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. Author manuscript; available in PMC: 2012 Sep 23.
Published in final edited form as: Chembiochem. 2011 Mar 4;12(6):932–941. doi: 10.1002/cbic.201000763

Unusual transglycosylation activity of Flavobacterium meningosepticum endoglycosidases enables convergent chemoenzymatic synthesis of core fucosylated complex N-glycopeptides

Wei Huang [a], Jie Li [a], Lai-Xi Wang [a],✉
PMCID: PMC3449086  NIHMSID: NIHMS379170  PMID: 21374780

Abstract

Structurally well-defined, homogeneous glycopeptides and glycoproteins are indispensable tools for functional glycomics studies. By screening of various endo-β-N-acetylglucosaminidases using an appropriate synthetic donor and acceptor substrates, we have found that the Flavobacterium meningosepticum endo-β-N-acetylglucosaminidases glycosidases (GH family 18), including Endo-F2 and Endo-F3, were able to glycosylate α-1,6-fucosylated GlcNAc derivative to provide natural, core-fucosylated complex type N-glycopeptides. The Endo-F2 and Endo-F3 were efficient for transferring both sialylated and asialylated glycans and were highly specific for an α-1,6-fucosyllated GlcNAc-peptide as acceptor for transglycosylation, as they showed only marginal activity on non-fucosylated GlcNAc-peptide. In contrast, we found that the commonly used endoglycosidases such as Endo-A and Endo-M, which belong to GH family 85, were unable to take α-1,6-fucosyl-GlcNAc derivative as acceptor for transglycosylation. The novel activity of Endo-F2 and Endo-F3 was successfully applied for a highly convergent chemoenzymatic synthesis of a full-size CD52 glycopeptide antigen carrying both terminal sialic acid and core fucose. This is the first report on endoglycosidases that are able to glycosylate α-1,6-fucosylated GlcNAc derivatives to form natural core-fucosylated glycopeptides.

Keywords: enzymatic transglycosylation, endoglycosidase, glycopeptides, chemoenzymatic synthesis, sugar oxazoline

Introduction

The oligosaccharide components of glycoproteins not only affect protein’s structure and stability, but also directly participate in many important biological recognition processes such as cell adhesion, cell differentiation, tumor metastasis, host-pathogen interactions, and immune responses.[1] Ample examples have shown that subtle changes in the glycan structures of glycoproteins can result in significant differences in functions.[2] Core fucosylation, the attachment of a fucose α-1,6-linked to the innermost GlcNAc moiety of the asparagine (N)-linked glycans, is a natural modification frequently found in natural and recombinant glycoproteins. It has been demonstrated that core fucosylation affects N-glycan’s conformations and regulates the interactions between N-glycan and various glycan-binding proteins.[3] On the other hand, significant increases in core fucosylation have been associated with various cancers and are correlated to tumor progression, suggesting that core-fucosylated N-glycans and glycoproteins could serve as novel biomarkers for diagnosis.[4, 5] However, functional glycomics studies are often hampered by the difficulties to obtain structurally well-defined complex oligosaccharides and glycoconjugates. Despite remarkable advance in chemical and chemoenzymatic synthesis of complex oligosaccharides and glycoconjugates in recent years,[6] synthesis of sialylated and fucosylated complex glycopeptides and glycoproteins is still a challenging task, because of the labile nature of the α-sialyl/α-fucosyl glycosidic linkages during multiple synthetic manipulations.[7] It should be noted that a late-stage enzymatic introduction of the core fucose by α-1,6-fucosyltransferase (Fut8) does not seem to be feasible for full-size N-glycans because Fut-8 only accepts the truncated N-glycan core as substrate for α-1,6-fucosylation.[5] In addition, a recent study on Fut8 showed an unusual substrate structural requirement for the Fut8-catalyzed α-1,6-fucosylation.[8] Thus, a convergent synthetic approach that permits a native ligation between a pre-assembled oligosaccharide and an independently generated Fucα1,6GlcNAc-peptide/protein would be highly desirable.

We and others have recently explored the transglycosylation activity of glycosyl hydrolase (GH) family 85 endo-β-N-acetylglucosaminidases (ENGases) for glycopeptide and glycoprotein synthesis.[9–17] It was shown that the Endo-A from Arthrobacter protophormiae[18] and the Endo-M from Mucor hiemalis,[19] could efficiently take synthetic sugar oxazolines as substrates for transglycosylation to form homogeneous glycopeptides/glycoproteins carrying truncated or modified N-glycans. In addition, glycosynthase mutants have been generated that are suitable for synthesizing glycoproteins carrying natural N-glycans without product hydrolysis.[12–14, 16] Despite these advances, this convergent chemoenzymatic method has not been tested for synthesizing core-fucosylated complex N-glycopeptides and/or N-glycoproteins. In fact, so far there were no reports on any endoglycosidases capable of transferring oligosaccharides to a GlcNAc acceptor capped with an α-1,6-fucose. We report in this paper the first discovery of novel transglycosylation activities of a class of endo-β-N-acetylglucosaminidases that recognize core-fucosylated GlcNAc acceptors. We have found that the GH family 18 endoglycosidases from Flavobacterium meningosepticum,[20, 21] particularly Endo-F2 and Endo-F3, [22] can specifically glycosylate the 4-OH of the GlcNAc moiety in an α-1,6-fucosyl-GlcNAc-peptide acceptor to form a homogeneous, core-fucosylated complex glycopeptide, whereas the GH family 85 endoglycosidases Endo-A and Endo-M, which were previously reported to efficiently glycosylate non-fucosylated GlcNAc, were unable to take Fucα1,6GlcNAc derivative for transglycosylation. This is the first report on the transglycosylation activity of Endo-F2 and Endo-F3 and the first discovery of endoglycosidases capable of recognizing core-fucosylated GlcNAc as substrate. We also describe here a convergent chemoenzymatic synthesis of a full-size, sialylated and core-fucosylated CD52 glycopeptide antigen by applying the novel transglycosylation activity.

Results and Discussion

Chemical synthesis of Asn-linked disaccharide derivative Fmoc-Asn(Fucα1,6GlcNAc)-OH

For screening the transglycosylation activity of ENGases on fucosylated GlcNAc acceptor, we synthesized an Asn-linked disaccharide derivative, Fmoc-Asn(Fucα1,6GlcNAc)-OH (9), using the peracetylated GlcNAc-azide (1)[23] as the starting material (Scheme 1). De-O-acetylation of 1, followed by selective tritylation at the 6-OH and benzoylation at the 3- and 4-OH gave compound 2. The 6-O-trityl group was then selectively removed by mild acid hydrolysis to give the glycosyl acceptor 3. Selective α-1,6-fucosylation of 3 with per-O-trimethylsilyl α-fucopyranosyl iodide (4) [24] as the glycosyl donor in the presence of 2,6-di-tert-butylpyridine, followed by in situ removal of the TMS protecting groups, gave the disaccharide derivative 5 in 61% yield. The newly formed glycosidic linkage was confirmed to be α-linkage as demonstrated by a relatively small coupling constant between H-1 and H-2 of the fucose (H-1, δ 4.82, d, J1,2 = 3.68 Hz). After changing the O-protecting groups to O-acetyl groups, the resulting glycosyl azide 6 was reduced with Me3P in THF and then coupled with Fmoc-Asp-OtBu to afford the Asn-linked derivative 7. The tert-butyl group was selectively removed with a mild acid condition (20% TFA in CH2Cl2) without cleavage of the acid-labile α-1,6-fucosidic linkage to give the Asn-linked disaccharide 8 in 95% yield. Finally selective de-O-acetylation of 8 was achieved by treatment of 8 with K2CO3 in MeOH/THF to provide the N-Fmoc-protected Asn-disaccharide (9) in 85% yield (Scheme 1).

Scheme 1.

Scheme 1

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Synthesis of Fmoc-Asn(Fucα1,6GlcNAc)-OH (9)

Screening of transglycosylation activity using the fucosylated GlcNAc-Asn as the acceptor substrate and two synthetic sugar oxazolines as the donor substrates

