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ISSN 1349-4848
number151
Contr ibution - The High Functionality of Glycosylation Using Plant Suspension Cells
Hiroki Hamada Professor of Department of Life Science, Faculty of Science, Okayama University of Science
Kei Shimoda Associate Professor of Department of Chemistry, Faculty of Medicine, Oita University
New Produc ts I nformation :- New Carbon Nanoring Material - An Enzyme that Adds Whole Sugar Chains without Breaking Down Products “Endo-M-N175Q”- Lysine Speci�c Demethylase 1-Selective Inhibitor
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CONTENTS
Plant Culture Cell
Capsaicin-O- -D-glucosideβ
Capsaicin
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1. Introduction
Because prodrugs and prosupplements (precursors of drugs and dietary supplements that are metabolized within the body to form the corresponding bioactive materials) have recently attracted a great deal of attention, there is a growing need for techniques that are capable of selective chemical modification of functional compounds. We have focused on glycosylation, particularly glucosylation, as one such type of chemical modification. There have been many studies on the production of useful substances by organic syntheses through routes that incorporate reactions induced by biocatalysts, such as cultured cells or enzymes, and the results of such studies have been applied in the production of a range of fine chemicals, including pharmaceuticals, aroma chemicals, and food additives. Among the biocatalysts that have been used are microorganisms, fungi, yeasts, animal cells, and enzymes extracted from these sources. In addition, biotransformations effected by using cultured plant cells as biocatalysts have recently attracted attention. Plants, which live on land and are generally incapable of movement, produce various secondary metabolites for the purposes of self-defense and signal transduction. As a result, plant cells contain a range of enzymes that have inherent abilities to transform or to synthesize organic substances. With the aim of utilizing the intrinsic ability of plant enzymes to effect biotransformations, we have studied biotransformations of exogenous substances by cultured plant cells and we have succeeded in effecting a range of reactions, including reduction, hydrolysis, isomerization, glycosylation, esterification, and hydroxylation reactions, by using cultured plant cells as biocatalysts. Glycosylation by plant cells is a particularly important reaction that is involved in the activation of metabolites in cells and might, therefore, be useful in stabilizing various biologically active compounds or in activating various physiological functions. A major advantage of using biocatalysts in organic synthesis is their high selectivity. Furthermore, glycosides, the chemical synthesis of which generally involves a complicated range of procedures, can be obtained by means of a one-step enzymatic reaction with a biocatalyst. For these reasons, the application of cultured plant cells that are capable of highly stereoselective glycosylation reactions in organic synthesis has
been eagerly anticipated. We have attempted to apply glycosylation reactions catalyzed by cultured plant cells to the transformation of bioactive compounds into synthetic compounds with improved stabilities and new bioactivities. This review introduces the results of our studies to date on the transformation and activation of bioactive compounds by means of cultured plant cells.
2. Glycosylation of tocophenols
Tocopherols have attracted clinical attention because of its potential to be a very useful medicine having effects on gynecological internal secretion control against sterility, heart circulation, liver diseases, aging, atherosclerosis, thrombosis, and carcinogenesis. Despite of such specific physiological and pharmacological activities, water-insolubility, instability, and light decomposition of these vitamins have been problems responsible for the poor absorption following oral administration and for the limit of their use as medicines. To synthesize water-soluble tocopherol glycosides, glycosylation of tocopherols with cultured plant cells was investigated. Just prior to use for this work, 50 g of cultured cells was transplanted to a 300 mL conical flask containing 100 mL of freshly prepared SH medium (pH 5.7) containing 3% sucrose and grown with continuous shaking for 1 week at 25 °C under illumination (4000 lux). A total of 90 mg of each vitamin substrates was administered to the 6 flasks (15 mg/flask) containing the suspension cultured cells and the cultures were incubated at 25 °C for 7 days on a rotary shaker (120 rpm). After incubation, the cells were harvested and extracted (×3) by homogenization with MeOH. The yield of the products was calculated on the basis of the peak area from HPLC using the calibration curves prepared by the HPLC analyses of authentic glycosides. The MeOH extract was concentrated and the residue was partitioned between H2O and EtOAc. The H2O layer was applied to a Diaion HP-20 column and the column was washed with H2O followed by elution with MeOH. The MeOH eluate was subjected to HPLC (column: 150×20 mm) to give products. No products were observed in the medium. The structures of the products were identified using HRFABMS, 1H and 13C NMR, H-H COSY, and C-H COSY.
