chains become
longer, the slopes become smaller. As expected, the water densities
between analogs 5 and 6 were different, supporting to our hy-
pothesis. The slope of the plots indicates a change in water density
accompanying increased analog concentration. In other words, the
slope is related to the change of water density of the hydration layer
surrounding the analogs. Considering the slope difference between
analogs 5 and 6, the density of hydration water surrounding analog
6 is small.
Based on molecular dynamics calculations,22 the molecular sizes
of analogs 1, 4e6, and 10 are as follows: 1: 0.57 nm, 4: 0.69 nm, 5:
0.82 nm, 6: 0.94 nm, and 10: 1.07 nm. Considering that the mo-
lecular sizes measured from DLS (average grain diameters for an-
alogs 1, 4, 5, and 6 are 0.80ꢀ0.04 nm, 1.01ꢀ0.08 nm, 0.98ꢀ0.04 nm,
and 1.18ꢀ0.04 nm at 300 mM, respectively) were considerably
larger than those calculated by molecular dynamics simulations,
this suggests the presence of a large hydration layer covering the
analogs.
Therefore, we then measured the osmolarity of the analog so-
lutions to estimate their hydration environment. In cells, betaines
are well-known osmoprotectants that regulate osmotic pressure by
binding water molecules to themselves.17e19 The osmolarities of
the analog solutions were plotted as a function of the analog con-
centrations (Fig. 7). As controls, DMSO, glycerol, and glucose were
selected because these are representative hydrophilic molecules. As
the results show, the osmolarities did not merge with each other,
indicating that their hydration environments were different.
According to a previous study,23,24 as molecules are more hydrated
their slopes tend to be larger. In Fig. 7, the slopes of glycerol and
glucose are larger than that of DMSO, indicating that the polyols
solvated more strongly than DMSO. In contrast, the slopes of the
analogs are bent more strongly than those of the controls, in-
dicating that the analogs were hydrated much more so than the
hydrophilic control molecules. The order of the slopes is as follows:
analogs 5¼6>4>1>10. Except for analog 10, this is in the same
order as the enzyme activation. Here, the osmolarity for analog 10
gradually decreased at concentrations above 500 mM. This was due
to molecular association, because when the molecules in-
termolecularly associate, the hydrated water molecules are re-
leased from the molecular surface, resulting in a decrease of
osmolarity. Compared with the osmolarities for analogs 1, 4e6,
analogs possessing longer aliphatic chains showed higher osmo-
larity. However, there was no significant difference between ana-
logs 5 and 6, although enzymatic activation in the presence of
analog 6 is higher than that of analog 5 (Fig. 3).
Fig. 8. Plots of water density of the analog-containing aqueous solution as a function
of the analog concentration for analogs 1 (filled circle), 4 (filled square), 5 (filled di-
amond), and 6 (filled triangle).
The present results on the osmolarity and water density of the
analog solutions suggest that the analogs, which strongly accelerate
a-glucosidase-mediated hydrolysis, possess large hydration layers
composed of a low density of hydration water. A large hydration
layer possessing low water density, corresponding to a large ex-
cluded volume, may eliminate the substrate and enzyme from the
area around the analogs, leading to increased collision frequency
between substrate and enzyme.
To understand the excluded volume effect, the average grain
diameter of a-glucosidase was measured as a function of analog 6
concentration. Fig. 9 shows plots of this. With increased analog
concentration, the average grain diameter of analog 6 itself did not
change significantly, even in the presence of
whereas that of -glucosidase increased as expected. The in-
termolecular association behavior of -glucosidase in the presence
a-glucosidase,
a
a
of analog 6 supports our hypothesis, i.e., analog 6 facilitates mo-
lecular interactions owing to the large excluded volume of the
hydrated analogs.
