Concise synthesis of glyconoamidines as affinity ligands for the purification of β-glucosidase involved in control of some biological events including plant leaf movement

Article abstract of DOI:10.1016/j.tetlet.2005.05.060

Glycosidases are involved in deactivation or storage of some endogenous bioactive substances through biologically intriguing processes. For example, nyctinastic leaf movement is controlled by a biological clock through the regulation of β-glucosidase activity. Ganem's glyconoamidine (1) is used as a micromolar inhibitor of glucosidase in biochemical studies and would be useful as an affinity ligand for purification of glycosidase. However, its use for the specific inhibition of glucosidase which is highly specific to a glycoside with voluntary aglycon is seriously restricted because no universal method for the synthesis of N-alkylated glyconoamidine has been reported. Here, we report a concise synthesis of N-alkylated Ganem's glyconoamidine with voluntary aglycon using a non-protected sugar derivative.

Full text of DOI:10.1016/j.tetlet.2005.05.060

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 4865–4869
Concise synthesis of glyconoamidines as affinity ligands for
the purification of b-glucosidase involved in control of
some biological events including plant leaf movement
Eisuke Kato, Tadahiro Kumagai and Minoru Ueda*
Tohoku University, Department of Chemistry, Aoba-ku, Sendai 980-8578, Japan
Received 22 April 2005; revised 12 May 2005; accepted 16 May 2005
Abstract—Glycosidases are involved in deactivation or storage of some endogenous bioactive substances through biologically
intriguing processes. For example, nyctinastic leaf movement is controlled by a biological clock through the regulation of b-gluco-
sidase activity. GanemÕs glyconoamidine (1) is used as a micromolar inhibitor of glucosidase in biochemical studies and would be
useful as an affinity ligand for purification of glycosidase. However, its use for the specific inhibition of glucosidase which is highly
specific to a glycoside with voluntary aglycon is seriously restricted because no universal method for the synthesis of N-alkylated
glyconoamidine has been reported. Here, we report a concise synthesis of N-alkylated GanemÕs glyconoamidine with voluntary agly-
con using a non-protected sugar derivative.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Glycosidases are involved in deactivation or storage of
some endogenous bioactive substances through biologi-
cally intriguing processes.1 In these processes, b-glucosi-
dase plays a key role in the control of intracellular
concentration of bioactive substances to convert its b-
glucoside (inactive form) into the corresponding aglycon
(active form). The glycoside hydrolases are classified
into 90 families according to their amino acid sequences,
and each family contains a number of glycosidases with
different aglycon substrate specificities. An inhibitor of
glycosidase is widely used for bioorganic studies of the
enzyme, and is also a good affinity ligand for the purifi-
cation of glycosidase.2 However, substrate specificity of
glycosidase with respect to the aglycon moiety usually
raises a serious problem in the molecular design of gly-
cosidase inhibitor.
An amidine-type b-glycosidase inhibitor, which was
found in nature as nagstatin,3 was developed by
Ganem,4 Tatsuta,5 Wong,6 Hiratake,7 et al. GanemÕs
glyconoamidine (1) is a micromolar inhibitor of b-gluco-
sidase. Several examples were reported on the synthesis
of this type of compounds.8–11 Most of these methods
require the benzyl-protected sugar derivatives, thus
some functional groups, such as olefin, cannot survive
in the deprotection conditions. We developed a novel
universal method for the synthesis of N-alkyl glycono-
amidine using a non-protected sugar derivative and syn-
thesized two inhibitors of glucosidase through the
internal processes of deactivation or storage of bioactive
substances in living cells.
Nyctinastic leaf movement is a typical example of a bio-
logical event in which b-glucosidase plays a key role.
Most leguminous plants close their leaves in the evening,
as if to sleep, and open them early in the morning
according to the circadian rhythm controlled by a bio-
logical clock. Leaf movement is controlled by a change
in the concentration of a glucoside-type leaf-movement
factor, which is induced by the circadian rhythmic regu-
lation of b-glycosidase activity by a biological clock.12,13
A biological clock maintains the rhythm of leaf move-
ment through the regulation of b-glycosidase activity.
