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Enzymatic synthesis of human blood group P1 pentasaccharide antigen
Carbohydrate Research 438 (2017) 39e43
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Enzymatic synthesis of human blood group P1 pentasaccharide antigen Dawa Tsering a, 1, Congcong Chen a, 1, Jinfeng Ye a, Zhipeng Han a, Bai-qian Jing b, **, Xian-wei Liu a, Xi Chen c, Fengshan Wang a, d, Peixue Ling a, e, Hongzhi Cao a, f, * a
National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, China Department of Pharmacy, Qilu Hospital, Shandong University, Jinan, 250012, China c Department of Chemistry, University of California, One Shields Avenue, Davis, CA, 95616, USA d Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drugs, Shandong University, Jinan, 250012, China e Shandong Academy of Pharmaceutical Science, Jinan, 250101, China f State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 July 2016 Received in revised form 30 November 2016 Accepted 30 November 2016 Available online 5 December 2016
The enzymatic synthesis of biologically important and structurally unique human P1PK blood group type P1 pentasaccharide antigen is described. This synthesis features a three-step sequential one-pot multienzyme (OPME) glycosylation for the stepwise enzymatic chain elongation of readily available lactoside acceptor with cheap and commercially available galactose and N-acetylglucosamine as donor precursors. This enzymatic synthesis provides an operationally simple approach to access P1 pentasaccharide and its structurally related Gb3 and P1 trisaccharide epitopes. © 2016 Elsevier Ltd. All rights reserved.
Keywords: P1 antigen Human blood group Glycosylation One-pot multienzyme Glycosyltransferase
The P1 antigen was discovered by Landsteiner and Levine [1] in 1927, and for a long time it was the only antigen belonging to previous human P blood group system. The P1 antigen determinant was identified as the trisaccharide 1 by Morgan and Watkins [2] in 1964, which shares the same terminal globobiose unit (Gala1e4Gal) as Pk trisaccharide antigen 2 (also known as Gb3 or CD77 belongs to previous human blood group GLOB system), and the P1 antigen was eventually elucidated as a pentasaccharide glycosphingolipid 3 (R' ¼ ceramide) by Naiki and Marcus [3] in 1974 (Fig. 1). Different hypotheses were proposed on the puzzling biosynthetic pathway of P1 antigen for many years, while, the results of recent studies showed that the P1/Pk synthase encoded by
* Corresponding author. National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, China. ** Corresponding author. E-mail addresses: [email protected] (B.-q. Jing), [email protected] (H. Cao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.carres.2016.11.019 0008-6215/© 2016 Elsevier Ltd. All rights reserved.
A4GALT gene is the only a1e4-galactosyltransferase responsible for the synthesis of terminal Gala1e4-linkage for both P1 and Pk antigens [4]. Based on their genetic contribution, the P1 and Pk antigens have been reassigned to the same blood group system (ISBT no. 003), and the system name has also been changed from P to the P1PK by the International Society of Blood Transfusion (ISBT) in 2011 [5]. Similar to other human blood group carbohydrate antigens, the P1 antigen mainly presents on red blood cells which may cause acute intravascular hemolytic transfusion reactions and recurrent spontaneous abortions [5b,6]. The P1 antigen was also identified on B lymphocytes, granulocytes and monocytes [7]. Most recently, Jacob et al. disclosed that the P1 antigen also over expressed on ovarian cancer cells and involved in cancer migration, thus it could be used as a new biomarker for ovarian cancer diagnosis [8]. The P1 and Pk antigens can also act as cellular receptors for several pathogens and toxins [9] such as Shigella dysenteriae, Escherichia coli O157, E. coli O104, Streptococcus suis, Pseudomonas aeruginosa, or HIV virus [10] and B19 parvovirus [11]. Every year,
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Fig. 1. Structure of human P1PK blood group system type Pk (P1PK3) and P1 (P1PK1) antigens (R' ¼ ceramide).