Finally, we measured the dielectric constants of the analog
solutions. Dielectric constants were measured using 8-
Fig. 7. Plots of osmolarity as a function of the analog concentration for analogs 1 (open
circle), 4 (open square), 5 (open diamond), 6 (open triangle), 10 (filled circle), DMSO
(open circle), glycerol (open square), and glucose (open diamond).
anilinonaphthalene sulfonic acid (ANS) as
a
fluorescence

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Y. Nakagawa et al. / Tetrahedron 70 (2014) 5895e5903
Although the changes in osmolarity and water density were too
small to compare the values directly, our proposed solvation dif-
ference can be observed. For example, a comparison of the values
for analogs 7 and 8, whose structural difference consists only of the
morpholine moiety being replaced with piperazine, indicates that
the polar oxygen atom located outside the molecule increases the
water density and decreases the osmolarity, indicating that hy-
drated water molecules tightly and narrowly packed around the
analogs. Also, a comparison of analogs 18 and 19, which possess
different numbers of OH groups, revealed that the osmolarity of
analog 19 is smaller than that of 18, and the water density of analog
19 is larger than that of 20. These behaviors are analogous. Con-
sidering these results, we concluded that solution property changes
are related to increased enzymatic activation.
2.7. Theoretical model of enzyme activation by the addition
of betaine-type metabolite analogs
Fig. 9. Plots of the average grain diameter of a-glucosidase (filled square) and analog 6
(filled circle) as a function of analog 6 concentration.
According to our previous study,21 betaine-type metabolite an-
alogs can facilitate the hydrolysis reaction, which is driven by the
enhancement of both the binding affinity between enzyme and
substrate (decrease in Km) and the catalytic activity of the enzyme
itself (increase in Vmax). In addition, the enzyme activation strongly
depends on the chemical structures of the analogs added into the
reaction buffer. In particular, analogs possessing relatively long al-
iphatic chains attached to the ammonium cation show profound
effects on enzyme activation.
In the present study, we expanded the analog library and found
the same structural dependency for enzyme activation as in pre-
vious results.21 Moreover, we previously showed that betaine-type
metabolite analogs facilitate different types of enzymatic reactions
probe.27e29 Protein structures are stabilized by Coulomb forces, and
therefore, a change in the dielectric constant in the bulk water
phase may have an impact on the stability and conformation of the
enzyme,30,31 resulting in altered enzymatic activity. We plotted the
relationship between 1/
3
r
and the activity of
a-glucosidase in
Fig. 10. As shown in the figure, there is no relationship between 1/
3
r
and activity, indicating that the change in the dielectric constant of
aqueous media, accompanied by the addition of analogs, does not
affect the enzymatic activity. In other words, analogs disperse in
aqueous media and are highly solvated with low density hydration
water, which is important in enzymatic activation.
catalyzed by a-glucosidases, b-glucosidases, alkaline phosphatases,
and DNA polymerases at almost the same concentrations.20,21 This
suggests that the facilitation effect is not related to specific in-
teractions between enzymes and analogs, but rather to indirect
effects. Therefore, we measured the solution properties of analog-
containing solutions. Here, we assumed that the hydration of the
analogs indirectly regulated the enzymatic reactions by changing
the solution properties. First, by measuring the average grain di-
ameters of analogs 1, 4e6, and 10, we revealed that the analogs
dissolved in aqueous media as monomers. Furthermore, the os-
molarity and water density measurements indicated that the ana-
logs that are good at facilitating enzymatic hydrolysis possess large
hydration layers with low water density. Given these results,
Scheme 1 represents the effect of analog addition on Km. The large
hydration layer of the analogs provides a large excluded volume in
the solution and eliminates the hydrophilic enzyme and substrate
from the surface, resulting in the enhancement of collision fre-
quency between substrate and enzyme. This causes an apparent
decrease of Km values in the presence of analogs.
Fig. 10. Plots of 1/ 3 as a function of the analog concentration for analogs 1 (open
circle), 4 (open square), 5 (open diamond), 6 (open triangle), and 10 (filled circle).
r
2.6. Influence of outside polar functional groups on the so-
lution properties
Concurrently, the analogs significantly decrease the water ac-
tivity in the solution, i.e., they enhance the osmolarity.23 Previous
studies have revealed that the activity and stability of enzymes are
highly sensitive to the hydration environment.32,33 The large de-
crease of water activity in the analog solutions alters the hydration
To address the question of why introduced polar functional
groups to the ammonium cation diminish the enzyme activity, we
compared the osmolarities and water densities for aqueous solu-
tions containing analogs 4, 7, 8, 18e21 (Table 2).