The High Functionality of Glycosylation Using Plant Suspension Cells
Hiroki Hamada1)* and Kei Shimoda2)
1) Department of Life Science, Faculty of Science, Okayama University of Science,1-1 Rida-cho Kita-ku Okayama 700-0005, Japan
*Corresponding author: e-mail:[email protected]) Department of Chemistry, Faculty of Medicine, Oita University,
1-1 Hasama-machi, Oita 879-5593, JapanE-mail: [email protected]
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On admin is t ra t ion o f 2 ,5 ,7 ,8 - te t ramethy l -2- (4-methylpentyl)-6-chromanol (tocopherol derivative, 1) to the cultured cells of Phytolacca americana, a product 4 (63%) was obtained (Fig. 1).1 The product 4 was identified as 2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-yl β-D-glucopyranoside, which was a new compound. Next, 2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)-6-chromanol (2) and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-6-chromanol (vitamin E, 3) with longer side chains were tested for the same biotransformation system. The structures of the isolated products 5 (35%) and 6 (7%) were determined as β-glucosides, i.e., 2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)chroman-6-yl β-D-glucopyranoside and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl β-D-glucopyranoside. On the other hand, two products, 4 (56%) and 7 (14%), were isolated from the cultured cells of C. roseus following administration of 1. The structure of 7 was determined to be 2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-yl 6-O-β-D-glucopyranosyl-β-D-glucopyranoside (β-gentiobioside), which was a new compound. On administration of 2, two
products were also isolated and identified as β-glucoside 5 (32%) and β-gentiobioside 8 (5%), i.e., 2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)chroman-6-yl 6-O-β-D-glucopyranosyl-β-D-glucopyranoside. When 3 was used as the substrate, only β-glucoside product 6 (8%) was obtained. Glycosylation of organic compounds often improves their bio- and pharmacological properties, e.g., glycosides of terpene alcohols have been widely used in folk medicines. Therefore, vitamin glycosides are expected to possess new physiological activities which can be of pharmacological interest. The suppressive action of the glycosides 4-8 on IgE antibody formation was examined according to the reported procedure (Table 1).2,3 As a result, 5 exerted the strongest action among the glycosides tested, whereas no actions were observed in the cases of 7 and 8. This shows that the β-glucosides of vitamin E and its homologues would be useful antiallergic drugs.
Compounds IgE level
4 184
5 170
6 195
7 366
8 353
Hydrocortisone 341
O n
C. roseus
OO
OHHO
HO
m
1: n=1; 2: n=2; 3: n=3 4: m=1, n=1; 5: m=1, n=2; 6: m=1, n=3; 7: m=2, n=1; 8: m=2, n=2
O n
HO
O n
P. americana
OO
OHHO
HO
1: n=1; 2: n=2; 3: n=3 4: n=1; 5: n=2; 6: n=3
O n
HO
HO
HO
12
34
56
7
89
10
11
12
13
Table 1. Anti-allergic activity of tocopheryl glycosides.
Figure 1. Glycosylation of tocopherols by cultured plant cells.