millions of people lose their lives caused by Shiga toxin-producing E. coli (STEC) infection. The Shiga toxins 1 and 2 (Stx1 and Stx2) are the major virulence of STEC and the leading cause of acute kidney failure resulting from life threatening hemolytic uremic syndrome (HUS) [9a,9c]. Currently no treatment is available for STEC infections, as conventional antibiotic treatment is reported to enhance toxin production and progression of HUS [12]. Accumulating evidence confirmed that the terminal Gala1e4Gal moiety of glycolipid is the functional toxin receptor on mammalian cells, which makes it an attractive target for the development of Shiga toxin neutralizer as an alternative approach for the treatment of STEC infections [9a-c,13]. The past few decades have seen tremendous achievements in the design and synthesis of various globobiose and Pk trisaccharide based inhibitors, especially the multivalent Pk trisaccharide based glycoconjugates [13,14]. One of the most successful multivalent inhibitor is a Pk trisaccharide based pentavalent ligand developed by Bundle and coworkers, which can neutralize Shiga toxin in vitro up to 10 million times more potently than monovalent ligand [15]. Apart from previous efforts on the development of Shiga toxin inhibitors mainly focused on the Pk trisaccharide, the P1 trisaccharide has been recognized as a better inhibitor than its counterpart in early 1990s [16], which has been used for selective affinity capture and purification of Shiga-like toxin 1 [17] and uropathogenic E. coli [18]. Most recently, Cherian et al. showed that multivalent P1 trisaccharide-decorated mucin-type protein bound with high avidity to Shiga toxin [19]. Gallegos et al. demonstrated that the P1 trisaccharide was 7-fold more potent binding to Shiga toxin 1 than Pk trisaccharide, and P1 pentasaccharide was over 30 times more potent than Pk trisaccharide [20]. Taking together, these results indicate that the P1 pentasaccharide can be utilized as a better lead compound for the development of more potent and selective ligands for the neutralization, detection or purification of Shiga toxins. Despite the potential utility of P1 pentasaccharide antigen, a highly efficient synthetic approach is still missing, as previous chemical [21] or whole cell synthesis [22] were only focused on the P1 trisaccharide. Herein, we report an operationally simple approach for the enzymatic synthesis of the P1 pentasaccharide 4 which was attached to a 3-azidopropyl aglycone as chemical handle for future application. As depicted in Fig. 2, our approach relies on
the one-pot multienzyme (OPME) [23] based sequential glycosylation of readily available lactoside 5. Therefore, the P1 pentasaccharide antigen 4 can be produced in 3 steps from 5 after b1e3N-acetyl-glucosaminylation, b1e4-galactosylation and a1e4galactosylation. Our synthesis commenced with the enzymatic synthesis of P1 trisaccharide determinant 8 from disaccharide 6 (Scheme 1). Previously, an a1e4-galactosyltransferase from Neisseria meningitidis (NmLgtC) has been successfully applied for the enzymatic synthesis of Pk trisaccharide and its various derivatives [24], and it also has been used for the preparation of P1 trisaccharide from recombinant E. coli cells [22]. To confirm whether NmLgtC is suitable for in vitro synthesis of non-reducing end a1e4-linked galactose unit of P1 antigen, the small scale pilot reaction (50 mL) in Tris-HCl buffer (100 mM, pH 7.5) was initially examined using LacNAc glycoside 6 as acceptor and UDP-Gal as donor, and the lactoside 5 was used as acceptor for positive control (Scheme 1a). These pilot reactions demonstrated that lactoside 5 was a better substrate as its reaction went faster than LacNAc glycoside 6 for NmLgtC-catalyzed a1e4-galactosylation, but both reactions could be completed in 1 h in over 80% yields as determined by thin-layer