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Reaction of sugar oxazolines with primary amines
Tetrahedron Letters 57 (2016) 5446–5448
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Reaction of sugar oxazolines with primary amines Minoru Suda ⇑, Wataru Sumiyoshi, Takashi Kinoshita, Shoko Ohno Fushimi Pharmaceutical Co. Ltd, 1676 Nakatsu-cho, Marugame, Kagawa 763-8605, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 18 August 2016 Revised 6 October 2016 Accepted 20 October 2016 Available online 24 October 2016
Sugar oxazolines, obtained by a dehydration reaction of 2-acetylamino pyranosides, reacted with primary amines in water to produce sugar imidazolines, which, when heated in water, were converted to sugar imidazoles by a dehydration reaction. The structures of these rather unexpected reaction products were determined by spectroscopic data. This offers a simple process to introduce a glycan onto peptides, proteins, and other biologically important compounds. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Sugar oxazoline Primary amine Imidazoline Glycation
Introduction
Reaction of sugar oxazolines with primary amines
Recently, an enzymatic transglycosylation of an oxazoline derivative (I),1 derived from a naturally occurring glycopeptide, has been extensively studied2 with an aim to produce homogeneous glycoproteins/peptides having improved biological activities. However, the chemical reactivity of oxazolines of this type has scarcely been investigated, probably due to its intrinsic instability toward hydrolysis. We were interested in studying the chemistry of sugar oxazolines of this type, and in this Letter we report our findings that sugar oxazolines react with primary amines to form a rather unexpected product.
In order to scrutinize the reaction pathway and structure of the reaction product, we started with a simple sugar oxazoline and a primary amine, carrying no other functional groups. A solution of sugar oxazoline (II), which was prepared by reacting N-acetylglucosamine with 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI), as reported in a literature3, and pentyl amine (in excess) in pH 9.0 buffer was kept at 30 °C. All II was consumed after 24 h. After chromatographic purification, a reaction product (III) was obtained. We assigned an imidazoline structure to this product from the following evidence. (See Supplementary information.) Its IR spectrum lacked amide absorption, excluding a simple N-glycoside structure. Its mass, 1H, 13C NMR, and HMBC spectra supported the structure. In its HMBC spectrum, (the numbering being based on the original N-acetylglucosamine), amide carbon showed correlations with H-1, H-2, and protons on the a-carbon in pentyl group, and C-1 did with H-1, H-2, H-3, and protons on the a-carbon in pentyl group. This indicated that amide carbon and pentyl amino group were linked to C-1. Also, no correlation was observed between H-1 and C-5, suggesting that the pyranoside structure was lost. A small coupling constant (J = 4.0 Hz) for the originally anomeric proton at d 5.44 indicated cis configuration, which was consistent with the reaction mechanism. Mechanistically, nucleophilic attack of amino group took place on the amide carbon, and subsequent ring opening produced an amidine intermediate, which, in turn, ring-closed to form the imidazoline structure with cis configuration.