Thus, b-glucosidase is a key enzyme of nyctinasty, and
Keywords: Nyctinasty; Glucosidase; Inhibitor.
*
Corresponding author. Tel./fax: +81 22 795 6553; e-mail: ueda@
mail.tains.tohoku.ac.jp
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.05.060

4866
E. Kato et al. / Tetrahedron Letters 46 (2005) 4865–4869
chemical studies on the rhythm of nyctinasty requires
purification of the enzyme.
Thus, we changed the amine from 5 into its correspond-
ing tert-butyl ester (7) to prevent self-condensation.
When 4 and 7 were coupled in pyridine–THF (9:1) by
using NBS (2 equiv), 8 was obtained in 87% yield.
Excess amount of 7 (ca. 10 equiv) was essential for
the good yield. And the yield was strongly affected by
the ratio of the mixed solvent.
In Lespedeza cuneata Don, change in the concentration of
a glucoside-type leaf-opening substance (potassium lespe-
dezate; 2),14 induces the rhythm of nyctinastic leaf move-
ment. Purification of b-glucosidase which has substrate
specificity to 2 is a key step in the mechanism of the circa-
dian rhythmic regulation of nyctinastic leaf movement.
Thus, we tried to synthesize an N-alkyl glyconoamidine
analog of 2 (3) which is expected to be a good ligand for
the affinity chromatography of the desired b-glucosidase.
Next, we examined the introduction of olefin in 8. DDQ
oxidation14 of 8 gave a decomposed product, and direct
bromination of 8 by NBS resulted in no reaction. Thus,
we synthesized fluorinated aglycon 9. Coupling of 9 with
4 and following defluorination would give the corre-
sponding olefin. The synthesis of 9 was carried out
according to Scheme 1.17
We examined coupling of 9 with 4. When the reaction was
carried out in DMF, coupling product 17 was directly ob-
tained in 90% yield with formation of olefin and depro-
tection of TBS yield together with 10% of TBS-
protected 17 (18) (Scheme 2). Compound 18 can also be
converted into 17 quantitatively by the treatment with
TBAF-AcOH. Long reaction time (ca. 5 h) was impor-
tant for direct formation of 17. Shorter reaction time gave
intermediates, such as 18. After purification by HPLC
(Cosmosil 5C18AR column, 30% MeOHaq containing
1% AcOH), 17 was treated with TFA to give 1918 quan-
titatively. The geometry of olefin and stereochemistry
of the alkyl amidine moiety in 19 was determined to be
We used thionolactam (4), which was synthesized
according to VasellaÕs method,15,16 as a sugar moiety
(Table 1). Non-protected thionolactam (4) was used in
the coupling with amine (5) because no protected 4
could give a coupling product: when we used acetyl-pro-
tected 4, the resulting glyconoamidine was completely
decomposed under the deprotection conditions because
the amidine group in the resulting glyconoamidine
(6) was very weak under the basic conditions. And the
use of benzyl or TMS-protected 4 resulted in no
reaction.
0
anti-(Z) by NOE experiment between H2 and H2 , and
0
H3 and H3 (Scheme 3). The resulting 19 was photoiso-
merized into a 1:1 mixture of (Z)-19 and (E)-3. And
(E)-319 was isolated by HPLC (Develosil C30-HG, H2O
0
containing 1% AcOH). Small NOE between H2 and ole-
We examined the coupling of 4 with 5 under several con-
ditions using some activators of 4. Alkaline condition
(K2CO3, pyridine), or the use of NBS, Hg(OAc)2, and
PbO gave no coupling product. Only HgO gave a small
amount (16%) of a coupling product (Table 1). In these
cases, the amino group of 5 would attack the carbonyl of
another 5, instead of the attack to 4, to give an oligomer.
finic H demonstrated that the geometry of olefin in 3 is
(E) and stereochemistry of the amidine moiety is anti.