balance of the enzyme. This should affect the conformation of
a-
glucosidase. However, according to our previous study,21 CD
Table 2
Comparison of osmolarity, water density, and molecular length of analogs 4, 7, 8, 18e21
Analogs
4
7
8
18
19
20
21
Osmolarity/mOsm kgꢁ1a
Water density/mg cmꢁ3a
Molecular size/nm
1235ꢀ4
1027ꢀ1
0.69ꢀ0.08
1162ꢀ1
1048ꢀ3
0.65ꢀ0.01
1313ꢀ1
1038ꢀ1
0.75ꢀ0.04
1315ꢀ7
1047ꢀ1
0.80ꢀ0.01
1232ꢀ1
1056ꢀ1
0.80ꢀ0.01
1280ꢀ3
1042ꢀ2
0.75ꢀ0.02
1362ꢀ2
1047ꢀ1
0.82ꢀ0.04
a
The values osmolarity and water density are those at [Analogs]¼1000 mM.

Y. Nakagawa et al. / Tetrahedron 70 (2014) 5895e5903
5901
Scheme 1. Schematic illustration of the addition effect of the betaine-type metabolite analogs on Km
.
spectra of
a
-glucosidase did not change at all even with increasing
the maximal activity showed a bell-shape curve with analog 6
(triplicate n-butyl chains) at the top.
the analog concentration. It suggests that the activity change is
related to the small conformational difference around at active site
Subsequent analyses of the solution properties revealed that
analogs, such as 5 and 6, which effectively facilitate the enzymatic
reaction, were dissolved as monomers and possess a large hydra-
tion layer with low water density. Such a specific hydration envi-
ronment is generated by the characteristic structure of the betaine-
type metabolite analogs, whose cationic charge is covered with
relatively long apolar hydrocarbons. The characteristic hydration
indirectly regulates enzyme activity and stability. These findings
not only increase our understanding enzyme activation by betaine-
type metabolite analogs, but also will contribute to the molecular
design of enzyme regulators.
of
of
a
-glucosidase. In fact, when we measured the activation energy
a
-glucosidase by Arrhenius plots in the presence of analog 6, the
activation energy was reduced by one-third at [analog 6]¼100 mM
(Fig. 11), at which CD spectrum of
a-glucosidase is completely
merged to that in the absence of analog.21 This suggests that a de-
crease in the water activity alters the conformational flexibility of
a
-glucosidase.
4. Experimental sections
4.1. Materials
All commercially available reagents and solvents were pur-
chased from Wako Pure Chemicals Ltd. (Osaka, Japan). a-Glucosi-
dases from Bacillus stearothermophillus (SIGMA: G3651-250UN)
was purchased from SIGMA. They were used without purification
unless otherwise noted. All compounds (analogs 10e21) synthe-
sized were synthesized according to
a previously reported
method21 and were characterized by means of 1H NMR, 13C NMR,
FTIR, and HR-ESI-MS spectroscopies. 1H NMR and 13C NMR spectra
were recorded on a JEOL ECA-500. Chemical shifts were reported
with respect to tetramethylsilane (TMS) as the internal standard.
FTIR spectra were recorded on a JASCO FT/IR-6300 Fourier trans-
form-infrared spectrometer. HR-ESI-MS spectra were measured on
a Fourier Transform Mass Spectrometry (Thermo Fisher Scientific
LTQ Orbitrap Discovery). The all spectra were shown in ESD.
Fig. 11. Plots of activation energy of hydrolysis reaction by
of analog 6 concentration.
a-glucosidase as a function
3. Conclusions
To understand the mechanism by which betaine-type metabo-
lite analogs facilitate enzymatic hydrolysis, in this study we ex-
panded the analog library and measured the properties of analog-
containing solutions. The synthetic analogs structure-
4.2. Syntheses
4.2.1. 2-N,N,N-Tri-n-pentylammonium acetate (10). To a solution of
tri-n-pentylamine (25 mL, 0.09 mol) in ethyl acetate (250 mL) was
added 2-bromoacetic acid ethyl ester (8.64 mL, 0.08 mol) and the
mixture was stirred at rt for 4 days. The reaction mixture was
concentrated to dryness under the reduced pressure and the resi-
due was purified with column chromatography (silicagel CHCl3/
methanol¼20:1). Thus obtained light yellow oil was dissolved in
H2O/methanol mixture and was passed through ion-exchange
dependently facilitated the hydrolysis reaction of
a-glucosidase,
similar to previous studies.21 Our results here indicated that suit-
able structures to facilitate the enzyme reaction efficiently should
have the ammonium cation in the betaine structure possess trip-
licate aliphatic chains from C1 to C7 without any polar functional
groups, as the effective concentration for enzyme activation shifts
to a lower range as the aliphatic chains become longer. In contrast,

5902
Y. Nakagawa et al. / Tetrahedron 70 (2014) 5895e5903
chromatography (Amberite IR-402CL). The eluting solution was
TMS, rt) d 13.75, 19.88, 24.54, 48.81, 61.90, 62.87, 164.54; HR-ESI-MS
evaporate to dryness and was dried in vacuo using P2O5 to give 10
(positive mode): m/z for C11H23NO2, calculated 202.1807 (MHþ),
(18.5 g, 83%) as a light yellow oil: FTIR (ATR)
n
/cmꢁ1 2955, 2929,
found 202.1801 (MHþ).