With the synthetic disaccharide acceptor (9) in hand, we screened a series of ENGases for their ability to transfer an oligosaccharide to the Fucα1,6GlcNAc moiety. These include Endo-A,[18] Endo-M,[19] Endo-H from Streptomyces plicatus,[25] Endo-D from Streptococcus pneumoniae,[26] and Endo-F1, Endo-F2, and Endo-F3 from Flavobacterium meningosepticum.[20–22] These enzymes all hydrolyze N-glycans at the N,N′-diacetylchitobiose core, but they have different substrate specificity. It is known that Endo-A, Endo-H, and Endo-F1 hydrolyze high-mannose type and/or hybrid type N-glycans;[18, 20, 25] Endo-M can hydrolyze high-mannose type, hybrid type, and non-fucosylated bi-antennary complex-type N-glycans;[19] Endo-D is specific for a truncated and core-fucosylated N-glycan;[26] and Endo-F2 and Endo-F3 are able to cleave both core-fucosylated and non-fucosylated complex-type N-glycans,[21, 22] but Endo-F3 prefers core-fucosylated N-glycans.[27] Two synthetic sugar oxazolines, the Man3GlcNAc-oxazoline (10)[10] and the sialylated N-glycan oxazoline (12),[14] were used as donor substrates for screening the enzymes. If there was a transglycosylation activity, a corresponding core-fucosylated glycopeptide (11 or 13) would be produced, as shown in Scheme 2. Thus, a mixture of oxazoline (10) or (12) and acceptor 9 (donor/acceptor, 4:1, molar ratio) was incubated with an appropriate amount of the respective ENGase at 30 °C in a phosphate buffer (50 mM, pH 7.0) containing 10% DMSO. Inclusion of 10% DMSO significantly enhanced the solubility of the Fmoc derivative 9 in the buffer. The course of transglycosylation reaction was monitored by RP-HPLC analysis of reaction aliquots taken at intervals, and new peaks (products) were subject to ESI-MS analysis to confirm their identity. The screening results were summarized in Figure 1. It was revealed that Endo-A, Endo-M, Endo-D, and Endo-H did not show any transglycosylation activity towards the α-1,6-fucosylated GlcNAc derivative, although the first three enzymes were previously shown to be able to transglycosylate non-fucosylated GlcNAc-Asn acceptor. In contrast, the Endo-F3 demonstrated remarkable transglycosylation activity toward the α-1,6-fucosylated GlcNAc acceptor, with both the truncated glycan oxazoline (10) and the full-size complex type glycan oxazoline (12). The reaction led to the formation of the corresponding core-fucosylated N-glycan (11) and (13) respectively, in over 70% yield within 1 h. The yield was based on the conversion of the acceptor. It should be mentioned that only a single transglycosylation product was formed when oxazoline 10 or 12 were used, indicating the specificity for the enzymatic transglycosylation. A HPLC profile of the reaction between oxazoline 10 and acceptor 9 in the presence of Endo-F3 was shown in Figure S1 (supporting information). It was found that a gradual hydrolysis of the product by Endo-F3 occurred when the mixture was incubated with the enzyme for a prolonged time. This was expected, due to the inherent hydrolytic activity of Endo-F3. However, the Endo-F3 catalyzed transglycosylation from the sugar oxazoline was surprisingly fast, allowing a significant accumulation of the transglycosylation product. These results suggest that the use of the highly active sugar oxazoline as donor substrate favours the transglycosylation. The Endo-F2 also showed transglycosylation activity on the 1,6-fucosylated GlcNAc acceptor, but the reaction was much slower than that catalyzed by Endo-F3. In comparison, the Endo-F1 only showed marginal transglycosylation activity when Man3GlcNAc-oxazoline (10) was used as donor substrate, and did not show any activity when the sialylated glycan oxazoline (12) was used as donor substrate (Figure 1). These results clearly demonstrate the distinct substrate specificity of these endoglycosidases in transglycosylation. Products 11 and 13 from the transglycosylation by Endo-F2 and Endo-F3 were purified by RP-HPLC and first characterized by mass spectrometric analysis. The MALDI-TOF MS data of 11 (calculated, M = 1392.50; found (m/z), 1416.12 [M + Na]+) indicated that the product was an adduct of the Man3GlcNAc-oxazoline and acceptor 9; the ESI-MS of 11 revealed two major m/z species at 1393.58 and 1247.60, which corresponded to [M + H]+ and [M − Fuc + H]+, respectively. The presence of fragment [M − Fuc + H]+ suggested that the transferred oligosaccharide was attached to the GlcNAc moiety rather than the Fuc moiety in the acceptor.

Scheme 2.

Scheme 2

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Screening transglycosylation activity using the Fmoc-Asn(Fucα1,6GlcNAc)-OH (9) as the acceptor and synthetic sugar oxazolines (10 and 12) as the donor substrates

Figure 1.

Figure 1

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The time courses of endoglycosidases-catalyzed transglycosylation on Fmoc-Asn(Fucα1,6GlcNAc)-OH (9) monitored by HPLC. Panel A: Man3GlcNAc oxazoline (10) as the donor substrate; Panel B: sialoglycan oxazoline (12) as the donor substrate.

Further characterization of product 11 was carried out by specific enzymatic transformations. Endo-D digestion of the isolated 11 gave tetrasaccharide Man3GlcNAc that was identical to an authentic sample, and the Fmoc-Asn(Fucα1,6GlcNAc)-OH. Since Endo-D is known to specifically cleave the β-1,4-glycosidic linkage in the N,N′-diacetylchitobiose unit of a core-fucosylated Man3GlcNAc2 moiety [26], the observed result indicated that the transferred Man3GlcNAc was attached specifically to the 4-OH of the GlcNAc moiety in acceptor 9 to form the desired β-1,4 glycosidic linkage. The sialylated product (13) was similarly characterized by MS analysis (See the experimental section in supporting information). To our knowledge, this is the first report showing a transglycosylation activity of Endo-F2 and Endo-F3 with a sugar acceptor.[28] The Endo-F2 and Endo-F3 also represent the first endoglycosidases capable of glycosylating core fucosylated GlcNAc acceptor to form a new core-fucosylated N-glycopeptide.

Endo-F2 and Endo-F3 do not accept non-fucosylated GlcNAc derivative as acceptor substrate for transglycosylation

The significant transglycosylation activity of Endo-F2 and Endo-F3 on Fmoc-Asn(Fucα1,6GlcNAc)-OH also prompted us to examine how these Flavobacterium enzymes act on non-fucosylated GlcNAc acceptor. We have previously shown that Endo-A and Endo-M are very efficient to transglycosylate non-fucosylated GlcNAc moiety.[9] Accordingly, we tested the transglycosylation of Endo-F1, Endo-F2, Endo-F3, and Endo-D on Fmoc-Asn(GlcNAc)-OH (14) using Man3GlcNAc oxazoline (10) as the donor substrate (Scheme 3). The results were summarized in Figure 2. Surprisingly, Endo-F2 and Endo-F3 did not exhibit transglycosylation activity on the non-fucosylated GlcNAc acceptor under the screening conditions. On the other hand, the Endo-F1 enzyme, which showed only marginal activity on the fucosylated GlcNAc acceptor as described above, demonstrated significant transglycosylation activity with the non-fucosylated GlcNAc (14) to lead to a rapid formation of the corresponding product (15). The yield reached 45% within 30 min. However, a prolonged incubation did not result in further increase in yield. HPAEC-PAD analysis of the reaction mixture revealed that the moderate yield was mainly due to the simultaneous hydrolysis of the sugar oxazoline (10), which was completely gone within 30 min. Interestingly, the product Man3GlcNAc2-Asn(Fmoc) (15) was not hydrolyzed even for a prolonged incubation, indicating that the truncated N-glycan product was a poor substrate of Endo-F1.

Scheme 3.

Scheme 3

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Screening transglycosylation activity using non-fucosylated GlcNAc-Asn-Fmoc (14) as the acceptor and Man3GlcNAc oxazolines (10) as the donor substrate

Figure 2.

Figure 2

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HPLC-monitored time courses of Endoglycosidases-catalyzed transglycosylation on Fmoc-Asn(GlcNAc)-OH using Man3GlcNAc oxazoline as the donor substrate.

The Endo-D is an interesting case. Our results indicated that Endo-D could use Man3GlcNAc-oxazoline (10) as donor substrate and GlcNAc-Asn derivative (14) as acceptor for transglycosylation, albeit at a low yield (Figure 2). This result is consistent with a recent report on Endo-D.[17] However, when the α1-6-fucosylated GlcNAc derivative (9) was used, no transglycosylation product was detected (Figure 1). Given the fact that Endo-D could efficiently hydrolyze α-1,6-fucosylated Man3GlcNAc2 core,[26] it is conceivable to think that Endo-D should be able to accommodate α1-6-fucosylated GlcNAc derivative (9) as an acceptor for transglycosylation. The failure to detect the transglycosylation product might be due to the quick Endo-D catalyzed hydrolysis of the product in situ before it was released from the catalytic site. Indeed, incubation of Endo-D and the purified core-fucosylated Man3GlcNAc2-Asn(Fmoc) (11) obtained from the Endo-F3 transglycosylation resulted in a very quick hydrolysis of 11 (see Experimental Section).

The presence of an α-1,6-linked fucose in the GlcNAc acceptor seems essential for recognition of the acceptor by Endo-F2 and Endo-F3 for an efficient transglycosylation. In contrast, attachment of a core fucose on the GlcNAc moiety completely blocked the accommodation of the acceptor by Endo-F1, Endo-A, and Endo-M for transglycosylation. It should be pointed out that the wild type Endo-F2 and Endo-F3 could still hydrolyze the transglycosylation product thus formed. One way to address this issue is to create glycosynthase mutants that is capable of working on the highly activated sugar oxazolines for transglycosylation but lacks product hydrolysis activity. We have previously shown that site-directed mutation at a specific Asn residue, the Asn175 in Endo-M and Asn171 in Endo-A, which promotes oxazoline formation during hydrolysis, could lead to glycosynthase mutants useful for synthesis.[12–14, 16] The crystal structure of Endo-F3 was solved and the two conserved active residues at the catalytic site were identified as Asp126 and Glu128. [29] In the substrate-assisted mechanism of the family 18 endoglycosidases,[30] the Glu128 of Endo-F3 serves as a general acid to protonate the glycosidic bond; whereas the Asp126 was assigned a secondary role, assumingly having the same function as the Asn171 of Endo-A to promote the formation of the oxazolinium ion intermediate, and to stabilize it, by interactions with the 2-acetamido group in the GlcNAc moiety.[29] Thus an interesting future experiment is to perform site-directed mutation at this aspartic acid residue to test whether efficient glycosynthase mutants can be generated.

