Synthesis of a high-mannose-type glycopeptide analog containing a glucose-asparagine linkage

BIOORGANIC Bioorganic & Medicinal Chemistry Letters 8 (1998) 1763-1766 Pergamon SYNTHESIS OF A HIGH-MANNOSE-TYPE CONTAINING GLYCOPEPTIDE A GLUCOSE-ASPARAGINE & MEDICINAL CHEMISTRY LEIIERS ANALOG LINKAGE Ina L. Deras’, Kaoru Takegawab, Akihiro Kondo’, Ikunoshin Katoc, Yuan C. Lee’* “Biology Department, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218 bDepartment of Bioresource Science, Faculty of Agriculture, Kagawa Givers@, Japan ‘Biotechnology Research Laboratory, Takara Shuzo Co., Ltd. Ohtsu, Shiga 520-21, Japan Received 30 April 1998; accepted 5 June 1998 Abstract: The title compound was prepared by enzymatic transfer of oligosaccharide to a synthetic pentapeptide containing the Glc-Asn linkage. The compound was not hydrolyzed by glycoamidases from plant and bacterial sources, but it inhibited both enzymes in the micromolar range. Its activity is compared to other potential inhibitors. 0 1998Elsevier Science Ltd. All rights reserved. Glycoamidase (peptide-N’-(N-acetyl-@D-glucosaminyl)-asparagine amidase, PNGase, GA) is found in a number of organisms across different kingdoms. lV3 The enzyme catalyzes the release of intact oligosaccharides from N-linked glycopeptides and glycoproteins via hydrolysis of the P-amide of the linking asparagine. To probe the mechanism and substrate requirements for glycoamidases, a number of natural and unnatural mono- and disaccharide glycopeptides were synthesized and tested for substrate activity. Cellobiose and lactose glycopeptides, in which GlcNAc-GlcNAc linked to Asn is replaced by a disaccharide of non-aminosugars, are not substrates for glycoamidases4 However, we wanted to examine the effects of changing only the innermost sugar, while retaining the second aminosugar and mannosyl residues, of a native substrate. Thus, we synthesized 1, a substrate analog in which the linking sugar, normally GlcNAc, is replaced with Glc. The unusual Glc-Asn linkage has been found in nature, H-Tyr-lie-fsn-Ala&x-NH2 Mana 1,2- Manal,6_ ?Cp 1 ,Mana1,6, ,ManP1,4-GlcNAcP1,4 Manal,Z-Manal, Manal,2-Manal,Z-Manal, 1 Figure 1. Structure of title compound. notably in Archaebacterias and laminin,e although the structure of the oligosaccharide beyond the linking Glc has not yet been elucidated. the a linkage glycopeptide.7 is found Additionally, Glc-Asn in in a nephritogenic Our compound serves further purpose as a model to study this class of glycoconjugate. Total organic synthesis of the target compound would be laborious, so instead we combined chemical and enzymatic methods to prepare the glycopeptide. The synthesis is based on the effective transglycosylation 0960-894X/98/$19.00 0 I998 Elsevier Science Ltd. All rights reserved. P/I: SO960-894X(98)00306-0 1764 I. L. Deras et al. / Bioorg. Med. Chem. Lett. 8 (1998) 1763-l 766 activity of endo-N-acetyl-S-D-glucosaminidase an endoglycosidase, from Arthrobacter protophormiae (Endo-A), normally acting as cleaving between the two GlcNAc residues of high-mannose-type N-glycans. In some aqueous organic solvents, however, the enzyme can mediate transfer of Mans-gGlcNAc to the equatorial 4-OH of an acceptor molecule.* In our case, MangGlcNAc&sn, isolated from soybean agglutinin,9 served as the donor and glucosyl pentapeptide (7) served as the acceptor. Synthesis of the enzymatic acceptor 7 is summarized in Scheme 1. Glucosyl azide tetra-O-acetate (3) was readily prepared from D-glucose, lo and protected glucosyl asparagine (4) could be prepared by two methods from 3. The first option was reduction of the azide to amine, followed by coupling with Boc-Asp0Bn.*1T12 Alternatively, 4 was prepared in one step from 3 and Boc-Asp-OBn Elongation to the pentapeptide l4 followed. coupling with H-Ala-Ser-OMe. via triethylphosphine.13 First, 4 was debenzylated by hydrogenolysis with subsequent The resultant tripeptide 5 was deprotected at the amino terminus under acidic conditions, followed by coupling with Boc-Tyr-Ile-OH. The pentapeptide 6 was then deprotected by sequential ammonolysis and acidolysis to yield 7 BOCNHJOR OH OAc 2 OAc 3 OAc 6 OH I OAc 5 i: (a) AclO, HBr-HOAc, rt, 2 h; (b) NaN3, DMF, rt, 16 h, 81%. ii: (a) HZ,PtOz, MeOH, rt, 16 h; (b) Boc-Asp-OBn, DCC, HOBt, CH2C12-DMF,rt, 2 h, 52%. iii: Boc-Asp-OBn, Et3P, CH& rt, 16 h, 54%. iv: (a) Hz, Pd/C, EtOH, rt, 6 h; (b) HAla-Ser-OMe, DCC, HOBt, DMF, rt, 16 h, 58%. v: (a) 55% TFA, 2% PhOH, CH$&, rt, 15 min; (b) Boc-Tyr-he-OH, DCC, HOBt, DMF, rt, 16 h, 58%. vi: (a) NI-Ix,THF-MeOH, rt, 2 d; (b) 55% TFA, 1% PhOH, HzO, rt, 15 mm, 