Inhibitory activities of 3 and 19 against various glycosi-
dases are shown in Table 1. In Table 1, b-glucosidase
activity was determined by measuring the absorbance at
405 nm of p-nitrophenol formed from p-nitrophenyl-b-
Table 1. Formation of glyconoamidine
Aglycon (equiv)
Activator
Solvent
Yield (%)
5 (2 equiv)
7 (1 equiv)
7 (2 equiv)
7 (2 equiv)
7 (10 equiv)
7 (2 equiv)
7 (10 equiv)
HgO (2 equiv)
NBS (2 equiv)
NBS (2 equiv)
NBS (2 equiv)
NBS (2 equiv)
NBS (2 equiv)
NBS (2 equiv)
MeOH/Pyr = 9:1
THF/Pyr = 1:1
THF/Pyr = 1:1
Pyr
16
9
38
Many spots
THF/Pyr = 1:1
THF/Pyr = 9:1
THF/Pyr = 9:1
59
46
87

E. Kato et al. / Tetrahedron Letters 46 (2005) 4865–4869
4867
Scheme 1. Synthesis of fluorinated aglycon (9).
Scheme 2. Synthesis of glyconoamidine-type inhibitor of b-glycosidase (3) concerning the control of nyctinastic leaf movement.
Scheme 3. Synthesis of glyconoamidine-type inhibitors of biological events in plant.
D-glucopyranoside by spectroscopic method.20 And
Interestingly, (E)-3 showed extremely high specific inhib-
itory activity against b-glucosidase, whereas (Z)-19
inhibited b-glucosidase as strong as a-glucosidase (Table
2). Both 3 and 19 did not inhibit b-galactosidase at all.
Here, we synthesized micro-molar b-glucosidase inhibi-
tor 3, which is expected to be a useful affinity ligand for
the purification of the key enzyme controlling nyctinasty.
inhibitory activities of synthetic N-alkyl glyconoamidines
against a few enzymes were determined by interpretation
of the Dixon plot.21 Ki values against a-glucosidase and b-
galactosidase were determined similarly. Interestingly, a
distinct difference was observed in the Ki values of (E)-3
and (Z)-19 against b-glucosidase from Aspergillus niger.
Ki value of (Z)-19 was 5.8 · 10ꢀ5 M, whereas that of
(E)-3 was 1.6 · 10ꢀ6 M (Table 2). Inhibitory activity of
3 was 40-fold as strong as that of 19.
According to our method, various glyconoamidine-type
glucosidase inhibitors can be obtained by using various

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E. Kato et al. / Tetrahedron Letters 46 (2005) 4865–4869
Table 2. Inhibitory activities of 3 and 19 against glycosidases
This work was financially supported by the Ministry of
Education, Science, Sports and Culture (Japan) Grant-
in-Aid for Scientific Research (No. 16073216 and No.
15681013) and JSPS Grant for EK. This work was also
supported by Grant-in-Aid for the 21st Century COE
program of Tohoku University from the Ministry of Edu-
cation, Culture, Sports, Science, and Technology, Japan.
Enzyme
Ki of 3 (lM) Ki of 19 (lM)
b-Glycosidase (Aspergillus niger)
b-Glycosidase (Almonds)
a-Glycosidase (Bacillus sp.)
1.6
255
436
—
58
540
52
b-Galactosidase (Aspergillus oryzae)
—
Ki values were measured at optimal pH of each enzyme [50 mM acetate
buffer (pH 5.0 for b-glucosidases and pH 6.8 for a-glucosidase)].
References and notes
1. Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry, 5th
ed.; W. H. Freeman and Company: New York, 2002.
2. Heightman, T. D.; Vasella, A. T. Angew. Chem., Int. Ed.
1999, 38, 750–770, and references cited therein.
3. Aoyagi, H.; Suda, S.; Uotani, K.; Kojima, F.; Aoyama,
T.; Horiguchi, K.; Hamada, M.; Takeuchi, T. J. Antibiot.
1992, 45, 1557–1558.