2872, 1635, 1472, 1340, 1313, 1049, 1033, 840, 717; 1H NMR
(500 MHz, CDCl3, TMS, rt)
d
0.91 (t, J¼7.0 Hz, 9H), 1.34 (m, J¼7.4 Hz,
4.2.8. 2-(N,N-Di-n-butyl-N-n-octylammonium)
Compound 17 was obtained in 88% yield as a colorless oil in the
same way as for the preparation of 10: FTIR (ATR)
/cmꢁ1 2957,
2925, 2858, 2359, 2342, 1635, 1467, 1352, 1311, 1045, 867, 717; 1H
NMR (500 MHz, DMSO-d6, TMS, rt)
0.87 (t, J¼6.8 Hz, 3H), 0.98 (t,
J¼7.5 Hz, 6H), 1.33 (m, 14H), 1.61 (m, J¼8.0 Hz, 6H), 3.48 (t, J¼8.5 Hz,
6H), 3.63 (s, 2H); 13C NMR (125 MHz, CDCl3, TMS, rt)
13.75, 14.75,
acetate
(17).
12H), 1.62 (m, J¼7.8 Hz, 6H), 3.48 (t, J¼8.3 Hz, 6H), 3.62 (s, 2H); 13
C
NMR (125 MHz, CDCl3, TMS, rt)
d
13.87, 21.86, 22.30, 28.58, 58.85,
n
59.87, 164.40; HR-ESI-MS (positive mode): m/z for C17H35NO2, cal-
culated 286.2746 (MHþ), found 286.2741 (MHþ).
d
4.2.2. 2-N,N,N-Tri-n-hexylammonium acetate (11). Compound 11
d
was obtained in 100% yield as a colorless oil in the same way as for the
19.93, 22.18, 22.64, 24.17, 26.55, 29.08, 29.13, 31.67, 58.70, 58.91,
59.93, 164.34; HR-ESI-MS (positive mode): m/z for C18H37NO2,
calculated 300.2903 (MHþ), found 300.2896 (MHþ).
preparation of 10: FTIR (ATR)
1363, 1049, 1033, 858, 748, 720; 1H NMR (500 MHz, CDCl3, TMS, rt)
0.88 (t, J¼5.8 Hz, 9H), 1.32 (m, 18H), 1.62 (m, 6H), 3.48 (t, J¼8.0 Hz,
6H), 3.62 (s, 2H); 13C NMR (125 MHz, CDCl3, TMS, rt)
13.94, 22.16,
n
/cmꢁ1 2955, 2924, 2858, 1635, 1457,
d
d
4.2.9. 2-(N,N-Dimethyl-N-2-hydroxyethylammonium) acetate (18).
Compound 18 was obtained in 88% yield as a white powder in the
same way as for the preparation of 4:20 Mp 165e167 ꢂC; FTIR (ATR)
22.48, 26.21, 31.27, 58.90, 59.90, 164.33; HR-ESI-MS (positive mode):
m/z for C20H41NO2, calculated 328.3216, found 328.3210 (MHþ).