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3. Glycosylationofflavones
Natural polyphenols such as quercetin, epicatechin, and catechin occur in plants and have diverse biological activities. Quercetin is a principal flavonoid in onion Allium cepa, and has anti-oxidative, anticarcinogenic, and anticancer activities. Epicatechin and catechin are main flavonoids found in tea Camellia seinensis, and have been reported on their anti-oxidative, anti-angiogenesis, anti-inflammatory, and anticancer effects. Irrespective of such physiological and pharmacological activities, the use of these polyphenols as drugs and food additives is limited because of their water-insolubility and low absorbability after oral administration. Quercetin 3-O-β-D-glucoside (10) was obtained as the major product in 51% yield together with 3-O-(6-O-malonyl)-β-D-glucoside (11, 10%), 3-O-[6-O-(α-L-rhamnosyl)]-β-D-glucoside (β-rutinoside, 12, 3%), 3,4'-O-β-D-diglucoside (13, 1%), and 3,7-O-β-D-diglucoside (14, 1%) after incubation of Nicotiana tabacum cells with quercetin (9) (Fig. 2).4 On the other hand, three products were isolated from the MeOH extracts of N. tabacum cells treated with epicatechin ((2R,3R)-15). The products were identified as 3'-O-β-D-glucoside ((2R,3R)-16, 38%), 5-O-β-D-glucoside ((2R,3R)-17, 7%), and 7-O-β-D-glucoside ((2R,3R)-18, 15%). No further glycosylation and acylation products were obtained. Similar tendency was found in the biotransformation of catechin ((2R,3S)-15) with N. tabacum cells. Three products, 3'-O-β-D-glucoside ((2R,3S)-16, 46%), 5-O-β-D-glucoside ((2R,3S)-17, 10%), and 7-O-β-D-glucoside ((2R,3S)-18, 17%), were isolated, and no further products were detected despite careful HPLC analyses.
4. Glycosylation of 4-phenylbutan-2-ones
4-(4-Hydroxyphenyl)butan-2-one (raspberry ketone) and 4-(4-hydroxy-3-methoxyphenyl)butan-2-one (zingerone) are major aromatic compounds of red raspberry Rubus idaeus and zinger Zingiber officinale, respectively, and have been used worldwide as food additives and spices. Recently, it has been reported that these compounds, the structures of which are similar to those of capsaicinoids, showed stronger anti-obesity activity than capsaicin (N-[(4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonamide) and synephrine (1-(4-hydroxyphenyl)-2-methylaminoethanol). Despite such bio- and physiological activities, their use as lipid degradation ingredients and medicines has been limited, due to their insolubility in water and decomposition under light. After cultured cells of P. americana were incubated with raspberry ketone (19) for three days, the glycosylated products 22-26 were isolated from the cells by extraction with MeOH. On the other hand, none were detected in the medium. On the basis of their HRFABMS, 1H and 13C NMR (Table 1), H-H COSY, C-H COSY, and NOE-spectroscopic analyses, the products were determined to be (2S)-4-(4-hydroxyphenyl)-2-butanol (20, 2%), (2S)-4-(3,4-dihydroxyphenyl)-2-butanol (21, 5%), 4-[4-(β-D-glucopyranosyloxy)phenyl]-2-butanone (22, 19%), 4-[(3S)-3-hydroxybutyl]phenyl-β-D-glucopyranoside (23, 23%), (2S)-4-(4-hydroxyphenyl)but-2-yl-β-D-glucopyranoside (24, 20%), 2-hydroxy-4-[(3S)-3-hydroxybutyl]phenyl-β-D-glucopyranoside (25, 12%), and 2-hydroxy-5-[(3S)-3-hydroxybutyl]phenyl-β-D-glucopyranoside (26, 11%) (Fig. 3).5
12
34
56
78
9 O
O
OH
OH
HO
OH
OH10
1'
2'3'
4'
5'6'
12
34
56
78
9 O
OH
OH
HO
OH
OH10
1'
2'3'
4'
5'6'
O
O
OH
OR1
R3O
OH
OR2
O
OR1
OH
R3O
OR2
OH
10: R1,R3 = H, R2 = Glc;
11: R1,R3 = H, R2
= MalonylGlc; 12: R1,R3
= H, R2 = GlcRham;
13: R1,R2 = Glc, R3
= H; 14: R1
= H, R2,R3 = Glc
16: R1 = Glc, R2,R3
= H; 17: R1,R3
= H, R2 = Glc;
18: R1,R2 = H, R3
= Glc
9
15
N. tabacum
N. tabacum
Figure 2. Glycosylation of flavones by cultured plant cells.