chromatography (TLC). This single enzyme (NmLgtC) catalyzed glycosylation provides an easy access to P1 trisaccharide determinant by using expensive UDP-Gal (1020 US$ per 100 mg from Sigma) which is not practical for large scale synthesis. For the large scale synthesis of P1 trisaccharide 8, a one-pot three-enzyme system (OPME 1) was developed to lower the cost which coupled two enzymes EcGalK [24a] and BLUSP [25]) for in situ generation of UDP-Gal directly from cheap galactose (36.8 US$ per 100 g from Sigma). As shown in Scheme 1b, the galactose was first converted to galactose-1-phosphate (Gal-1-P) by an E. coli galactokinase (EcGalK) [24a] in Tris-HCl buffer (100 mM, pH 7.5) in the presence of adenosine 5'-triphosphate (ATP, 1.3 equiv) and MgCl2 (20 mM). The resulting Gal-1-P was utilized by a Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP) [25] in the presence of uridine 5'-triphosphate (UTP, 1.3 equiv) to form the UDP-Gal, and the in situ generated UDP-Gal was taken by NmLgtC as a donor to provide the P1 trisaccharide 8 (46 mg) in 92% yield after purification. Following the same approach, the Pk trisaccharide 7 (65 mg) was also prepared in one-pot in 94% yield after purification using lactoside 5 as acceptor. The formation of a1e4linked galactoside of P1 trisaccharide 8 was confirmed by 1H, 13C, and 2D NMR spectroscopies (see ESIy for details). Taking advantage of the substrate promiscuity of NmLgtC, the one-pot three-enzyme a1e4-galactosylation (OPME 1) was demonstrated as a practical and highly efficient approach for the preparative-scale synthesis of P1 tirsaccharide 8 and Pk trisaccharide 7 from readily available disaccharide acceptors and cheap galactose in one pot. Encouraged by these results, the total synthesis of P1 pentasaccharide 4 from lactoside 5 was explored through a sequential three-step OPME chain elongation as depicted in Scheme 2. A one-pot three-enzyme system (OPME 2) was
Fig. 2. Retrosynthetic analysis of P1 pentasaccharide 4.
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Scheme 1. a) Small scale pilot reaction to confirm the feasibility of synthesis of P1 trisaccharide using a1e4-galactosyltransferase NmLgtC; b) One-pot three-enzyme preparative scale synthesis of P1 trisaccharide 8 and Pk trisaccharide 7. NmLgtC, N. meningitides a1e4-galactosyltransferase; EcGalK, E. coli galactokinase; BLUSP, B. longum UDP-sugar pyrophosphorylase; Reagents and conditions: a) The total volume of small scale reaction is 50 mL which contains UDP-Gal (13 mM), Tris-HCl buffer (100 mM, pH 7.5), MgCl2 (20 mM), NmLgtC (5 mg), disaccharide 5 (10 mM, 1 equiv), 37 C, disaccharide 6 (10 mM, 1 equiv), 1 h, over 80% for 7 and 8 based on TLC estimation, respectively; b) Galactose (20 mg, 1.3 equiv), disaccharide 6 (37 mg, 0.08 mM), ATP (1.3 equiv), UTP (1.3 equiv), Tris-HCl buffer (100 mM, pH 7.5), MgCl2 (20 mM), EcGalK (1.5 mg), BLUSP (2.0 mg), NmLgtC (1.5 mg), 37 C, 8 h, 92% for 8 (46 mg) in onepot; 94% for 7 (65 mg) in one-pot from disaccharide 5 (50 mg).
employed for the synthesis of trisaccharide 9 from lactoside 5 (Scheme 2). In this system, N-acetylglucosamine (GlcNAc) was converted to uridine 5'-diphosphate-N-acetylglucosamine (UDPGlcNAc) in Tris-HCl buffer (100 mM, pH 8.0) in the presence of ATP (1.3 equiv), UTP (1.3 equiv), MgCl2 (20 mM) and a fusion enzyme, NahK-GlmU [26] constructed from B. longum N-acetylhexosamine1-kinase (NahK) and E. coli N-acetylglucosamineuridylyltransferase (GlmU). The in situ generated UDP-GlcNAc was utilized by a H. pylori b1e3-N-acetylglucosaminyltransferase (HpLgtA) [27] as a donor to furnish the trisaccharide 9 (105 mg) in 89% yield. The b1e4-galactosylation of trisaccharide 9 was performed by using a one-pot three-enzyme b1e4-galactosylation system (OPME 3). The OPME 3 shares same two enzymes (EcGalK and BLUSP) in OPME 1 for in situ generation of UDP-Gal from galactose but using a different galactosyltransferase from N. meningitides (NmLgtB) [28] to form the desired b1e4-linked galactose unit of 10 (Scheme 2). The tetrasaccharide 10 (94 mg) was obtained in 93% yield after