Convergent chemoenzymatic synthesis of a sialylated and core-fucosylated complex-type glycoform of CD52 antigen

The remarkable transglycosylation activity of Endo-F2 and Endo-F3 with α-1,6-fucosylated GlcNAc acceptor, together with their flexibility in accepting different sugar oxazolines as donor substrates, opens a new avenue to a highly convergent chemoenzymatic synthesis of core-fucosylated complex type N-glycans, glycopeptides, and/or glycoproteins. As a test case, we chose to synthesize a sialylated and core-fucosylated complex-type glycoform of the CD52 glycopeptide antigen. CD52 is a GPI-anchored glycopeptide antigen found on human lymphocytes and sperm cells. It is a glycopeptide consisting of only 12 amino acid residues but carrying a large complex-type N-glycan at the Asn-3 residue.[31] Several truncated CD52 glycoforms were made previously by chemical methods.[32] We have previously used CD52 antigen as a model glycopeptide to test our chemoenzymatic method.[15, 16, 33] However, a full-size complex-type CD52 antigen carrying both terminal sialic acid residues and core fucose moiety had not been made by either chemical or chemoenzymatic method. A convergent chemoenzymatic synthesis of the target CD52 antigen required the preparation of a CD52 polypeptide containing a Fucα1,6GlcNAc moiety at the Asn-3. To avoid a strong acidic condition for retrieving the polypeptide from the solid support in the Fmoc approach, we performed the solid-phase peptide synthesis on an acid-labile O-link TGT-resin, which can be cleaved by mild acid treatment (Scheme 4). Fmoc-Asn(Ac5Fucα1-6GlcNAc)-OH (8) was used as a building block to replace the residues at Asn-3 during the synthesis to introduce a Fucα1,6GlcNAc moiety. HATU (0.5 M in DMF) and DIPEA (1.0 M in DMF) (1:1, v/v) were used as the coupling reagent and piperidine (20% in DMF) was used as the deblocking reagent. 4-fold excess of Fmoc-protected building blocks were used for each coupling reaction cycle. The N-terminus amino group was protected with an acetyl group by treatment with Ac2O/DIPEA. The disaccharide-containing polypeptide was released from the resin (16) by treatment with 20% TFA in CH2Cl2 at r.t. for 5 h, with simultaneous removal of the side-chain protecting groups (Trt and t-Bu). The Ac groups on the disaccharide were removed by treatment with 5% aqueous hydrazine at r.t. for 1 h. The crude product was then purified by preparative HPLC to give the Fucα1,6GlcNAc-CD52 (17) in an excellent overall yield (90%) (Scheme 4). The product was characterized by ESI-MS (calculated, M = 1598.64; found (m/z), 1599.16 [M + H]+) and a detailed NMR analysis (see supporting information).

Scheme 4.

Scheme 4

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Chemoenzymatic synthesis of sialylated and core-fucosylated CD52 antigen

With the Fucα1,6GlcNAc-CD52 (17) in hand, we next performed the transglycosylation with Endo-F3, using sialoglycan oxazoline (12) as donor substrate, which was efficiently synthesized from a free N-glycan by a single-step conversion in water.[14] Thus, incubation of 12 and 17 (donor/acceptor, 2:1, molar ratio) with Endo-F3 in a phosphate buffer (pH 7.0) led to the formation of the CD52 glycoform (18) carrying a sialylated and core-fucosylated bi-antennary N-glycan. The reaction was monitored by HPLC. When the formation of 18 reached a plateau, the reaction was stopped and the product was isolated in 55% yield by HPLC. The efficiency of the transglycosylation was quite impressive, given the fact that only two-fold of the donor substrate was used. Using the Endo-F2 to replace Endo-F3 for the transglycosylation gave a 25% yield of 18 after a longer period (6 h) of incubation (data not shown). ESI-MS of 18 gave the expected m/z species (calculated for C137H221N21O90, M = 3600.34; found (m/z), 1201.66 [M + 3H]3+). Further characterization of the product was carried out by specific enzymatic transformations. Digestion of 18 with peptide-N4-(N-acetyl-β-D-glucosaminyl)asparagine amidase F (PNGase F), which specifically hydrolyzes the β-aspartylglucosylamine bond of N-glycopeptides and N-glycoproteins,[34] yielded the expected sialoglycan 19 (ESI-MS: calculated for C90H148N6O66, M = 2368.84; found (m/z), 1185.82 [M + 2H]2+) and the expected aspartic acid-containing CD52 peptide (20) (ESI-MS: calculated for C47H74N14O26, M = 1250.49; found (m/z), 1251.56 [M + H]+, 626.38 [M + 2H]2+ (Figure S2, supporting information). On the other hand, incubation of purified 18 with large amount of Endo-F3 gave Fucα1,6GlcNAc-CD52 (17) and the corresponding N-glycan 21 (Figure S3, supporting information). These results suggest that the transferred glycan was attached to the GlcNAc in the peptide through the GlcNAc-β-1,4-GlcNAc linkage. Finally, a detailed 1H-13C 2D HSQC NMR analysis of 18 permitted the assignment of all the sugar anomeric protons and anomeric carbons, as well as all the amino acid residues in the product (Figure S9, supporting information). These experiment data confirmed that the product obtained was the expected full-size, sialylated and core-fucosylated CD52 antigen.

Conclusions

We have demonstrated that the GH family 18 Flavobacterium meningosepticum endoglycosidases Endo-F2 and Endo-F3 possess significant transglycosylation activities and are able to specifically glycosylate α-1,6-fucosylated GlcNAc-peptide by using sugar oxazoline as donor substrate. The novel activity of Endo-F2 and Endo-F3 was successfully applied for a convergent chemoenzymatic synthesis of a full-size CD52 glycopeptide antigen carrying both terminal sialic acid and core fucose. This is the first discovery of transglycosylation activity of Endo-F1, Endo-F2 and Endo-F3 that show distinct acceptor substrate specificity. This study also represents the first report on endoglycosidases capable of taking core-fucosylated GlcNAc-peptides for transglycosylation. In addition to the usefulness for complex glycopeptide synthesis, the unusual transglycosylation activity discovered may be particularly useful for glycosylation remodelling of monoclonal antibodies that usually carry core fucosylated N-glycans at the Fc domain but can be specifically de-glycosylated by an endoenzyme to provide a core-fucosylated GlcNAc acceptor at the glycosylation site. Work along this line is currently undergoing in our laboratory and will be reported in due course.

Experimental Section

Materials

The Fmoc-protected amino acids and Fmoc-Ser(tBu)-O-TGT resin were purchased from Novabiochem Corporation (San Diego, CA). 2-(1-H-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) was purchased from GenScript Corp (Piscataway, NJ). Diisopropylethylamine (DIPEA) was purchased from Applied Biosystems (Carlsbad, CA). Piperidine (20% in DMF) was purchased from American Bioanalytical (Natick, MA). N,N-Dimethylformamide sequencing grade was purchased from Fisher Biotech (Pittsburgh, PA). Acetonitrile HPLC grade was purchased from Fisher Scientific (Pittsburgh, PA). 3,4,6-Tri-O-acetyl-1β-azido-GlcNAc (1) was synthesized following reference.[23] 2,3,4-Tri-O-acetyl-α-L-fucosyl iodide (4) was synthesized by reported method.[24] Man3GlcNAc oxazoline (10) was synthesized as reported.[10] The bi-antennary complex-type sialoglycan oxazoline (12) was prepared following the reported procedure.[14] Fmoc-Asn(GlcNAc)-OH (14) was prepared by the reported procedure.[35] Endo F1, Endo F2, and Endo F3 were purchased from CalBioChem (San Diego, CA). Endo H was purchased from New England Biolabs (Ipswich, MA). Endo D was purchased from United States Biological (Swampscott, MA). Endo-A was overproduced in E. coli and purified following the previously reported procedure,[36] using the plasmid pGEX-2T/Endo-A that was kindly provided by Prof. Kaoru Takegawa. Endo-M was overproduced according to the previously reported method [12]. PNGase F was purchased from New England Biolabs (Ipswich, MA). All other reagents were purchased from Sigma/Aldrich and used as received.

High-performance liquid chromatography (HPLC)

Analytical RP-HPLC was performed on a Waters 626 HPLC instrument with a Symmetry300™ C18 column (5.0 μm, 4.6 × 250 mm) or a XBridge™ BEH130 C18 column (3.5 μm, 4.6 × 250 mm) at 40 °C. The Symmetry300 column was eluted with a linear gradient of 0–90% MeCN containing 0.1% TFA within 30 min at a flow rate of 1 mL/min (Method A). The XBridge column was eluted with a linear gradient of 0–20% MeCN containing 0.1% TFA within 30 min at a flow rate of 0.5 mL/min (Method B). Preparative HPLC was performed on a Waters 600 HPLC instrument with a preparative Symmetry300™ C18 column (7.0 μm, 19 × 250 mm) or a XBridge™ Prep ShieldRP18 column (5.0 μm, 10 × 250 mm). These columns were eluted with a suitable gradient of aqueous acetonitrile containing 0.1% TFA at a flow rate of 12 mL/min (for Symmetry300 column) or 4 mL/min (for XBridge column).

