2
E. Kato et al. / Tetrahedron Letters xxx (2016) xxx–xxx
Ph
O
OH
HO
OTIPS
6
4
HO
HO
O
HO
a-e
g,h
RO
PMBO
RO
HN
RO
PMBO
O
O
O
HN
HN
HO
PMBO
HO
RO
OCH3
PMBO
OCH3
OCH3
methyl acarviosin
(α,β mixture)
4
2: R=H
f
3
: R=PMB
OH
O
HO
HO
OH
O
i,j
HO
O
n
O
O
O
HO
O
HO
HN
HO
O
O
O
I
n
HO
OCH3
5a c
-
(n=4,6,9)
6: n=4
7: n=6
8: n=9
Scheme 1. Reagents and conditions: (a) 2,2-Dimethoxypropane, DMF, CSA; (b) separation of the
a-anomer, 18% in two steps; (c) 80% AcOH aq, 1,4-dioxane; (d) a,a-
dimethoxytoluene, CSA, DMF; (e) 40% AcOH aq, 1,4-dioxane, 42% in three steps; (f) PMBCl, NaH, DMF, 56%; (g) 80% AcOH aq, 1,4-dioxane, 50 °C; (h) TIPSCl, imidazole, CH2Cl2,
65% in two steps; (i) 5, NaH, DMF, THF, 15–25%; (j) TFA, MeOH, quant.
Table 1
to yield the desired products (6–8). Additionally, compound 9 was
synthesized from 4, which has no glucose moiety and the shortest
a-Amylase inhibitory activity of glucose–acarviosin conjugates
alkyl chain (Scheme 2).
Compound Inhibitory activity (%)
The
are summarized in Table 1. Methyl
-amylase inhibitory activity at 500
2 mM. In contrast, the synthetic compounds (6–8) showed scarce
or no inhibition at 500 M and mild inhibitory activity at 2 mM.
Unlike the previously synthesized glucose–deoxynojirimycin
conjugate, no enhancement of -amylase inhibitory activity was
a
-amylase inhibitory activities of the synthetic compounds
-acarviosin (1) showed mild
M and strong inhibition at
0.5 mM
2.0 mM
a
l
Methyl
a
-acarviosin (1)
65
4
2
5
0
85
45
43
43
5
a
6 (n = 4)
7 (n = 6)
8 (n = 9)
9 (n = 1, without glucose)
Acarbose (5 M)
l
l
55
a
observed for any of the synthetic compounds when compared with
the inhibitory activity of 1. However, when the activity of 6–8 is
compared with 9, clear enhancement is observed, indicating the
From these results,
pounds is summarized as the following. Methyl
itself has low affinity toward -amylase. However, during the
enzyme reaction, 1 undergoes the transglycosylation reaction to
form maltose or glucose attached products. These products mimic
trisaccharides or tetrasaccharides and occupy one or two addi-
a
-amylase inhibition of the tested com-
effect of glucose addition. Thus, our concept to enhance a-amylase
a-acarviosin (1)
inhibitory activity through conjugation of glucose is still effective.
Therefore, we postulate that for the activity of 1, the free hydroxyl
group at the C-4 position of acarviosin may have some function in
a
the
a-amylase inhibitory activity, and derivatization using this
residue decreases its activity.
In the case of acarbose, the hydroxyl group at the C-4 position is
known to participate in the transglycosylation reaction and pro-
tional subsites of
is a part of the substrate binding site of
with a single glucose unit.15
-Amylase shows strong affinity
a
-amylase when compared with 1. The subsite
a-amylase that interacts
a
duces a product with stronger affinity to
a-amylase during the
toward starch by interacting with multiple glucose units with mul-
enzyme reaction.14 To test if a similar reaction occurs for 1, we ana-
lyzed the enzyme reaction mixture. Analysis of the reaction mixture
by UPLC-Tof-MS (Supplementary Fig. 1) showed two peaks, the
minor peak with m/z 498.2179 ([1+glucoseÀH2O+H]+, C20H36NO13
requires m/z 498.2187) and the major peak with 660.2714 ([1+mal-
toseÀH2O+H]+, C26H46NO18 requires m/z 660.2715), which corre-
sponds to the transglycosylation products of 1 (Fig. 1A). Similar
transglycosylation products were also detected from the analysis
of the enzyme reaction mixture of 6–8, except that glucose trans-
ferred products were more abundant (Supplementary Figs. 2–4).
No transglycosylation products were detected for 9 (Supplementary
Fig. 5).
tiple subsites and therefore, the transglycosylation products show
higher affinity to
a-amylase and possess higher inhibitory activi-
ties. Thus, the transglycosylation process of 1 can yield products
that show stronger inhibitory activity (Fig. 1A). In comparison,
compound 9 does not receive transglycosylation reaction because
the C-4 hydroxyl group is not readily accessible, and the inhibitory
activity of this compound results from their original structure
(Fig. 1C). Compounds 6–8 are susceptible to transglycosylation
reaction. However, the reaction has lower effect on the inhibitory
activity since there are only two subsites above the acarviosin
binding position,14 and additional glucose is not recognized by
a-
amylase enzyme (Fig. 1B). Therefore, the glucose–acarviosin conju-
gates (6–8) or their transglycosylation products interact with three
subsites, thus bypassing one in the middle and 9 interacts with two
subsites. Thus glucose–acarviosin conjugates show higher activity
compared with 9; however, all synthetic compounds show lower
activity compared with 1, because the transglycosylation products
of 1 interact with more subsites.
OH
H3CO
k,l
HO
4
HO
HN
HO
O
HO
In conclusion, we have synthesized glucose–acarviosin conju-
OCH3
9
gates and tested their
to enhance -amylase inhibitory activity by conjugating glucose
to -glucosidase inhibitors was proven to have some effect when
a-amylase inhibitory activity. Our concept
a
Scheme 2. Reagents and conditions: (k) CH3I, NaH, DMF, THF, 75%; (l) TFA, MeOH,
92%.
a