HO HO AcHN
OH
CO2 H O O OH
HO HO
O OH
HO HO AcHN
OH
NHAc
HO O HO
O
O
HO HO HO
O
HO
CO2 H O O OH
HO HO
O OH
HO O HO
HO HO HO
O O
O O
O
NHAc
(II ) ⇑ Corresponding author. Tel.: +81 877 22 6283. E-mail address: [email protected] (M. Suda). http://dx.doi.org/10.1016/j.tetlet.2016.10.074 0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
OH O
HO O HO
O O N C H3
5447
M. Suda et al. / Tetrahedron Letters 57 (2016) 5446–5448
HO
HO HO HO
H
O O
pH 9.0
C H3
+ H 2N
HO
OH
N
N C H3
N
OH C H3
C H3
(II I)
(II II) (Vb), having NH2 group at the N-terminus of a peptide, and aniline (Vc), an aromatic primary amine, were treated with II, individually, and addition products (VIa, b, c) with imidazoline structure were obtained. Their structures were supported by the spectroscopic data. The imidazolines (VIa, b, c) were then dehydrated by heating to 50 °C in water. The product from VIa was a sugar imidazole (VIIa) as judged by its NMR spectra. On the other hand, the products from VIb, c were found to be bicyclic imidazolines (VIIIb, c), as judged by their NMR spectra. When VIIIb, c were individually heated to 70 °C in water, they were converted to the corresponding imida-
Although this compound (III) was stable toward hydrolysis at ambient temperature in the pH range of 3.0 to 9.0, we found it to be labile to heat. When III was heated to 50 °C in water, it changed to another compound (IV). Its mass, 1H and 13C NMR, and UV spectra suggested it to be an imidazole derivative. The 1H and 13C NMR spectra showed the presence of one olefinic CH unit. The UV spectrum had an absorption peak at kmax 211 nm, which was close to that of a similar imidazole derivative in a literature.4 Dehydration took place either directly or via a bicyclic imidazoline. (See below.) Similar transformation to form a sugar imidazole has been reported in a literature.4
HO
HO H HO
H
HO
OH
H
HO N
N
HO
water
OH
N
50C
C H3
OH HO N
C H3
C H3
C H3
(II II)
(II V)
We then subjected primary amines having other functional groups to this reaction. Fmoc-L-lysine (Va), having a primary amino group in the side chain of an amino acid, glycyl-L-phenylalanine
zoles (VIIb, c). Hydrogen migration to form imidazole moiety was retarded probably due to the steric hindrance.
OH OH
HO HO
N
N
Ra
C H3 HO HO HO
HO H
O O
+ H 2N Ra
N CH3
(II I)
HO
OH
N
(V V II)
H
HO N
OH Ra
CH 3
(V V)
(V V I)
HO HO HO
O N
N C H3
(V V III) Va, VIa, VIIa ; Ra = CH2 CH2 CH2 CH2-CH(CO2 H)-NH-Fmoc Vb, VIb, VIIb, VIIIb ; Ra = CH2 CONHCH(CO2H)-CH2Ph Vc, VIc, VIIc, VIIIc ; Ra = Ph
Ra
5448
M. Suda et al. / Tetrahedron Letters 57 (2016) 5446–5448
Above transformation shows that by using this reaction, one can attach a sugar chain, via its oxazoline form, to peptides and proteins on the primary amino group of lysine residue and/or Nterminus. We then studied the reactivity of a complex glycan. A sialylglycan (IX), obtained by treating a naturally occurring sialylglycopeptide (SGP) with peptide-N-glycosidase (PNGase), was converted to its oxazoline (X) by reacting with CDMBI. X was treated with Fmoc-L-lysine (Va) in pH 9.0 buffer at 30 °C, and after chromatographic purification, an addition product (XIa) was obtained. Its 1H and 13C NMR spectra supported the imidazoline structure. When this was heated to 40 °C for 7 days, dehydration reaction proceeded smoothly and a sugar-imidazolyl lysine derivative (XIIa) was isolated.