Nuclear magnetic resonance (NMR)

The 1H NMR spectra were measured on JEOL ECX 400 MHz or Inova 500 MHz or Bruker DRX 500 MHz NMR spectrometers. All chemical shifts were assigned in ppm. The 13C NMR was measured at 125 MHz.

Mass spectrometry (MS)

The ESI-MS Spectra were measured on a Waters Micromass ZQ-4000 single quadruple mass spectrometer. MALDI-TOF MS measurement was performed on an Autoflex II MALDI-TOF mass spectrometer (Bruker Daltonics). The instrument was calibrated by using ProteoMass Peptide MALDI-MS calibration kit (MSCAL2, Sigma/Aldirich). The matrix used for glycans was 2,5-dihydroxybenzoic acid (DHB) and/or alpha-cyano-4-hydroxycinnamic acid (ACHA) (10 mg/mL in 50% acetonitrile containing 0.1% trifluoroacetic acid). The measuring conditions: 337 nm nitrogen laser with 100 μJ output; laser frequency 50.0 Hz; laser power 30–45%; linear mode; positive polarity; detection range 500–5000; pulsed ion extraction: 70ns; high voltage: on; realtime smooth: high; shots: 500–2000.

2-Acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-trityl-β-D-glucopyranosyl azide (2)

3,4,6-Tri-O-acetyl-1β-azido-GlcNAc (1) (610 mg, 1.64 mmol) was dissolved in anhydrous MeOH (15 mL) and NaOMe (wt. 25% in MeOH) (2.05 mL, 35.8 mmol) was added. The reaction mixture was stirred for 2 hr under argon. Then it was treated with Dowex 50WX8-100 ion-exchange resin H+ form and filtered. The filtrate was concentrated to dryness under vacuum to give the crude β-glycosyl azide.

A mixture of the crude glycosyl azide, pyridine (10 mL) and trityl chloride (0.91 g, 3.25 mmol) was stirred at 60 °C overnight. Then it was cooled to rt and benzoyl chloride (0.47 mL, 4.06 mmol) was added. The solution was continued to stir at rt overnight. The residue was diluted with EtOAc and washed with 1N HCl, saturated NaHCO3, water, and brine. The organic layer was dried over Na2SO4 and concentrated. The syrup was subject to a silica gel column chromatography (EtOAc/hexane, 1:2, v/v) to give product 2 as a white foam (810 mg, 70%, three steps). 1H NMR (400 MHz, CDCl3) δ 7.91 (dd, J = 8.24, 0.92 Hz, 2H), 7.65 (dd, J = 8.24, 1.36 Hz, 2H), 7.32 – 7.49 (m, 11H), 7.08 – 7.16 (m, 10H), 5.84 (d, J = 8.72 Hz, 1H), 5.72 (dd, J = 10.08, 9.6 Hz, 1H), 5.53 (dd, J = 10.56, 10.52 Hz, 1H), 4.78 (d, J = 9.16 Hz, 1H), 4.30 (dd, J = 10.52, 9.16 Hz, 1H), 3.89 (m, 1H), 3.38 (dd, J = 10.56, 2.28 Hz, 1H), 3.16 (dd, J = 10.52, 4.12 Hz, 1H), 1.88 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 170.62, 167.17, 164.69, 143.52, 133.77, 133.24, 130.06, 129.66, 129.11, 128.65, 128.31, 127.85, 127.02, 88.59, 86.79, 73.29, 68.75, 62.06, 54.67, 23.35

2-Acetamido-3,4-di-O-benzoyl-2-deoxy-β-D-glucopyranosyl azide (3)

A solution of compound 2 (810 mg, 1.16 mmol) in 80% aqueous AcOH (40 mL) was stirred at 60 °C overnight. Then it was concentrated under vacuum. The residue was diluted with EtOAc and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated. The resulted syrup was subject to silica gel column chromatography (EtOAc/hexane, 1:1, v/v) to give product 3 as a white foam (410 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 7.89 – 7.92 (m, 4H), 7.48 – 7.52 (m, 2H), 7.33 – 7.37 (m, 4H), 6.02 (d, J = 9.16 Hz, 1H), 5.78 (dd, J = 10.56, 9.16 Hz, 1H), 5.50 (dd, J = 10.08, 9.64 Hz, 1H), 4.97 (d, J = 9.16 Hz, 1H), 4.17 (dt, J = 10.52, 9.16 Hz, 1H), 3.84 – 3.89 (m, 2H), 3.73 (dd, J = 12.84, 4.6 Hz, 1H), 1.89 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 170.76, 166.80, 166.01, 133.86, 133.80, 129.94, 128.63, 128.54, 88.76, 77.43, 72.55, 69.08, 61.31, 54.61, 23.29.

2-Acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-(α-L-fucopyranosyl)-β-D-glucopyranosyl azide (5)

To a stirring solution of tetra-O-trimethylsilyl-α-L-fucopyranose (1.23 g, 2.72 mmol) in anhydrous CH2Cl2 (8 mL) was added iodotrimethylsilane (0.37 mL, 2.72 mmol). The mixture was stirred at rt for 20 min to give the 2,3,4-Tri-O-acetyl-α-L-fucopyranosyl iodide (4) in situ. Then, a solution of compound 3 (0.62 g, 1.34 mmol) and 2,6-di-tert-butylpyridine (0.6 mL, 2.72 mmol) in anhydrous CH2Cl2 (7 mL) was added to the reaction solution and the resulting mixture was stirred at rt for 5 h. MeOH (5 mL) was added and the resulting mixture was stirred for 20 min to remove the TMS group. Then it was treated with Dowex 1X2-100 ion-exchange resin (OH− form). And filtered. The filtrate was concentrated under vacuum and the residue was subject to a silica gel column chromatography (CH2Cl2/MeOH, 15:1, v/v) to provide compound 5 as a white solid (0.50 g, 61%). 1H NMR (400 MHz, CDCl3) δ 7.86 – 7.89 (m, 4H), 7.48 (dt, J = 7.32, 1.36 Hz, 2H), 7.25 – 7.34 (m, 4H), 6.30 (d, J = 8.72 Hz, 1H), 5.68 – 5.75 (m, 2H), 4.93 (d, J = 9.64 Hz, 1H), 4.82 (d, J = 3.68 Hz, 1H), 4.22 (dd, J = 10.08, 9.16 Hz, 1H), 4.09 (dd, J = 13.28, 6.4 Hz, 1H), 3.94 – 4.04 (m, 2H), 3.78 – 3.84 (m, 2H), 3.58 (dd, J = 11.48, 3.68 Hz, 1H), 3.46 (broad s, 1H), 3.10 (d, J = 10.52 Hz, 1H), 2.90 (broad s, 1H), 1.87 (s, 3H), 1.26 (d, J = 6.44 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 170.96, 166.83, 165.86, 133.98, 133.84, 129.92, 128.65, 128.50, 128.46, 98.44, 88.44, 75.10, 72.64, 71.76, 71.51, 69.49, 68.58, 66.45, 65.05, 54.5, 23.24, 16.20.

2-Acetamido-3,4-di-O-acetyl-2-deoxy-6-O-(tri-O-acetyl-α-L-fucopyranosyl)-β-D-glucopyranosyl azide (6)

A solution of compound 5 (0.50 g, 0.83 mmol) and NaOMe in MeOH (0.5 M, 50 μL, 50 μmol) in anhydrous MeOH/THF (1/1, v/v, 20 mL) was stirred at rt under argon for 1 h. The reaction mixture was treated with Dowex 50WX8-100 ion-exchange resin (H+ form) and filtered. The filtrate was concentrated to dryness under vacuum. Pyridine (5 mL) and Ac2O (5 mL) were added to the residue and the mixture was stirred at rt overnight. Then it was diluted with EtOAc and washed with saturated NaHCO3, water, and brine. The organic layer was dried over Na2SO4 and concentrated under vacuum. The residue was subject to silica gel column chromatography using EtOAc as the eluent to give compound 6 as a white solid (0.46 g, 92%). 1H NMR (400 MHz, CDCl3) δ 5.72 (d, J = 8.68 Hz, 1H), 5.28 – 5.33 (m, 2H), 5.24 (dd, J = 10.52, 9.16 Hz, 1H), 5.03 – 5.10 (m, 3H), 4.75 (d, J = 9.64 Hz, 1H), 4.16 (dt, J = 6.88, 6.4 Hz, 1H), 3.86 (dt, J = 10.52, 9.2 Hz, 1H), 3.71 – 3.79 (m, 2H), 3.51 (dd, J = 11.48, 5.04 Hz, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 171.07, 170.80, 170.70, 170.49, 170.12, 169.22, 96.76, 88.17, 75.07, 72.33, 71.11, 68.59, 68.04, 68.00, 66.36, 64.64, 54.33, 23.34, 20.82, 20.79, 20.72 (2C), 20.70, 15.96.