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Glycoside products 29-32 were obtained from the MeOH extracts of the cells treated with zingerone (27), whereas product 28 was isolated from EtOAc extract of the medium. The products were identified as (2S)-4-(4-hydroxy-3-methoxyphenyl)-2-butanol (28, 15%), 4-[4-(β-D-glucopyranosyloxy)-3-methoxyphenyl]-2-butanone (29, 21%), 4-[(3S)-3-hydroxybutyl]-2-methoxyphenyl-β-D-glucopyranoside
(30 , 24%), (2S)-4-(4-hydroxy-3-methoxyphenyl)but-2-yl-β-D-glucopyranoside (31, 17%), and (2S)-2-(β-D-glucopyranosyloxy)-4-[4-(β-D-glucopyranosyloxy)-3-methoxyphenyl]butane (32, 16%).
HO
O
GlcO
O
HO
GlcO
HO
GlcO
HO
HO
GlcO
HO
HO
OH
OGlc
OH
OH
OH
OH
1922
23
24
25
26
12
34
56
7
89
10
20
21
P. americana
Figure 3. Glycosylation of 4-phenylbutan-2-ones by cultured plant cells.
HO
O
GlcO
O
HO
GlcO
HO
GlcO
H3CO
H3CO H3CO
H3CO
H3CO
H3COOH
OH
OGlc
OGlc
27
29
30
31
32
12
34
56
7
89
10
28
P. americana
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5. Glycosylation of curcumin
Curcuma longa Linn. has been used as a spice for centuries worldwide. Also it has been used in folk medicines for the treatment of a variety of inflammatory conditions. Its intake reduces the risk of certain kinds of cancers and renders other protective pharmacological effects in human. These medicinal properties have been attributed mainly to the curcuminoids, and the main component present in C. longa L. is curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] that has been widely studied for its anticancer, anti-inflammatory, antiaging, antiangiogenic, wound healing, and antioxidant effects. Irrespective of such pharmacological activities, its use as a medicine has been limited, because of its water insolubility and poor absorption after oral administration. Incubation of Strophanthus gratus suspension cells with curcumin (33) gave glucoside 34 as the sole product in 68% yield.6 Cultured cells of P. americana converted 33 into 34 (27%) and doubly glucosylated product (5%). On the other hand, no glycosylation products were isolated from the cultured N. tabacum cells which had been treated with 33. The difference in the ability of glucosylation of curcumin between the three plant species is probably due to the substrate specificity of glucosyltransferases in these plant cells. Glucosylation of curcumin with S. gratus described here is considerably efficient method to give curcumin β-D-glucoside rather than chemical glucosylation. Biocatalytic glycosylation of curcumin β-D-glucoside with CGTase was attempted to synthesize curcumin β-maltooligosides. As a result, products 35 (28%), 36 (20%), 37 (14%), and 38 (10%) were obtained after 24 h incubation of curcumin β-D-glucoside (34), which had been prepared by glucosylation of curcumin (33) with S. gratus, with CGTase in the presence of soluble starch (Fig. 4).
6. Glycosylation of capsaicin
Capsaicin, N-[(4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-(E)-6-nonenamide, is the most pungent principle among naturally occurring capsaicinoids. Capsaicin has also been reported to decrease adipose tissue weight and serum triacylglycerol content in rats by enhancing energy metabolism. Capsaicin has shown a wide range of pharmacological properties, such as analgesic, antigenotoxic, antimutagenic, and anticarcinogenic effects, and has been used to treat various peripheral painful conditions, including rheumatoid arthritis and diabetic neuropathy. However, capsaicinoids possess extensive neurological toxicity, and direct irritant effects on skin and mucous membrane. Furthermore, capsaicinoids are scarcely soluble in water and poorly absorbed after oral administration. These disadvantages prevent capsaicinoids from being used as food additives and medicines. Glycoside product 40 was isolated from cultured cells of P. americana that had been previously treated with capsaicin (39) by extraction with MeOH.7 The substrate, capsaicin (39), was detected in both the media and cell extracts. The glycoside product was purified and isolated by preparative HPLC for structure identification by spectroscopic methods. The product 40 was identified as capsaicin 4-O-β-D-glucopyranoside by comparison of its 1H and 13C NMR data with previously reported data (Fig. 5).