n
/cmꢁ1 3149, 2362, 2342,1617,1390,1337, 1322, 1063, 990, 922, 894,
4.2.3. 2-N,N,N-Tri-n-heptylammonium acetate (12). Compound 12
786, 768, 715; 1H NMR (500 MHz, DMSO-d6, TMS, rt)
d 3.11 (s, 6H),
was obtained in 85% yield as a colorless oil in the same way as for
3.54 (s, 2H), 3.57 (t, J¼5.3 Hz, 2H), 3.73 (br, 2H), 5.21 (br, 1H); 13C
the preparation of 10: FTIR (ATR)
n
/cmꢁ1; 1H NMR (500 MHz, CDCl3,
NMR (125 MHz, methanol-d4, TMS, rt) d 51.61, 55.88, 64.56, 65.02,
TMS, rt)
d
0.88 (t, J¼5.8 Hz, 9H), 1.32 (m, 18H), 1.62 (m, 6H), 3.48 (t,
167.66; HR-ESI-MS (positive mode): m/z for C6H13NO3, calculated
J¼8.0 Hz, 6H), 3.62 (s, 2H); 13C NMR (125 MHz, CDCl3, TMS, rt)
148.0974 (MHþ), found 148.0965 (MHþ).
d
14.09, 22.20, 22.53, 26.52, 28.84, 31.58, 58.90, 59.90, 164.31; HR-
ESI-MS (positive mode): m/z for C23H47NO2, calculated 370.3685
4.2.10. 2-[N-Methyl-N,N-bis(2-hydroxyethyl)ammonium]
acetate
(MHþ), found 370.3682 (MHþ).
(19). Compound 19 was obtained in 88% yield as a white powder in
the same way as for the preparation of 4:20 Mp 91e92 ꢂC; FTIR
4.2.4. 2-N,N,N-Tri-n-octylammonium acetate (13). Compound 13
(ATR)
1340, 1289, 1095, 1008, 910, 872, 819, 726; 1H NMR (500 MHz,
DMSO-d6, TMS, rt) 3.15 (s, 3H), 3.60 (s, 2H), 3.66 (m, 4H), 3.74 (br,
4H), 5.25 (t, 2H); 13C NMR (125 MHz, methanol-d4, TMS, rt)
50.04,
n
/cmꢁ1 3399, 3032, 2900, 2839, 2779, 2355, 1600, 1485, 1402,
was obtained in 84% yield as a colorless solid in the same way as for
the preparation of 10: Mp 32e33 ꢂC; FTIR (ATR)
n
/cmꢁ1 2954, 2919,
d
2872, 2853, 2360, 1635, 1472, 1363, 1339, 852, 721; 1H NMR
d
(500 MHz, CDCl3, TMS, rt)
d
0.87 (t, J¼6.8 Hz, 9H),1.28 (m, 30H),1.61
55.73, 62.72, 64.34, 167.92; HR-ESI-MS (positive mode): m/z for
(m, 6H), 3.47 (t, J¼7.5 Hz, 6H), 3.62 (s, 2H); 13C NMR (125 MHz,
C7H15NO4, calculated 177.1079 (MHþ), found 178.1074 (MHþ).
CDCl3, TMS, rt)
d 14.09, 22.20, 22.53, 26.52, 28.84, 31.58, 58.90,
59.90, 164.31; HR-ESI-MS (positive mode): m/z for C26H53NO2,
4.2.11. 2-(N-Pyridinium) acetate (20). Compound 20 was obtained
calculated 412.4155 (MHþ), found 412.4148 (MHþ).
in 60% yield as a white powder in the same way as for the prepa-
ration of 4:20 Mp > 300 ꢂC; FTIR (ATR) /cmꢁ1 2967, 2362, 2337,
n
4.2.5. 2-(N-n-Butyl-N,N-di-n-hexylammonium)
Compound 14 was obtained in 96% yield as a colorless oil in the
same way as for the preparation of 10: FTIR (ATR)
/cmꢁ1 2956,
2928, 2859, 2363, 2355, 2333, 1635, 1467, 1352, 1313, 1043, 862,
726, 716; 1H NMR (500 MHz, DMSO-d6, TMS, rt)
0.88 (t, J¼6.0 Hz,
6H), 0.98 (t, J¼7.0 Hz, 3H), 1.31 (m, 14H), 1.67 (t, 6H), 3.47 (t, 6H),
3.62 (s, 2H); 13C NMR (125 MHz, CDCl3, TMS, rt)
13.75,13.94,19.93,
acetate
(14).