Nϖ-(2-Acetamido-3,4-di-O-acetyl-2-deoxy-6-O-(tri-O-acetyl-αL-fucopyranosyl)-Nα-(9-fluorenylmethyloxycarbonyl)-L-asparagine tert-butyl ester (7)

To a solution of compound 6 (0.46 g, 0.76 mmol) in THF (10 mL) was slowly added trimethylphosphine in toluene (1.0 M, 1.15 mL, 1.15 mmol) and the mixture was stirred at rt for 2 h until TLC showed the complettion of the reaction. The solvent was removed under vacuum and the residue was dissolved in DMF (2 mL). Then, Nα-(9-Fluorenylmethyloxycarbonyl)-L-aspartic acid α-tert-butyl ester (350 mg, 0.85 mmol), HATU (0.5 M in DMF, 4.5 mL, 2.25 mmol), and DIPEA (1.0 M in DMF, 3.0 mL, 3.0 mmol) were added. The resulting mixture was stirred at rt overnight under argon. The reaction solution was diluted with EtOAc and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated to dryness under vacuum. The residue was subject to silica gel column chromatography using EtOAc as the eluent to give compound 7 as a white solid (570 mg, 77%, two steps). 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 7.8 Hz, 2H), 7.63 (t, J = 7.32 Hz, 2H), 7.36 (t, J = 7.8 Hz, 2H), 7.26 – 7.31 (m, 2H), 7.15 (broad d, J = 8.24 Hz, 1H), 6.35 (d, J = 9.16 Hz, 1H), 5.93 (d, J = 7.76 Hz, 1H), 5.34 (dd, J = 11, 3.64 Hz, 1H), 5.26 (d, J = 2.72 Hz, 1H), 5.16 (dd, J = 10.96, 3.68 Hz, 1H), 4.98 – 5.06 (m, 4H), 4.96 (dd, J = 9.6, 8.24 Hz, 1H), 4.53 (m, 1H), 4.37 (dd, J = 10.52, 7.8 z, 1H), 4.32 (dd, J = 10.08, 7.36 Hz, 1H), 4.24 (t, J = 7.32 Hz, 1H), 4.06 (dt, J = 9.6, 8.24 Hz, 1H), 3.68 (m, 1H), 3.52 (dd, J = 11.92, 5.04 Hz, 1H), 2.87 (dd, J = 16.52, 4.6 Hz, 1H), 2.68 (dd, J = 16.48, 3.64 Hz, 1H), 2.12 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.96 (s, 9H), 1.93 (s, 3H), 1.42 (s, 9H), 1.07 (d, J = 6.44 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 172.32, 172.11, 171.17, 170.93, 170.71, 170.25, 170.10, 169.32, 156.56, 144.10, 141.31, 127.69, 127.19, 127.14, 125.46, 125.42, 119.98, 96.84, 82.00, 75.25, 73.13, 71.15, 68.30, 68.02, 67.79, 67.25, 65.47, 64.66, 53.44, 50.94, 47.19, 38.69, 38.03, 27.99 (3C), 23.16, 20.89, 20.81, 20.79, 20.72, 20.56, 15.92.

Nϖ-(2-Acetamido-3,4-di-O-acetyl-2-deoxy-6-O-(tri-O-acetyl-αL-fucopyranosyl)-Nα-(9-fluorenylmethyloxycarbonyl)-L-asparagine (8)

Compound 7 (100 mg, 0.1 mmol) was dissolved in a solution of TFA in CH2Cl2 (20%, 2 mL) and the mixture was stirred at room temperature for 1h. The residue was co-evaporated with toluene (3 mL) under vacuum and the resulted white solid was washed with Et2O (5 mL) to give the pure compound 8 (90 mg, 95%). 1H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 9.2 Hz, 1H, 1-NH of GlcNAc), 7.84 (m, 3H, 2-NHAc of GlcNAc, Ar-H), 7.68 (d, J = 7.6 Hz, 2H, Ar-H), 7.45 (d, J = 8.8 Hz, 1H, FmocNH), 7.38 (m, 2H, Ar-H), 7.28 (m, 2H, Ar-H), 5.19-5.15 (m, 3H, H1 of GlcNAc, H3 and H4 of Fuc), 5.03 (t, J = 9.6 Hz, 1H, H3 of GlcNAc), 4.91-4.80 (m, 3H, H1 and H2 of Fuc, H4 of GlcNAc), 4.34 (dd, J =7.2, 13.2 Hz, αH of Asn), 4.26-4.15 (m, 4H, H5 of Fuc, CH2 and H9 of Fmoc), 3.86 (q, J =9.6 Hz, 1H, H2 of GlcNAc), 3.66 (m, 2H, H5 and H6a of GlcNAc), 3.45 (m, 1H, H6b of GlcNAc), 2.65-2.49 (m, 2H, βH of Asn), 2.06 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.93 (s, 3H, Ac), 1.93 (s, 3H, Ac), 1.90 (s, 3H, Ac), 1.86 (s, 3H, Ac), 1.68 (s, 3H, NHAc), 0.98 (d, J =6.8 Hz, 1H, CH3 of Fuc); 13C NMR (100 MHz, CDCl3) δ 173.5, 170.8, 170.6, 170.3, 170.2, 170.1, 169.9, 169.7, 156.3, 144.3, 141.2, 128.1, 127.6, 125.8, 120.6, 96.5, 78.5, 74.2, 74.0, 71.1, 69.2, 67.9, 67.6, 66.4, 66.2, 64.5, 52.5, 50.5, 47.1, 37.4, 23.1, 21.0, 20.9, 20.8, 16.0. ESI-MS: calculated for C43H51N3O19, M = 913.31, found, 914.47 [M+H]+.

Nϖ-(2-Acetamido-6-O-(α-L-fucopyranosyl)-Nα-(9-fluorenylmethyloxycarbonyl)-L-asparagine (9)

A mixture of compound 8 (90 mg, 0.1 mmol) and K2CO3 (87 mg, 0.63 mmol) in MeOH/THF (3/1, v/v, 4 mL) was stirred at rt. The reaction was carefully monitored by ESI-MS until all the acetyl groups were selectively removed. Then a HCl solution (1N) was added to neutralize the solution to pH 2–3. The residue was subject to a preparative HPLC to give compound 9 as a white solid (60 mg, 85%). 1H NMR (400 MHz, DMSO-d6 + 1% D2O) δ 8.20 (broad d, J = 9.16 Hz, 1H), 7.85 (d, J = 7.8 Hz, 2H), 7.67 (d, J = 5.92 Hz, 2H), 7.39 (t, J = 7.8 Hz, 2H), 7.30 (t, J = 7.32 Hz, 2H), 4.80 (dd, J = 9.2, 6.44 Hz, 1H), 4.59 (d, J = 3.2 Hz, 1H), 4.31 (dd, J = 7.8, 5.52 Hz, 1H), 4.15 – 4.25 (m, 3H), 3.83 (dd, J = 13.28, 6.4 Hz, 1H), 3.44 – 3.54 (m, 6H), 3.30 (dd, J = 9.64, 9.16 Hz, 2H), 3.09 (t, J = 9.16 Hz, 1H), 2.57 (dd, J = 16.04, 5.04 Hz, 1H), 2.44 (dd, J = 16.04, 7.32 Hz, 1H), 1.74 (s, 3H), 1.03 (d, J = 6.88 Hz, 3H). 13C NMR (100 MHz, DMSO-d6 + 1% D2O) δ 173.38, 171.71, 170.33, 156.56, 144.10, 144.05, 141.12, 128.33, 127.75, 125.70, 120.61, 100.03, 78.96, 77.33, 74.29, 71.95, 70.58, 69.91, 68.51, 67.95, 66.46, 66.38, 54.82, 50.5, 46.98, 37.19, 23.03, 16.81. ESI-MS: calculated for C33H41N3O14, M = 703.25, found, 702.31 [M−H]−.

Chemoenzymatic transglycosylation assay of various endoglycosidases using Man3GlcNAc oxazoline (10) as donor substrate and Fmoc-Asn(Fucα1,6GlcNAc)-OH (9) as acceptor substrate

A mixture of acceptor 9 (25 μg, 36 nmol) and Man3GlcNAc oxazoline (10) (100 μg, 145 nmol) in a phosphate buffer (50 mM, pH 7.0, 12 μL) was incubated at 30 °C with individual Endo-β-N-acetylglucosaminidase (4 μg). The reaction was monitored by analytic HPLC by taking reaction aliquots at 10 min, 20 min, 30 min, 1 h, 2 h, 4h and 20 h. The yields were calculated based on the peak area of product and starting material acceptor. seven ENGases, including Endo-F1, Endo-F2, Endo-F3, Endo-A, Endo-M, Endo-D, and Endo-H, were measured for their transglycosylation activity in this assay. The time-course was shown in Figure 1. Panel A. The product (11) was purified by RP-HPLC and confirmed by MS analysis. Fmoc-Asn(Man3GlcNAc2Fuc)-OH (11): tR = 16.8 min (analytical HPLC, method A). MALDI-TOF MS: Calculated for C59H84N4O34, M = 1392.50; found, 1416.12 [M + Na]+; ESI-MS: found 1393.58 [M + H]+, 1247.60 [M − Fuc + H]+, 1085.43 [M − Fuc − Man + H]+, 923.45 [M − Fuc − 2Man + H]+, 761.39 [M − Fuc − 3Man + H]+.