HN
O
HO
H3COHN
O
O
H3CO
O
OHHO
HOOH
39 40
P. americana
34
H3CO
HO
OCH3
OH
O OH
H3CO
O
OCH3
OH
O OH
O
OHHO
HOHO
S. gratus1 3 5 72 4 6
1' 3'1''3''
6' 4'6''4''
2'' 2'
5'' 5'1a
2a3a
5a6a
4a
33
35: n=1; 36: n=2; 37: n=3; 38: n=4
H3CO
O
OCH3
OH
O O
O
OHHO
HOO
OHO
HOHO
CGTase
n
HO
Figure 5. Glycosylation of capsaicin by cultured plant cells.
Figure 4. Glycosylation of curcumin by cultured plant cells and CGTase.
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7. Summary
Biocatalytic glycosylation using plant cultured cells and enzymes is a convenient and useful method to prepare costly and scarce glycosides from inexpensive and plentiful organic compounds. It was demonstrated that cultured plant cells and enzymes such as CGTase can glycosylate exogenously added compounds. The diverse glycosylation of exogenous compounds was observed between the cultured cells. Purification of the glucosyltransferases which catalyze the biotransformation of exogenous compounds and studies on the pharmacological activities of the glycosides are now in progress in our laboratory. In the future we strongly believe that this process and method will play an important role in pharmaceutical and supplement industry.
References
1) K. Shimoda, Y. Kondo, K. Abe, H. Hamada, H. Hamada, Tetrahedron Lett. 2006, 47, 2695-2698.
2) R. K. Uhrig, M. A. Picard, K. Beyreuther, M. Wiessler, Carbohydr. Res. 2000, 325, 72-80.
3) T. Satoh, H. Miyataka, K. Yamamoto, T. Hirano, Chem. Pharm. Bull. 2001, 49, 948-953.
4) K. Shimoda, T. Otsuka, Y. Morimoto, H. Hamada, H. Hamada, Chem. Lett. 2007, 36, 1292-1293.
5) K. Shimoda, T. Harada, H. Hamada, H. Hamada, Phytochemistry 2007, 68, 487-492.
6) K. Shimoda, T. Hara, H. Hamada, H. Hamada, Tetrahedron Lett. 2007, 48, 4029-4032.
7) H. Hamada, S. Ohiwa, T. Nishida, H. Katsuragi, T. Takeda, H. Hamada, N. Nakajima, K. Ishihara, Plant Biotechnol. 2003, 20, 253-255.
Introduction of the authors :
Hiroki Hamada Professor, Department of Life Science, Faculty of Science, Okayama University of Science, Japan
He was born in August 3, 1952. He completed his master course in 1982 and joined to Faculty of Science in Okayama University of Science in 1983. He received his Ph.D. degree from Hiroshima University in 1987. From 1988 to 1990, he worked as a postdoctoral fellow in Oklahoma State University and Texas A&M University. In 1992, he was promoted to an assistant professor in Okayama University of Science and in 1998, he was appointed as a professor. His research interests include bioorganic chemistry, biotechnology, and synthetic organic chemistry. Specifically, his current research involves development of novel biocatalysts including microbes, plants, and algae. Recently he is interested in glycosylation of biological active compounds using the living cells (plant and glycosyltransferase so on).
Kei Shimoda Associate Professor, Department of Chemistry, Faculty of Medicine, Oita University, Japan
He finished his master course in Hiroshima University in 1994. He received his Ph.D. degree from Hiroshima University in 1999. He worked as a postdoctoral fellow at Liverpool University, UK from 1999 to 2000. In 2002, he was promoted as an associate professor of Oita University.
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TCI Related Compounds: Contribution (pp. 2-7)
CH3CH3
HOCH3
O CH3
CH3 CH3 CH32
O
O
HO
OH
OH
OH
OH
xH2O.
OOH
HOHO
HOO
HN
OCH3
CH3
CH3O
CH3CH3
HOCH3
O CH3
CH3 CH3 CH32
O
OH
OH
OH
OH
HO
xH2O.