30%. Scheme 1. Synthesis of glucosyl pentapeptide acceptor. Enzymatic transfer of the MansGlcNAc structure to 7 was performed according to literature procedure.8 Briefly, MangGlcNAclAsn, 7, and Endo-A, in ammonium acetate, pH 6, containing 35% acetone, were incubated at 37 “C for 20 min and boiled for 3 min to stop the reaction. The product was purified by HPLC on a Spherisorb SS ODS semiprep column (1 x 25 cm) with 9% aqueous acetonitrile containing 0.05% trifluoroacetic acid (3.5 mL/min) as eluant. The product eluted at 9.5 mitt, and excess acceptor eluted at 17.9 min. Both 1765 I. L. Deras et al. / Bioorg. M ed. Chem. Lett. 8 (1998) 1763- l 766 product and acceptor were collected and recovered. Eluant containing product was lyophilized to yield the desired product as a white solid in 39% yield. H-Tyr-lie-Asn-Ala-Ser-NH2 ~ ~ Endo-A OH ManpGlcNAc+Asn 1 H-Tyr-Ile-Asn-Ala-Ser-NH2 MansGlcNAcOH&k OH GlcNAcAsn 1 Scheme 2. Enzymatic transfer of MangGlcNAc to glucosyl pentapeptide acceptor ‘H NMR and amino acid and monosaccharide composition analyses were consistent with the expected structure of the product.15 The ‘H NMR chemical shift values of the anomeric protons beyond the linking Glc were very similar to those reported for known glycopeptides containing the MangGlcNAc structure. l6 The synthetic product 1 was tested as a substrate for commercially available glycoamidases from almond (GAA) or from Ffavobacterium meningosepticum (GAF). 1 was incubated overnight with either enzyme in ammonium acetate buffer and analyzed by HPLC. No change was observed by HPLC in the presence or absence of either enzyme. 1 was then tested as an inhibitor for glycoamidases. Using glycopeptide isolated from thermolysin digest of bovine asialofetuin as substrate, a mixture containing 10 mM ammonium acetate (pH 5 and 8 for GAA and GAP, respectively), 12.5 pM glycopeptide, 0.5-1000 pM 1, and glycoamidase was incubated at 37 “C for 10 min, and the reaction was stopped by boiling for 3 min. The mixture was then analyzed by HPLC on a Shimadzu CLC-ODS column (60 x 150 mm) with 6% aqueous ?? Glycoanidase A 0 Gtycoanidase F acetonitrile trifluoroacetic Substrate, acid hydrolysis containing (1 r&/mm) product, 0.05% as eluant. and inhibitor eluted at 6.7, 9.8, and 13.1 min, respectively. Peak area of the product was measured and compared to a control containing no inhibitor to 0.2- -6.5 determine relative activity. Results in the figure -6.0 -5.5 -5.0 4 -4.5 111 show an I& -4.0 -3.5 of 8 pM for GAA and 62 pM for -3.0 Figure 2. Studies of inhibition of glycoamidases by 1. GAP. The K, values are 4 uM and 75 uM for GAA and GAP, respectively. Correlating ICJO and K, with Ki,l7 the Ki values for 1 were determined to be 2 I.IM for GAA and 53 l.tM for GAP. The lack of turnover and the inhibition results suggest that the acetamido group of the innermost sugar plays an important role in recognition and cleavage of the natural GlcNAc-Asn bond. 1766 I. L. Deras et al. / Bioorg. Med. Chem. Lett. 8 (I 998) 1763-I 766 The inhibition by 1 of glycoamidases is comparable to a C-glycopeptide analog, in which a methylene bridge is inserted between the high-mannose-type glycan and asparagine g-amide of a pentapeptide (1 and 43 @I for GAA and GAF, respectively). l4 Initial inhibition studies by other nonsubstrate compounds for GAA or GAF demonstrated different activities. MangGlcNAczAsn, MangGlcNAcGlcAsn (a truncated form of l), and the glycan from fetuin glycopeptide inhibited GAF at 1000 pM. On the other hand, the same compounds showed no inhibition of GAA. Furthermore, neither enzyme was inhibited by 500 pM 7, demonstrating a role for the oligosaccharide chain for inhibition. Acknowledgments. The authors thank Dr. Mei Tang for the gift of fetuin glycopeptide. supported in part by NIH Research grant DK09970. This work was REFERENCES 1. Suzuki, T.; Kitajima, K.; Emori, Y.; Inoue, Y.; Inoue, S. Proc. Natl. Acad. Sci. U.S.A. 1997,94,6244. 2. Berger, S.; Menudier, A.; Julien, R.; Karamanos, Y. Biochimie 1995, 77, 75 1, 3. Kitajima, K.; Suzuki, T.; Kouchi, 2.; Inoue, S.; Inoue, Y. Arch. Biochem. Biophys. 1995,319,393. 4. Fan, J.-Q.; Lee, Y. C. J. Biol. Chem. 1997,272,27058. 5. Messner, P. Glycoconj. J. 1997,14, 3. 6. Schreiner, R.; Schnabel, E.; Wieland, F. J. Cell Biol. 1994, 124, 1071. 7. Shibata, S.; Takeda, T.; Natori, Y. J. Biol. Chem. 1988,263,2483. 8. Fan, J.-Q.; Takegawa, K.; Iwahara, S.; Kondo, A.; Kato, I.; Abeygunawardana, C.; Lee, Y. C. J. Biol. Chem. 1995,270, 17723. 9. Fan, J.-Q,; Kondo, A.; Kato, I.; Lee, Y. C. Anal. 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