Endo-D digestion of compound 11

A solution of 11 (50 μg) in a phosphate butter (50 mM, pH 7.0, 20 μL) was incubated with Endo-D (1 μg) at 30 °C for 2 h. The digestion was monitored by RP-HPLC combined with ESI-MS analysis. Endo-D digestion resulted in complete hydrolysis of 11 and the formation of Fmoc-Asn(Fucα1,6GlcNAc)-OH which was confirmed by ESI-MS analysis. This digestion data validated the β1,4-linkage of the N,N′-diacetylchitobiose unit in compound 11.

Chemoenzymatic transglycosylation assay of various Endoglycosidases using complex-type sialoglycan oxazoline (12) as donor substrate and Fmoc-Asn(Fucα1,6GlcNAc)-OH (9) as acceptor substrate

A mixture of acceptor 9 (25 μg, 36 nmol) and sialoglycan oxazoline 12 (290 μg, 145 nmol) in phosphate buffer (50 mM, pH 7.0, 12 μL) was incubated at 30 °C with individual endo-β-N-acetylglucosaminidase (4 μg). The reaction was monitored by analytic HPLC by taking reaction aliquots at 10 min, 20 min, 30 min, 1 h, 2 h, 4h and 20 h. The yields were calculated based on the peak area of product and starting material acceptor. Seven ENGases, including Endo-F1, Endo-F2, Endo-F3, Endo-A, Endo-M, Endo-D, and Endo-H, were measured for their transglycosylation activity in this assay. The time-course was shown in Figure 1. Panel B. The product (13) was purified and confirmed by MS analysis. Fmoc-Asn(Sia2Gal2GlcNAc2Man3GlcNAc2Fuc)-OH (13):, tR = 16.3 min (analytical HPLC, method A). MALDI-TOF MS: Calculated for C107H161N7O69, M = 2704.95; found, 2728.48 [M + Na]+; ESI-MS: found 1353.98 [M + 2H]2+, 1208.19 [M − Sia + 2H]2+.

Endo-F3 digestion of compound 13

A solution of 13 (10 μg) in a phosphate butter (50 mM, pH 7.0, 20 μL) was incubated with Endo-F3 (2 μg) at 30 °C for 20 h. The digestion was monitored by RP-HPLC combined with ESI-MS analysis. The Endo-F3 digestion led to complete hydrolysis of 13 and the formation of the expected complex type N-glycan and the Fmoc-Asn(Fucα1,6GlcNAc)-OH as confirmed by MS analysis.

Chemoenzymatic transglycosylation assay of various endoglycosidases using Man3GlcNAc oxazoline (10) as donor substrate and Fmoc-Asn(GlcNAc)-OH (14) as acceptor substrate

A mixture of acceptor 14 (20 μg, 36 nmol) and Man3GlcNAc oxazoline (100 μg, 145 nmol) in phosphate buffer (50 mM, pH 7.0, 12 μL) was incubated at 30 °C with individual Endo-β-N-acetylglucosaminidase (4 μg). The reaction was monitored by analytic HPLC by taking reaction aliquots at 10 min, 20 min, 30 min, 1 h, 2 h, 4h and 20 h. The yields were calculated based on the peak area of product and starting material acceptor. The time-course was shown in Figure 2. The product was purified by RP-HPLC to give Fmoc-Asn(Man3GlcNAc2)-OH (15): tR = 17.0 min (analytical HPLC, method A). MALDI-TOF MS: Calculated for C53H74N4O30, M = 1246.44; found, 1270.25 [M + Na]+; ESI-MS: found 1247.68 [M + H]+.

Endo-M digestion of compound 15

A solution of 15 (10 μg) in a phosphate butter (50 mM, pH 7.0, 20 μL) was incubated with Endo-M (1 μg) at 30 °C for 2 h. The digestion was monitored by RP-HPLC combined with ESI-MS analysis. The Endo-M digestion of compound 15 gave the glycan Man3GlcNAc and the Fmoc-Asn(GlcNAc)-OH as confirmed by MS analysis.

Solid-phase synthesis of the CD52 peptide bearing Fucα1-6GlcNAc (17)

The CD52 peptide was synthesized manually by the Fmoc-chemistry using Fmoc-protected amino acid derivatives. An O-link TGT resin (Novabiochem Corp) was used as the solid support, in which the first amino acid (Ser) was attached through the acid-labile ester linkage. To introduce a Fucα1-6GlcNAc residue at the N-glycosylation site, Fmoc-Asn(Ac5Fucα1-6GlcNAc)-OH (8) was used as building blocks to replace the residues at Asn-3 in the solid-phase peptide synthesis. HATU (0.5 M in DMF) and DIPEA (1.0 M in DMF) (1:1, v/v) were used as the coupling activator and piperidine (20% in DMF) was used as the deblocking reagent. Synthesis was carried out on a 10 μmol scale and 4-fold excess of Fmoc-protected building blocks were used for each coupling reaction cycle. The N-terminus amino group was protected with acetyl group by treatment with Ac2O/DIPEA. The resulted resin (16) was cleaved by treatment with TFA/CH2Cl2 (1/4, v/v) at r.t. for 5 h to release the crude peptide and simultaneously remove the protecting groups of Trt and t-Bu. The Ac groups on the disaccharide were removed by treatment with 5% aqueous hydrazine at rt for 1h. The residue was subject to preparative HPLC purification to give the glycopeptide Fucα1-6GlcNAc-CD52 (17) as a white powder (14.4 mg, overall yield 90%). Analytical HPLC of 17 (Method B): tR = 23.8 min; 1H NMR (D2O, 400 MHz): δ 4.91 (d, J = 9.6 Hz, 1H, H1 of GlcNAc), 4.76 (d, J = 3.6 Hz, 1H, H1 of Fuc), 4.71-4.58 (m, 3H, αH of Ser, αH of Asp, αH of Asn), 4.39-4.25 (m, 7H, αH of Ser x 3, αH of Pro, αH of Gln x 2, αH of Thr), 4.19-4.10 (m, 3H, αH of Thr, βH of Thr x 2), 3.99 (q, J = 6.8 Hz, 1H, H5 of Fuc), 3.86-3.60 (m, 18H), 3.52-3.38 (m, 3H), 2.82-2.64 (m, 4H, βH of Asn x 2, βH of Asp x 2), 2.27-2.15 (m, 5H, Gln-H, Pro-H), 2.08-1.82 (m, 13H, Ac, Gln-H, Pro-H), 1.08 (m, 9H, Thr-CH3 x 2, Fuc-CH3); 1H-13C HSQC NMR (13C 125 MHz, 1H 500 MHz, D2O) δ 4.91/78.4 (GlcNAc-1), 4.76/99.2 (Fuc-1), 4.69/53.1 (Ser-α), 4.68/49.8 (Aspα), 4.60/49.6 (Asn-α), 4.39/56.1 (Ser-α), 4.35/61.8 (Pro-α), 4.31/56.3 (Ser-α), 4.30/56.0 (Gln-α), 4.28/53.3 (Gln-α), 4.26/58.9 (Ser-α), 4.25/59.1 (Thr-α), 4.18/53.5 (Thr-α), 4.16/67.8 (Thr-β), 4.13/67.9 (Thr-β), 3.99/67.5 (Fuc-5), 3.85/68.3 (GlcNAc-6), 3.64/68.3 (GlcNAc-6), 2.80/35.8 (Asn-β), 2.74/36.4 (Asp-β), 2.25/31.4 (Gln-β), 2.21/30.7 (Pro), 2.02/22.1 (Ac), 1.95/22.8 (Ac), 1.89/30.6 (Pro), 1.83/27.2 (Ac), 1.10/16.2 (Thr-CH3), 1.08/19.7 (Fuc-CH3), 1.06/16.2 (Thr-CH3); MALDI-TOF MS: Calculated for C61H98N16O34, M = 1598.64; found, 1622.32 [M + Na]+; ESI-MS: 1599.16 [M + H]+, 800.27 [M + 2H]2+.