CH3
O
HO
CH3O
OHC
CH2
C OH
O
O
CH CH
CH CH
OCH3
OCH3
CH3O
HO
NH
OCH3
3H2O.
O
O OH
OHHO
OH
OCH3HOHO OH
OO
HOHO
HOO
CH3
HO
O
NH
O
HO
CH3O
D-α-Tocopherol25g
[T2309]
Quercetin Hydrate25g
[P0042]
Capsaicin β-D-Glucopyranoside200mg[C2548]
DL-α-Tocopherol25g, 250g
[T0251]
(+)-Catechin Hydrate1g, 10g[C0705]
4-(4-Hydroxy-3-methoxyphenyl)-2-butanone (= Zingerone)
25g[H1314]
Curcumin (Synthetic)5g, 25g[C2302]
N-Vanillylnonanamide [= Capsaicin (Synthetic)]
10g[M0900]
Rutin Trihydrate25g
[R0035]
4-(4-Hydroxyphenyl)-2-butanone (= Raspberry Ketone)
25g, 250g[H0604]
Capsaicin (Natural)1g
[M1149]
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New Carbon Nanoring Material
C2449 [12]Cycloparaphenylene (1) 10mg
Cycloparaphenylenes (CPPs), in which benzene rings are bound to each other at their para positions, represent the shortest sidewall segment (carbon nanoring) of single-walled carbon nanotubes (SWCNTs). In 2009, Itami et al. have reported the first synthesis of [12]cycloparaphenylene (1), which is composed of twelve benzene rings.1a,b) Various applications using 1 are expected. Moreover, by using 1 as a “template” to grow a nanotube, the selective synthesis of structurally uniform SWCNTs is also expected in the future.2)
References1) Selective synthesis of [12]cycloparaphenylene a) H. Takaba, H. Omachi, Y. Yamamoto, J. Bouffard, K. Itami, Angew. Chem. Int. Ed. 2009, 48, 6112. b) H. Omachi, S.
Matsuura, Y. Segawa, K. Itami, Angew. Chem. Int. Ed. 2010, 49, 10202.2) The amplification strategy using CPPs for the selective synthesis of SWNTs E. H. Fort, P. M. Donovan, L. T. Scott, J. Am. Chem. Soc. 2009, 131, 16006.
1
growth
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No.151
An Enzyme that Adds Whole Sugar Chains without Breaking Down ProductsEndo-M-N175Q
G0365 Glycosynthase (= Endo-M-N175Q) Recombinant: from Mucor hiemalis expressed in Escherichia coli (100m units/vial) 1vial
Glycosynthase (Endo-M-N175Q, 1) is an enzyme developed by Yamamoto, Umekawa, et al. through site-directed mutation of areas around the active center of Endo-M1) which is already marketed. Since the feature of Glycosynthase is efficient transglycosylation activity by using oxazoline derivatives as glycosyl donors while suppressing sugar hydrolysis activity, the resulting glycosylated products are obtained in high yield with less digestion of the products by the enzyme. Due to this feature Glycosynthase is expected to be applied as useful tool in glycotechnology.
Umekawa and her colleagues caused transglycosylation reactions at the GlcNAc site of sperm antigen CD52 using oxazoline derivatives of the high-mannose type sugar chains or the complex type sugar chains as glycosyl donors.2) They succeeded in obtaining glycosylated products in high yield of 84% and 76%, respectively. Moreover, they also achieved transglycosylation reactions using two biologically active blood-pressure-lowing peptides, PAMP12 and Substance P, as glycosyl acceptors and the oxazoline derivative of complex type sugar chain containing sialic acids as a glycosyl donor in 95% and 98% yield, respectively.3) The articles in 2009 describe the advantages of this glycosylation method using sugar-oxazoline derivatives.4,5)
Practical realization of efficient transglycosylation reactions would also be useful for expansion into glycoprotein synthesis, such as the area of biosimilars, and creation of new functional sugar complexs can be expected.