Figure 1. Release of bioactive aglycon from corresponding
b-glycosides.
4. Tong, M. K.; Papandreous, G.; Ganem, B. J. Am. Chem.
Soc. 1990, 112, 6137–6139; Ganem, B.; Papandreous, G.
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Lett. 1995, 36, 6721–6724.
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Ichikawa, Y.; Porco, J. A.; Wong, C.-H. J. Am. Chem.
Soc. 1991, 113, 6187–6196.
aglycons containing an amino group. The use of non-
protected 4 would serve for both shortening of the syn-
thetic route and expanding versatility of this reaction:
applicable for a wide range of aglycons with various
functional groups which would be decomposed in the
deprotection conditions.
7. Guo, W.; Hiratake, J.; Ogawa, K.; Yamamoto, M.; Ma,
S.-J.; Sakata, K. Bioorg. Med. Chem. Lett. 2000, 11, 467–
470; Inoue, K.; Hiratake, J.; Mizutani, M.; Takada, M.;
Yamamoto, M.; Sakata, K. Carbohydr. Res. 2003, 338,
1447–1490.
Glycoside 20, which is stored or deactivated as a glyco-
side, is known to be released as an active form by the ac-
tion of specific b-glucosidase. 2-Phenylethanol (21), that
is stored as an aroma precursor of 2-phenylethyl b-D-
glucopyranoside (20), is one of the dominant floral scent
compounds emitted from roses.22 b-Glucosidase is in-
volved in the emission of 21 when the rose flower opens
(Fig. 1). We synthesized glyconoamidines (22) from cor-
responding glucoside 20 by using phenylethylamine (23)
and 4 by using NBS in 27% yield (Scheme 3). The yield
of this coupling reaction seems to be low. However, this
is because most of 22 was lost in repeated purification by
silica gel or ODS chromatography even in acidic condi-
tions. The Ki value of 22 against b-glucosidase (Aspergil-
lus niger) was determined to be 1 nM according to the
same method as described above. And the inhibitory
activity of 22 was specific to b-glucosidase: 22 did not
show any inhibitory activity against a-glucosidase
(Bacillus sp.).
8. Papandreou, G.; Tong, M. K.; Ganem, B. J. Am. Chem.
Soc. 1993, 115, 11682–11690.
9. Bluriot, Y.; Genre-Grandpierre, A.; Tellier, C. Tetrahe-
¨
dron Lett. 1994, 35, 1867–1870.
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11. Heck, M.-P.; Vincent, S. P.; Murray, B. W.; Bellamy, F.;
Wong, C.-H.; Mioskowskii, C. J. Am. Chem. Soc. 2004,
126, 1971–1979.
12. Ohnuki, T.; Ueda, M.; Yamamura, S. Tetrahedron 1998,
54, 12173–12184.
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1400–1414.
14. Shigemori, H.; Sakai, N.; Miyoshi, E.; Shizuri, Y.;
Yamamura, S. Tetrahedron 1990, 46, 383–394.
15. Hoos, R.; Naughton, A. B.; Vasella, A. Helv. Chim. Acta
1993, 76, 1802–1807.
Compound 2223 would be a useful inhibitor of this bio-
logical event in biochemical studies of this process.
Inhibitors of b-glucosidases which are specific to 20,
would be a useful tool for biochemical studies of this
biological event, and can be used as a ligand in affinity
chromatography for the purification of the glucosidase.
16. Hoos, R.; Naughton, A. B.; Thiel, W.; Vasella, A.; Weber,
W.; Rupitz, K.; Withers, S. G. Helv. Chim. Acta 1993, 76,
2666–2686.
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1995, 36, 6373–6374.