Chemoenzymatic synthesis of sialylated and core-fucosylated complex-type glycoform of CD52 (18)

A mixture of CD52 peptide 17 (2 mg, 1.25 μmol) and sialoglycan oxazoline 12 (5 mg, 2.5 μmol) in a phosphate buffer (50 mM, pH 7.0, 250 μL) was incubated at 30 °C with Endo-F3 (80 μg) for 1h. The reaction was quenched with 1% TFA (10 μL) and the residue was subject to RP-HPLC to give Sia2Gal2GlcNAc2Man3(Fuc)GlcNAc2-CD52 (18) as a white powder (2.5 mg, 55%). tR = 21.5 min (analytical HPLC, method B). 1H NMR (D2O, 400 MHz): δ 5.00 (s, 1H, H1 of Man4), 4.89 (d, J = 9.6 Hz, 1H, H1 of GlcNAc1), 4.81 (s, 1H, H1 of Man4′), 4.75 (s, 1H, H1 of Fuc), 4.72-4.58 (m, 4H, αH of Ser, H1 of Man3, αH of Asp, αH of Asn), 4.56 (d, J = 7.6 Hz, H1 of GlcNAc2), 4.48 (d, J = 7.2 Hz, H1 of GlcNAc5, H1 of GlcNAc5′), 4.41-4.24 (m, 9H, αH of Ser x 3, αH of Pro, αH of Gln x 2, H1 of Gal6, H1 of Gal6′, αH of Thr), 4.18-4.14 (m, 3H, αH of Thr, βH of Thr x 2), 4.12 (m, 1H), 4.07 (m, 1H), 3.98 (m, 2H), 3.89-3.35 (m, 82H), 2.87-2.61 (m, 4H, βH of Asn x 2, βH of Asp x 2), 2.54 (m, 2H, H3ax of Sia x 2), 2.27-2.18 (m, 5H, Gln-H, Pro-H), 2.07-1.83 (m, 29H, Ac, Gln-H, Pro-H), 1.60 (t, J = 12.0 Hz, 2H, H3eq of Sia x 2), 1.07 (m, 9H, Thr-CH3 x 2, Fuc-CH3); 1H-13C HSQC NMR (13C 125 MHz, 1H 500 MHz, D2O) δ 5.00/99.6 (Man4-1), 4.89/78.7 (GlcNAc1-1), 4.81/97.5 (Man4′-1), 4.75/99.8 (Fuc-1), 4.68/54.4 (Ser-α), 4.66/51.3 (Asp-α), 4.65/101.3 (Man3-1), 4.59/51.5 (Asn-α), 4.56/101.7 (GlcNAc2-1), 4.48/100.1 (GlcNAc5-1, GlcNAc5′-1), 4.38/56.3 (Ser-α), 4.35/61.5 (Pro-α), 4.31/56.8 (Ser-α), 4.31/56.3 (Gln-α), 4.30/104.4 (Gal6-1, Gal6′-1), 4.27/53.7 (Gln-α), 4.26/59.8 (Ser-α), 4.24/60.1 (Thr-α), 4.18/54.2 (Thr-α), 4.15/67.8 (Thr-β), 4.13/67.8 (Thr-β), 3.98/67.5 (Fuc-5), 2.83/35.8 (Asn-β), 2.74/36.2 (Asp-β), 2.54/40.3 (Sia-3ax), 2.25/31.3 (Gln-β), 2.02/22.3 (Ac), 1.95/22.4 (Ac), 1.87/26.8 (Ac), 1.60/40.3 (Sia-3eq), 1.08/15.9 (Thr-CH3), 1.07/19.7 (Fuc-CH3), 1.05/15.9 (Thr-CH3); ESI-MS: Calculated for C137H221N21O90, M = 3600.34; found 1201.66 [M + 3H]3+

Supplementary Material

Supporting information

Acknowledgments

This work was supported by the National Institutes of Health (NIH grant R01 GM080374.

Footnotes

Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. ((Please delete if not appropriate))