References1) K. Yamamoto, S. Kadowaki, J. Watanabe, H. Kumagai, Biochem. Biophys. Res. Commun. 1994, 203, 244.2) M. Umekawa, C. Li, T. Higashiyama, W. Huang, H. Ashida, K. Yamamoto, L.-X. Wang, J. Biol. Chem. 2010, 285, 511.3) M. Umekawa, T. Higashiyama, T. Tanaka, M. Noguchi, A. Kobayashi, S. Shoda, W. Huang, L.-X. Wang, H. Ashida, K.
Yamamoto, Biochim. Biophys. Acta 2010, 1800, 1203.4) P. Bojarova, V. Kren, Trends Biotechnol. 2009, 27, 199.5) J. R. Rich, S. G. Withers, Nature Chem. Biol. 2009, 5, 206.
Related CompoundA1651 endo-β-N-Acetylglucosaminidase (= Endo-M) Recombinant: from Mucor hiemalis expressed in Candida boidinii [Purity: single band by SDS-PAGE(85KDa)] 1vial
SG-GlcNAc-Oxa
GlcNAc-Racceptor
SG-GlcNAc-GlcNAc-R
Transglycosylationtransglycosylation product
Glycosynthase(Endo-M-N175Q)G0365
Fig. 2 Experiment example of the transglycosylation
O O
HOAcHN
HO
OHOO
OHO
HOHO
OHO
OHOHO
O
HO
O
HO
OHO
OH
O
OO
HOAcHN
OH
HO OH
HO
COONa
OAcHN
HO
HO
OHO
OH
O
HO OH
HO
COONa
OAcHN
HO
O
O
HO
AcHN
OH
OO
HO
AcHN
OHHN
O
FRKKWNKWALSR
O O
HOAcHN
HO
OHOO
OHO
HOHO
OHO
OHOHO
O
HO
O
HO
OHO
OH
O
OO
HOAcHN
OH
HO OH
HO
COONa
OAcHN
HO
HO
OHO
OH
O
HO OH
HO
COONa
OAcHN
HO
O
O
HO
AcHN
OH
OO
HO
AcHN
OHHN
O
RPKP
QQFFGL
M
Glycosynthase (Endo-M-N175Q)G036510min.
Glycosynthase (Endo-M-N175Q)G036510min.
GlcNAc-Asn-PAMP12(Acceptor)
Sialo-complex-type glycoform of PAMP12(Yield: 95%)
Sialo-complex-type glycoform of Substance P(Yield: 98%)
Sialo-CT sugar oxazoline(Donor)
GlcNAc-Gln-Substance P(Acceptor)
Sialo-CT sugar oxazoline(Donor)
No.151 No.151
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Lysine Specific Demethylase 1-Selective Inhibitor
A2411 N-[(1S)-3-[3-(trans-2-Aminocyclopropyl)phenoxy]-1-(benzylcarbamoyl)propyl]- benzamideHydrochloride(=NCL-1•HCl) 5mg
NCL-1, developed by Suzuki, Mizukami, Miyata et al., inhibits lysine specific demethylase 1 (LSD1) which plays a key role in the regulation of gene expression by removing the methyl groups from methylated Lys4 of histone H3 (H3K4) (Fig. 1).1) LSD1 inhibitory activity of NCL-1 was more potent than that of a known LSD1 inhibitor, trans-2-phenylcyclopropylamine. NCL-1 showed H3K4-methylating activity and antiproliferative activity in cellular assays. This product is a 1:1 mixture of two diastereomers (1R,2S) and (1S,2R) at the trans-cyclopropane ring.
References1) Identification of cell-active lysine specific demethylase 1-selective inhibitors a) R. Ueda, T. Suzuki, K. Mino, H Tsumoto, H. Nakagawa, M. Hasegawa, R. Sasaki, T. Mizukami, N. Miyata,
J. Am. Chem. Soc. 2009, 131, 17536. b) N. Miyata, T. Suzuki, R. Ueda, T. Mizukami, R. Sasaki, PCT Int. Appl. WO 2010143582, 2010.
HN
O NH2
O
HN O
· HCl
NCL-1· HCl (1)
NH
N
O
CH3H3C NCL-1
NH
N
OH3K4me2 H3K4
LSD1
HH
Fig. 1
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