2165, 2155, 1628, 1485, 1341, 1181, 1051, 1033, 893, 787, 716, 604; 1H
NMR (500 MHz, DMSO-d6, TMS, rt)
d
4.85 (t, 1H), 8.02 (d, J¼6.5 Hz,
n
2H), 8.46 (m, J¼8.0 Hz, 1H), 8.81 (m, J¼6.0 Hz, 2H); 13C NMR
(125 MHz, methanol-d4, TMS, rt)
d 63.51, 127.34, 145.00, 145.65,
d
168.55; HR-ESI-MS (positive mode): m/z for C7H7NO2, calculated
138.0555 (MHþ), found 138.0547 (MHþ).
d
22.15, 22.48, 24.17, 26.21, 31.28, 58.69, 58.91, 59.92, 164.32; HR-ESI-
MS (positive mode): m/z for C18H37NO2, calculated 300.2903
(MHþ), found 300.2897 (MHþ).
4.2.12. 2-(N0-Methyl-N-imidazolium) acetate (21). Compound 21
was obtained in 73% yield as a white powder in the same way as for
the preparation of 4:20 Mp 276e277 ꢂC; FTIR (ATR) /cmꢁ1 3033,
n
2356, 1619, 1562, 1456, 1359, 1294, 1173, 1038, 928, 892, 817, 782,
694, 631; 1H NMR (500 MHz, DMSO-d6, TMS, rt)
3.80 (s, 3H), 4.34
(s, 2H), 7.53 (s, 2H), 8.94 (s, 1H); 13C NMR (125 MHz, methanol-d4,
4.2.6. 2-(N,N-Di-n-hexyl-N-n-octylammonium)
Compound 15 was obtained in 16% yield as a colorless oil in the
same way as for the preparation of 10: FTIR (ATR)
/cmꢁ1 2954,
2924, 2857, 1635, 1472, 1340, 1033, 856, 720; 1H NMR (500 MHz,
acetate
(15).
d
n
TMS, rt) d 34.97, 51.96, 122.65, 123.55, 137.51; HR-ESI-MS (positive
mode): m/z for C6H8N2O2, calculated 141.0664 (MHþ), found
DMSO-d6, TMS, rt)
d
0.88 (t, 6H), 1.32 (m, 22H), 1.62 (m, 6H), 3.48 (t,
141.0661 (MHþ).
J¼6.8 Hz, 6H), 3.62 (s, 2H); 13C NMR (125 MHz, CDCl3, TMS, rt)
d
13.94, 14.14, 22.14, 22.48, 22.64, 26.21, 26.55, 29.07, 29.12, 31.28,
4.3. Enzymatic reactions21
31.68, 58.90, 59.90, 164.34; HR-ESI-MS (positive mode): m/z for
22H45NO2, calculated 356.3529 (MHþ), found 356.3522 (MHþ).
C
Enzymatic reactions were performed as follows: a 1 mL aliquot of
a
-glucosidase stock solution (2.5ꢃ10ꢁ5 mg/mL) was placed onto
4.2.7. 2-(N,N-Di-n-butyl-N-methylammonium)
Compound 16 was obtained in 84% yield as a colorless oil in the
same way as for the preparation of 10: FTIR (ATR)
/cmꢁ1 2961,
2875, 2361, 2342, 1628, 1459, 1378, 1318, 1034, 880, 717; 1H NMR
(500 MHz, DMSO-d6, TMS, rt)
acetate
(16).
the wall of each well of a 96-well plate containing 199 mL substrate
(0e2.0 mM), metabolite analogs (0e1000 mM) and 100 mM
phosphate buffer solution (pH 7.0), and incubated at 37 ꢂC for
3 min. The hydrolysis reaction was started by shaking the plate,
thus mixing together the enzyme and buffer. The initial slopes of
the absorbance at 405 nm against incubation time were converted
into initial velocities using the molar extinction coefficient of p-
n
d
0.98 (t, J¼7.5 Hz, 6H), 1.39 (m,
J¼7.4 Hz, 4H), 1.66 (m, J¼8.0 Hz, 4H), 3.20 (s, 3H), 3.48 (t, J¼6.1 Hz,
2H), 3.56 (t, J¼6.0 Hz, 2H), 3.71 (s, 2H); 13C NMR (125 MHz, CDCl3,

Y. Nakagawa et al. / Tetrahedron 70 (2014) 5895e5903
5903
nitrophenol at 405 nm (10,900 cmꢁ1
M
ꢁ1). The initial velocities
References and notes
were estimated as the average of five measurements.
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Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2014.06.028.