References

  • 1.a) Dwek RA. Chem Rev. 1996;96:683–720. doi: 10.1021/cr940283b. [DOI] [PubMed] [Google Scholar]; b) Helenius A, Aebi M. Science. 2001;291:2364–2369. doi: 10.1126/science.291.5512.2364. [DOI] [PubMed] [Google Scholar]; c) Haltiwanger RS, Lowe JB. Annu Rev Biochem. 2004;73:491–537. doi: 10.1146/annurev.biochem.73.011303.074043. [DOI] [PubMed] [Google Scholar]; d) Dube DH, Bertozzi CR. Nat Rev Drug Discov. 2005;4:477–488. doi: 10.1038/nrd1751. [DOI] [PubMed] [Google Scholar]; e) Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA. Annu Rev Immunol. 2007;25:21–50. doi: 10.1146/annurev.immunol.25.022106.141702. [DOI] [PubMed] [Google Scholar]
  • 2.a) Dwek RA, Butters TD, Platt FM, Zitzmann N. Nat Rev Drug Discov. 2002;1:65–75. doi: 10.1038/nrd708. [DOI] [PubMed] [Google Scholar]; b) Walsh G, Jefferis R. Nat Biotechnol. 2006;24:1241–1252. doi: 10.1038/nbt1252. [DOI] [PubMed] [Google Scholar]; c) Kaneko Y, Nimmerjahn F, Ravetch JV. Science. 2006;313:670–673. doi: 10.1126/science.1129594. [DOI] [PubMed] [Google Scholar]; d) Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV. Science. 2008;320:373–376. doi: 10.1126/science.1154315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.a) Andre S, Kozar T, Kojima S, Unverzagt C, Gabius HJ. Biol Chem. 2009;390:557–565. doi: 10.1515/BC.2009.072. [DOI] [PubMed] [Google Scholar]; b) Andre S, Kozar T, Schuberth R, Unverzagt C, Kojima S, Gabius HJ. Biochemistry. 2007;46:6984–6995. doi: 10.1021/bi7000467. [DOI] [PubMed] [Google Scholar]; c) Unverzagt C, Andre S, Seifert J, Kojima S, Fink C, Srikrishna G, Freeze H, Kayser K, Gabius HJ. J Med Chem. 2002;45:478–491. doi: 10.1021/jm0110237. [DOI] [PubMed] [Google Scholar]; d) Jefferis R. Biotechnol Prog. 2005;21:11–16. doi: 10.1021/bp040016j. [DOI] [PubMed] [Google Scholar]
  • 4.Barrabes S, Pages-Pons L, Radcliffe CM, Tabares G, Fort E, Royle L, Harvey DJ, Moenner M, Dwek RA, Rudd PM, De Llorens R, Peracaula R. Glycobiology. 2007;17:388–400. doi: 10.1093/glycob/cwm002. [DOI] [PubMed] [Google Scholar]
  • 5.Takahashi M, Kuroki Y, Ohtsubo K, Taniguchi N. Carbohydr Res. 2009;344:1387–1390. doi: 10.1016/j.carres.2009.04.031. [DOI] [PubMed] [Google Scholar]
  • 6.a) Nicolaou KC, Mitchell HJ. Angew Chem. 2001;113:1624–1672. [PubMed] [Google Scholar]; Angew Chem Int Ed. 2001;40:1576–1624. [PubMed] [Google Scholar]; b) Grogan MJ, Pratt MR, Marcaurelle LA, Bertozzi CR. Annu Rev Biochem. 2002;71:593–634. doi: 10.1146/annurev.biochem.71.110601.135334. [DOI] [PubMed] [Google Scholar]; c) Buskas T, Ingale S, Boons GJ. Glycobiology. 2006;16:113R–136R. doi: 10.1093/glycob/cwj125. [DOI] [PubMed] [Google Scholar]; d) Bennett CS, Wong CH. Chem Soc Rev. 2007;36:1227–1238. doi: 10.1039/b617709c. [DOI] [PubMed] [Google Scholar]; e) Wang LX, Huang W. Curr Opin Chem Biol. 2009;13:592–600. doi: 10.1016/j.cbpa.2009.08.014. [DOI] [PMC free article] [PubMed] [Google Scholar]; f) Gamblin DP, Scanlan EM, Davis BG. Chem Rev. 2009;109:131–163. doi: 10.1021/cr078291i. [DOI] [PubMed] [Google Scholar]; g) Rich JR, Withers SG. Nat Chem Biol. 2009;5:206–215. doi: 10.1038/nchembio.148. [DOI] [PubMed] [Google Scholar]
  • 7.a) Unverzagt C, Kunz H. Bioorg Med Chem. 1994;2:1189–1201. doi: 10.1016/s0968-0896(00)82070-2. [DOI] [PubMed] [Google Scholar]; b) Eller S, Schuberth R, Gundel G, Seifert J, Unverzagt C. Angew Chem. 2007;119:4251–4253. doi: 10.1002/anie.200604788. [DOI] [PubMed] [Google Scholar]; Angew Chem Int Ed. 2007;46:4173–4175. doi: 10.1002/anie.200604788. [DOI] [PubMed] [Google Scholar]; c) Sun B, Srinivasan B, Huang X. Chem Eur J. 2008;14:7072–7081. doi: 10.1002/chem.200800757. [DOI] [PMC free article] [PubMed] [Google Scholar]; d) Wang P, Zhu J, Yuan Y, Danishefsky SJ. J Am Chem Soc. 2009;131:16669–16671. doi: 10.1021/ja907136d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ihara H, Hanashima S, Okada T, Ito R, Yamaguchi Y, Taniguchi N, Ikeda Y. Glycobiology. 2010;20:1021–1033. doi: 10.1093/glycob/cwq064. [DOI] [PubMed] [Google Scholar]
  • 9.Wang LX. Carbohydr Res. 2008;343:1509–1522. doi: 10.1016/j.carres.2008.03.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Li B, Zeng Y, Hauser S, Song H, Wang LX. J Am Chem Soc. 2005;127:9692–9693. doi: 10.1021/ja051715a. [DOI] [PubMed] [Google Scholar]
  • 11.a) Li H, Li B, Song H, Breydo L, Baskakov IV, Wang LX. J Org Chem. 2005;70:9990–9996. doi: 10.1021/jo051729z. [DOI] [PubMed] [Google Scholar]; b) Li B, Song H, Hauser S, Wang LX. Org Lett. 2006;8:3081–3084. doi: 10.1021/ol061056m. [DOI] [PubMed] [Google Scholar]; c) Ochiai H, Huang W, Wang LX. J Am Chem Soc. 2008;130:13790–13803. doi: 10.1021/ja805044x. [DOI] [PMC free article] [PubMed] [Google Scholar]; d) Wei Y, Li C, Huang W, Li B, Strome S, Wang LX. Biochemistry. 2008;47:10294–10304. doi: 10.1021/bi800874y. [DOI] [PMC free article] [PubMed] [Google Scholar]; e) Huang W, Wang D, Yamada M, Wang LX. J Am Chem Soc. 2009;131:17963–17971. doi: 10.1021/ja9078539. [DOI] [PMC free article] [PubMed] [Google Scholar]; f) Schwarz F, Huang W, Li C, Schulz BL, Lizak C, Palumbo A, Numao S, Neri D, Aebi M, Wang LX. Nat Chem Biol. 2010;6:264–266. doi: 10.1038/nchembio.314. [DOI] [PMC free article] [PubMed] [Google Scholar]; g) Rising TW, Claridge TD, Moir JW, Fairbanks AJ. Chem Bio Chem. 2006;7:1177–1180. doi: 10.1002/cbic.200600183. [DOI] [PubMed] [Google Scholar]; h) Heidecke CD, Ling Z, Bruce NC, Moir JW, Parsons TB, Fairbanks AJ. Chem Bio Chem. 2008;9:2045–2051. doi: 10.1002/cbic.200800214. [DOI] [PubMed] [Google Scholar]; i) Rising TW, Heidecke CD, Moir JW, Ling Z, Fairbanks AJ. Chem Eur J. 2008;14:6444–6464. doi: 10.1002/chem.200800365. [DOI] [PubMed] [Google Scholar]; j) Parsons TB, Moir JW, Fairbanks AJ. Org Biomol Chem. 2009;7:3128–3140. [Google Scholar]
  • 12.Umekawa M, Huang W, Li B, Fujita K, Ashida H, Wang LX, Yamamoto K. J Biol Chem. 2008;283:4469–4479. doi: 10.1074/jbc.M707137200. [DOI] [PubMed] [Google Scholar]
  • 13.Huang W, Li C, Li B, Umekawa M, Yamamoto K, Zhang X, Wang LX. J Am Chem Soc. 2009;131:2214–2223. doi: 10.1021/ja8074677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Huang W, Yang Q, Umekawa M, Yamamoto K, Wang LX. Chem Bio Chem. 2010;11:1350–1355. doi: 10.1002/cbic.201000242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Huang W, Zhang X, Ju T, Cummings RD, Wang LX. Org Biomol Chem. 2010;8:5224–5233. doi: 10.1039/c0ob00341g. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Umekawa M, Li C, Higashiyama T, Huang W, Ashida H, Yamamoto K, Wang LX. J Biol Chem. 2010;285:511–521. doi: 10.1074/jbc.M109.059832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Parsons TB, Patel MK, Boraston AB, Vocadlo DJ, Fairbanks AJ. Org Biomol Chem. 2010;8:1861–1869. doi: 10.1039/b926078a. [DOI] [PubMed] [Google Scholar]
  • 18.Takegawa K, Yamabe K, Fujita K, Tabuchi M, Mita M, Izu H, Watanabe A, Asada Y, Sano M, Kondo A, Kato I, Iwahara S. Arch Biochem Biophys. 1997;338:22–28. doi: 10.1006/abbi.1996.9803. [DOI] [PubMed] [Google Scholar]
  • 19.Yamamoto K, Kadowaki S, Fujisaki M, Kumagai H, Tochikura T. Biosci Biotechnol Biochem. 1994;58:72–77. doi: 10.1271/bbb.58.72. [DOI] [PubMed] [Google Scholar]
  • 20.Trimble RB, Tarentino AL. J Biol Chem. 1991;266:1646–1651. [PubMed] [Google Scholar]
  • 21.Tarentino AL, Plummer TH., Jr Methods Enzymol. 1994;230:44–57. doi: 10.1016/0076-6879(94)30006-2. [DOI] [PubMed] [Google Scholar]
  • 22.a) Reddy A, Grimwood BG, Plummer TH, Tarentino AL. Glycobiology. 1998;8:633–636. doi: 10.1093/glycob/8.6.633. [DOI] [PubMed] [Google Scholar]; b) Tarentino AL, Quinones G, Plummer TH., Jr Glycobiology. 1995;5:599–601. doi: 10.1093/glycob/5.6.599. [DOI] [PubMed] [Google Scholar]
  • 23.Kirsch P, Kusunose N, Aikawa J, Kigawa T, Yokoyama S, Ogawa T. Bioorg Med Chem. 1995;3:1631–1636. doi: 10.1016/0968-0896(95)00148-4. [DOI] [PubMed] [Google Scholar]
  • 24.Uchiyama T, Hindsgaul O. Synlett. 1996:499–501. [Google Scholar]
  • 25.Rao V, Guan C, Van Roey P. Structure. 1995;3:449–457. doi: 10.1016/s0969-2126(01)00178-2. [DOI] [PubMed] [Google Scholar]
  • 26.Muramatsu H, Tachikui H, Ushida H, Song X, Qiu Y, Yamamoto S, Muramatsu T. J Biochem. 2001;129:923–928. doi: 10.1093/oxfordjournals.jbchem.a002938. [DOI] [PubMed] [Google Scholar]
  • 27.Plummer TH, Jr, Phelan AW, Tarentino AL. Anal Biochem. 1996;235:98–101. doi: 10.1006/abio.1996.0096. [DOI] [PubMed] [Google Scholar]
  • 28.An Endo-F preparation was first shown to have transglycosylation activity on glycerol when the later was used as a stabilizing reagent, but the enzymes in the preparation were not defined and were likely to be a mixture of several Flavobacterium endoglycosidases. See: Trimble RB, Atkinson PH, Tarentino AL, Plummer TH, Jr, Maley F, Tomer KB. J Biol Chem. 1986;261:12000–12005.
  • 29.Waddling CA, Plummer TH, Jr, Tarentino AL, Van Roey P. Biochemistry. 2000;39:7878–7885. doi: 10.1021/bi0001731. [DOI] [PubMed] [Google Scholar]
  • 30.a) Tews I, Terwisscha van Scheltinga AC, Perrakis A, Wilson KS, Dijkstra BW. J Am Chem Soc. 1997;119:7954–7959. [Google Scholar]; b) Brameld KA, Shrader WD, Imperiali B, Goddard WA., 3rd J Mol Biol. 1998;280:913–923. doi: 10.1006/jmbi.1998.1890. [DOI] [PubMed] [Google Scholar]
  • 31.a) Treumann A, Lifely MR, Schneider P, Ferguson MA. J Biol Chem. 1995;270:6088–6099. doi: 10.1074/jbc.270.11.6088. [DOI] [PubMed] [Google Scholar]; b) Schroter S, Derr P, Conradt HS, Nimtz M, Hale G, Kirchhoff C. J Biol Chem. 1999;274:29862–29873. doi: 10.1074/jbc.274.42.29862. [DOI] [PubMed] [Google Scholar]; c) Domagala A, Kurpisz M. Med Sci Monit. 2001;7:325–331. [PubMed] [Google Scholar]
  • 32.a) Shao N, Xue J, Guo Z. J Org Chem. 2003;68:9003–9011. doi: 10.1021/jo034773s. [DOI] [PubMed] [Google Scholar]; b) Shao N, Xue J, Guo Z. Angew Chem. 2004;116:1595–1599. [Google Scholar]; Angew Chem Int Ed. 2004;43:1569–1573. doi: 10.1002/anie.200353251. [DOI] [PubMed] [Google Scholar]; c) Pratt MR, Bertozzi CR. J Am Chem Soc. 2003;125:6149–6159. doi: 10.1021/ja029346v. [DOI] [PubMed] [Google Scholar]
  • 33.Li H, Singh S, Zeng Y, Song H, Wang LX. Bioorg Med Chem Lett. 2005;15:895–898. doi: 10.1016/j.bmcl.2004.12.066. [DOI] [PubMed] [Google Scholar]
  • 34.a) Kuhn P, Tarentino AL, Plummer TH, Jr, Van Roey P. Biochemistry. 1994;33:11699–11706. doi: 10.1021/bi00205a005. [DOI] [PubMed] [Google Scholar]; b) Tarentino AL, Quinones G, Trumble A, Changchien LM, Duceman B, Maley F, Plummer TH., Jr J Biol Chem. 1990;265:6961–6966. [PubMed] [Google Scholar]
  • 35.Haneda K, Inazu T, Yamamoto K, Kumagai H, Nakahara Y, Kobata A. Carbohydr Res. 1996;292:61–70. doi: 10.1016/s0008-6215(96)91025-3. [DOI] [PubMed] [Google Scholar]
  • 36.Fujita K, Tanaka N, Sano M, Kato I, Asada Y, Takegawa K. Biochem Biophys Res Commun. 2000;267:134–138. doi: 10.1006/bbrc.1999.1963. [DOI] [PubMed] [Google Scholar]

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