18. 19: H NMR (300 MHz, CD3OD, rt): 7.59 (1H, s), 7.43
1
(2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.4 Hz), 4.32 (1H, d,
J = 9.6 Hz), 3.75 (1H, dd, J = 7.2, 9.3 Hz), 3.69 (1H, dd,
J = 3.0, 11.7 Hz), 3.61 (1H, dd, J = 9.3, 9.6 Hz), 3.52 (1H,
dd, J = 3.6, 11.7 Hz), 3.21 (1H, ddd, J = 3.0, 3.6, 7.2 Hz)
ppm; 13C NMR (75 MHz, CD3OD, rt): 170.0, 165.3,
160.6, 136.4, 132.9, 125.9, 125.5, 116.8, 74.2, 70.4, 69.7,
62.3, 61.9 ppm; HR ESI MS (positive): [M+H]+ Found
m/z 339.1187, C15H19N2O7 requires m/z 339.1192; IR
Purification of the b-glycosidase concerning nyctinasty
is now in progress. Details on this enzyme will be
reported in due course.
Acknowledgements
18
(film) m: 3207, 1668, 1606, 1558, 1514, 1379 cmꢀ1; ½aꢁD
ꢀ21.6 (c 0.50, CH3OH).
19. 3: 1H NMR (300 MHz, D2O, rt): 7.40 (2H, d, J = 8.4 Hz),
6.88 (2H, d, J = 8.4 Hz), 6.73 (1H, s), 4.48 (1H, d,
J = 8.8 Hz), 3.891 (1H, dd, J = 8.8, 9.5 Hz), 3.893 (1H,
dd, J = 3.0, 12.4 Hz), 3.84 (1H, dd, J = 9.0, 9.5 Hz), 3.75
We thank Professor Jun Hiratake and Professor Kanzo
Sakata (Institute for Chemical Research, Kyoto Univer-
sity) for helpful discussions and suggestions about this
work.

E. Kato et al. / Tetrahedron Letters 46 (2005) 4865–4869
4869
(1H, dd, J = 4.4, 12.4 Hz), 3.59 (1H, ddd, J = 3.0, 4.4,
9.0 Hz) ppm; 13C NMR (75 MHz, D2O, rt): 172.0, 166.0,
158.2, 133.0, 132.5, 128.0, 126.6, 117.0, 73.5, 70.3, 68.9,
61.7, 61.6 ppm; HR ESI MS (positive): [M+H]+ Found
m/z 339.1188, C15H19N2O7 requires m/z 339.1187; IR
Takada, M.; Ogawa, K.; Watanabe, N. Tetrahedron 2004,
60, 7005–7013.
23. 22: H NMR (300 MHz, CD3OD, rt): 7.35–7.21 (5H, m),
1
4.12 (1H, d, J = 9.6 Hz), 3.78 (1H, dd, J = 3.0, 11.7 Hz),
3.72 (1H, br t, J = 9.5 Hz), 3.70 (1H, dd, J = 3.5, 11.6 Hz),
3.64–3.57 (3H, m), 3.50 (1H, br t, J = 9.6 Hz), 2.95 (2H, br
t, J = 7.0 Hz); 13C NMR (75 MHz, CD3OD, 30 ꢁC): 166.1,
139.0, 130.0, 129.8, 128.0, 74.2, 70.0, 69.4, 62.3, 61.4, 44.1,
35.0 ppm; HR ESI MS (positive): [M+H]+ Found m/z
281.1496, C14H21N2O4 requires m/z 281.1496; IR (film) m:
26
(film) m: 3246, 1664, 1609, 1560, 1514, 1411 cmꢀ1; ½aꢁD
ꢀ26.1 (c 0.40, H2O).
20. Collins, R. A.; Ng, T. B.; Fong, W. P.; Wan, C. C.; Yeung,
H. W. Biochem. Mol. Biol. Int. 1997, 42, 1163–1169.
21. Dixon, M. Biochem. J. 1953, 55, 170–171.
22. Hayashi, S.; Yagi, K.; Ishikawa, T.; Kawasaki, M.; Asai,
T.; Picone, J.; Turnbull, C.; Hiratake, J.; Sakata, K.;
25
3238, 2926, 2858, 1672, 1560, 1412 cmꢀ1; ½aꢁD +9.42 (c
0.10, CH3OH).