As an example of a large peptide, we treated insulin (Vd)5 with X in a similar way, and an addition product (XId) was obtained. Its mass spectrum showed that one glycan unit was attached. Insulin has two S–S bridged chains, thus two N-termini, and one lysine residue. We guessed that the sugar chain was attached to the lysine residue. When XId, in pH 9.0 buffer, was kept at 40 °C for 7 days, it changed to the corresponding imidazole derivative (XIId) after dehydration reaction. Conclusion In the above, we showed that sugar oxazolines reacted with various types of primary amines to form imidazolines,6 which were then smoothly converted to imidazole derivatives by heating in water. These reactions are versatile as it proceeds in water in neutral to weakly alkali condition, without protecting OH groups, and at around ambient temperature. By using this reaction, complex Nglycan can easily be attached to proteins and peptides at the side chain of lysine or N-terminus. It is widely known that attachment of glycan, particularly sialylglycan, on peptides and proteins, increases the solubility in water and stability in blood circuitry system.5 Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.10. 074. References and notes 1. Noguchi, M.; Tanaka, T.; Gyakushi, H.; Kobayashi, A.; Shoda, S. J. Org. Chem. 2009, 74, 2210. 2. (a) Umekawa, M.; Huang, W.; Li, B.; Fujita, K.; Ashida, H.; Wang, L. X.; Yamamoto, K. J. Biol. Chem. 2008, 283, 4469; (b) Umekawa, M.; Higashiyama, T.; Koga, Y.; Tanaka, T.; Noguchi, M.; Kobayashi, A.; Shoda, S.; Huang, W.; Wang, L. X.; Ashida, H.; Yamamoto, K. Biochem. Biophys. Acta 2010, 1800, 1203. 3. Noguchi, M.; Fujieda, T.; Huang, W. C.; Ishihara, M.; Kobayashi, A.; Shoda, S. Helv. Chem. Acta 2012, 95, 1928. 4. Kato, M.; Uno, T.; Hiratake, J.; Sakata, K. Bioorg. Med. Chem. 2005, 13, 1563. 5. Glycosylation of insulin has been reported in an attempt to prolong the glucoselowering effect. Sato, M.; Furuike, T.; Sadamoto, R.; Fujiani, N.; Nakahara, T.; Niikura, K.; Kondo, H.; Nishimura, S. J. Am. Chem. Soc. 2004, 126, 14013. 6. Davis et al.7 recently reported a non-enzymatic glycation of an antibody, as an undesirable reaction, when treated with sugar oxazoline under slightly alkali conditions, without defining the exact nature of the reaction product. We assume imidazoline forming reaction on the side chain of lysine residues and/or on the N-termini, as we report here, is the nature of their findings. 7. Parsons, T. B.; Struwe, W. B.; Gault, J.; Yamamoto, K.; Taylor, T. A.; Raj, R.; Wals, K.; Mohammed, S.; Robinson, C. V.; Benesch, J. L. P.; Davis, B. G. Angew. Chem., Int. Ed. 2016, 55, 2361.
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Reaction of sugar oxazolines with primary amines Minoru Suda ⇑, Wataru Sumiyoshi, Takashi Kinoshita, Shoko Ohno Fushimi Pharmaceutical Co. Ltd, 1676 Nakatsu-cho, Marugame, Kagawa 763-8605, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 18 August 2016 Revised 6 October 2016 Accepted 20 October 2016 Available online 24 October 2016
Sugar oxazolines, obtained by a dehydration reaction of 2-acetylamino pyranosides, reacted with primary amines in water to produce sugar imidazolines, which, when heated in water, were converted to sugar imidazoles by a dehydration reaction. The structures of these rather unexpected reaction products were determined by spectroscopic data. This offers a simple process to introduce a glycan onto peptides, proteins, and other biologically important compounds. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Sugar oxazoline Primary amine Imidazoline Glycation
Introduction
Reaction of sugar oxazolines with primary amines
Recently, an enzymatic transglycosylation of an oxazoline derivative (I),1 derived from a naturally occurring glycopeptide, has been extensively studied2 with an aim to produce homogeneous glycoproteins/peptides having improved biological activities. However, the chemical reactivity of oxazolines of this type has scarcely been investigated, probably due to its intrinsic instability toward hydrolysis. We were interested in studying the chemistry of sugar oxazolines of this type, and in this Letter we report our findings that sugar oxazolines react with primary amines to form a rather unexpected product.
In order to scrutinize the reaction pathway and structure of the reaction product, we started with a simple sugar oxazoline and a primary amine, carrying no other functional groups. A solution of sugar oxazoline (II), which was prepared by reacting N-acetylglucosamine with 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI), as reported in a literature3, and pentyl amine (in excess) in pH 9.0 buffer was kept at 30 °C. All II was consumed after 24 h. After chromatographic purification, a reaction product (III) was obtained. We assigned an imidazoline structure to this product from the following evidence. (See Supplementary information.) Its IR spectrum lacked amide absorption, excluding a simple N-glycoside structure. Its mass, 1H, 13C NMR, and HMBC spectra supported the structure. In its HMBC spectrum, (the numbering being based on the original N-acetylglucosamine), amide carbon showed correlations with H-1, H-2, and protons on the a-carbon in pentyl group, and C-1 did with H-1, H-2, H-3, and protons on the a-carbon in pentyl group. This indicated that amide carbon and pentyl amino group were linked to C-1. Also, no correlation was observed between H-1 and C-5, suggesting that the pyranoside structure was lost. A small coupling constant (J = 4.0 Hz) for the originally anomeric proton at d 5.44 indicated cis configuration, which was consistent with the reaction mechanism. Mechanistically, nucleophilic attack of amino group took place on the amide carbon, and subsequent ring opening produced an amidine intermediate, which, in turn, ring-closed to form the imidazoline structure with cis configuration.
HO HO AcHN
OH
CO2 H O O OH
HO HO
O OH
HO HO AcHN
OH
NHAc
HO O HO
O
O
HO HO HO
O
HO
CO2 H O O OH
HO HO
O OH
HO O HO
HO HO HO
O O
O O
O
NHAc
(II ) ⇑ Corresponding author. Tel.: +81 877 22 6283. E-mail address: [email protected] (M. Suda). http://dx.doi.org/10.1016/j.tetlet.2016.10.074 0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
OH O
HO O HO
O O N C H3
5447
M. Suda et al. / Tetrahedron Letters 57 (2016) 5446–5448
HO
HO HO HO
H
O O
pH 9.0
C H3
+ H 2N
HO
OH
N
N C H3
N
OH C H3
C H3
(II I)
(II II) (Vb), having NH2 group at the N-terminus of a peptide, and aniline (Vc), an aromatic primary amine, were treated with II, individually, and addition products (VIa, b, c) with imidazoline structure were obtained. Their structures were supported by the spectroscopic data. The imidazolines (VIa, b, c) were then dehydrated by heating to 50 °C in water. The product from VIa was a sugar imidazole (VIIa) as judged by its NMR spectra. On the other hand, the products from VIb, c were found to be bicyclic imidazolines (VIIIb, c), as judged by their NMR spectra. When VIIIb, c were individually heated to 70 °C in water, they were converted to the corresponding imida-
Although this compound (III) was stable toward hydrolysis at ambient temperature in the pH range of 3.0 to 9.0, we found it to be labile to heat. When III was heated to 50 °C in water, it changed to another compound (IV). Its mass, 1H and 13C NMR, and UV spectra suggested it to be an imidazole derivative. The 1H and 13C NMR spectra showed the presence of one olefinic CH unit. The UV spectrum had an absorption peak at kmax 211 nm, which was close to that of a similar imidazole derivative in a literature.4 Dehydration took place either directly or via a bicyclic imidazoline. (See below.) Similar transformation to form a sugar imidazole has been reported in a literature.4
HO
HO H HO
H
HO
OH
H
HO N
N
HO
water
OH
N
50C
C H3
OH HO N
C H3
C H3
C H3
(II II)
(II V)
We then subjected primary amines having other functional groups to this reaction. Fmoc-L-lysine (Va), having a primary amino group in the side chain of an amino acid, glycyl-L-phenylalanine
zoles (VIIb, c). Hydrogen migration to form imidazole moiety was retarded probably due to the steric hindrance.
OH OH
HO HO
N
N
Ra
C H3 HO HO HO
HO H
O O
+ H 2N Ra
N CH3
(II I)
HO
OH
N
(V V II)
H
HO N
OH Ra
CH 3
(V V)
(V V I)
HO HO HO
O N
N C H3
(V V III) Va, VIa, VIIa ; Ra = CH2 CH2 CH2 CH2-CH(CO2 H)-NH-Fmoc Vb, VIb, VIIb, VIIIb ; Ra = CH2 CONHCH(CO2H)-CH2Ph Vc, VIc, VIIc, VIIIc ; Ra = Ph
Ra
5448
M. Suda et al. / Tetrahedron Letters 57 (2016) 5446–5448
Above transformation shows that by using this reaction, one can attach a sugar chain, via its oxazoline form, to peptides and proteins on the primary amino group of lysine residue and/or Nterminus. We then studied the reactivity of a complex glycan. A sialylglycan (IX), obtained by treating a naturally occurring sialylglycopeptide (SGP) with peptide-N-glycosidase (PNGase), was converted to its oxazoline (X) by reacting with CDMBI. X was treated with Fmoc-L-lysine (Va) in pH 9.0 buffer at 30 °C, and after chromatographic purification, an addition product (XIa) was obtained. Its 1H and 13C NMR spectra supported the imidazoline structure. When this was heated to 40 °C for 7 days, dehydration reaction proceeded smoothly and a sugar-imidazolyl lysine derivative (XIIa) was isolated.
As an example of a large peptide, we treated insulin (Vd)5 with X in a similar way, and an addition product (XId) was obtained. Its mass spectrum showed that one glycan unit was attached. Insulin has two S–S bridged chains, thus two N-termini, and one lysine residue. We guessed that the sugar chain was attached to the lysine residue. When XId, in pH 9.0 buffer, was kept at 40 °C for 7 days, it changed to the corresponding imidazole derivative (XIId) after dehydration reaction. Conclusion In the above, we showed that sugar oxazolines reacted with various types of primary amines to form imidazolines,6 which were then smoothly converted to imidazole derivatives by heating in water. These reactions are versatile as it proceeds in water in neutral to weakly alkali condition, without protecting OH groups, and at around ambient temperature. By using this reaction, complex Nglycan can easily be attached to proteins and peptides at the side chain of lysine or N-terminus. It is widely known that attachment of glycan, particularly sialylglycan, on peptides and proteins, increases the solubility in water and stability in blood circuitry system.5 Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.10. 074. References and notes 1. Noguchi, M.; Tanaka, T.; Gyakushi, H.; Kobayashi, A.; Shoda, S. J. Org. Chem. 2009, 74, 2210. 2. (a) Umekawa, M.; Huang, W.; Li, B.; Fujita, K.; Ashida, H.; Wang, L. X.; Yamamoto, K. J. Biol. Chem. 2008, 283, 4469; (b) Umekawa, M.; Higashiyama, T.; Koga, Y.; Tanaka, T.; Noguchi, M.; Kobayashi, A.; Shoda, S.; Huang, W.; Wang, L. X.; Ashida, H.; Yamamoto, K. Biochem. Biophys. Acta 2010, 1800, 1203. 3. Noguchi, M.; Fujieda, T.; Huang, W. C.; Ishihara, M.; Kobayashi, A.; Shoda, S. Helv. Chem. Acta 2012, 95, 1928. 4. Kato, M.; Uno, T.; Hiratake, J.; Sakata, K. Bioorg. Med. Chem. 2005, 13, 1563. 5. Glycosylation of insulin has been reported in an attempt to prolong the glucoselowering effect. Sato, M.; Furuike, T.; Sadamoto, R.; Fujiani, N.; Nakahara, T.; Niikura, K.; Kondo, H.; Nishimura, S. J. Am. Chem. Soc. 2004, 126, 14013. 6. Davis et al.7 recently reported a non-enzymatic glycation of an antibody, as an undesirable reaction, when treated with sugar oxazoline under slightly alkali conditions, without defining the exact nature of the reaction product. We assume imidazoline forming reaction on the side chain of lysine residues and/or on the N-termini, as we report here, is the nature of their findings. 7. Parsons, T. B.; Struwe, W. B.; Gault, J.; Yamamoto, K.; Taylor, T. A.; Raj, R.; Wals, K.; Mohammed, S.; Robinson, C. V.; Benesch, J. L. P.; Davis, B. G. Angew. Chem., Int. Ed. 2016, 55, 2361.