41
Scheme 2. Sequential three-step OPME synthesis of P1 pentasaccharide 4. NahKGlmU, B. longum N-acetylhexosamine-1-kinase (NahK) and E. coli N-acetylglucosamineuridylyltransferase (GlmU) fusion enzyme; HpLgtA, Helicobacter pylori b1e3-Nacetylglucosaminyltransferase; NmLgtB, N. meningitides b1e4-galactosyltransferase; Reagents and conditions: OPME 2, GlcNAc (54 mg, 1.3 equiv), 5 (80 mg, 0.19 mM, 1.0 euiv), ATP (1.3 equiv), UTP (1.3 equiv), Tris-HCl buffer (100 mM, pH 8.0), MgCl2 (20 mM), NahK-GlmU (1.2 mg), HpLgtA (0.6 mg), 37 C, 16 h, 89% for 9 (105 mg) in onepot; OPME 3, Gal (30 mg, 1.3 equiv), 9 (80 mg, 0.13 mM, 1.0 euiv), ATP (1.3 equiv), UTP (1.3 equiv), Tris-HCl buffer (100 mM, pH 7.5), MgCl2 (20 mM), EcGalK (2.5 mg), BLUSP (3.0 mg), NmLgtB (1.3 mg), 37 C, 12 h, 93% for 10 (94 mg) in one-pot; OPME 1, Gal (23 mg, 1.3 equiv), 10 (79 mg, 0.1 mM, 1.0 euiv), ATP (1.3 equiv), UTP (1.3 equiv), TrisHCl buffer (100 mM, pH 7.5), MgCl2 (20 mM), EcGalK (1.5 mg), BLUSP (2.0 mg), NmLgtC (2.0 mg), 37 C, 12 h, 55% for 4 (52 mg) in one-pot.
purification. With tetrasaccharide 10 in hand, the aforementioned one-pot three-enzyme a1e4-galactosylation (OPME 1) system for the synthesis of P1 trisaccharide 8 was adapted to finish the total synthesis of P1 pentasaccharide 4 (Scheme 2). Although the NmLgtCcatalyzed a1e4-galactosylation of tetrasaccharide 10 was much slower than using disaccharide 5 as acceptor, the tetrasaccharide 10 was still well tolerated by a1e4-galactosyltransferase NmLgtC to afford P1 pentasaccharide 4 (52 mg) in 55% yield. In summary, a highly efficient three-step sequential one-pot multienzyme glycosylation approach was developed for the enzymatic synthesis of human blood P1PK system P1 pentasaccharide antigen 4. The three OPME systems comprised four enzymes for the in situ generation of two UDP-sugar donors (two for UDP-GlcNAc and two for UDP-Gal, respectively), and three glycosyltransferases for the introducing three different glycosidic linkages, respectively. Taking advantage of the substrate promiscuity of all seven bacterial enzymes, the P1PK pentasaccharide antigen 4 was synthesized from readily available lactoside 5 along with cheap galactose and GlcNAc as donor precursors under operationally simple three steps with an overall yield of 46%. This biologically important pentasaccharide can be used as probes for bacterial infection detection and development of antigen-based multivalent inhibitors for neutralization of bacterial toxins.
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1. Experimental 1.1. General methods All chemicals were obtained from commercial suppliers and used without further purification unless noted. Thin layer chromatography (TLC) was performed on silica gel plates 60 F254 (Merck, Billerica MA). Plates were visualized under UV light and/or by treatment with 5% sulfuric acid in ethanol or p-anisaldehyde sugar stain followed by heating. Silica gel 60 (300e400 mesh, Haiyang, Qingdao, China) was used for flash column chromatography. Gel filtration chromatography was performed using a column (100 cm 2.5 cm) packed with Bio-Gel P-2 Fine resins (BioRad, Hercules, CA). 1H NMR (600 MHz) and 13C NMR (150 MHz) spectra were recorded on Bruker AVANCE-600 spectrometer at 25 C. NMR spectra were calibrated using solvent signals (1H: d 7.26 for CDCl3, d 3.34 for CD3OD or d 4.79 for D2O, 13C: d 77.0 for CDCl3). High resolution electrospray ionization (ESI) mass spectra were obtained at the National Glycoengineering Research Center and Drug Testing and Analysis Center in Shandong University. 1.2. Gala1e4Galb1e4GlcbProN3 (7) LactosebProN3 5 [29] (50 mg, 0.12 mmol), Gal (28 mg, 0.16 mmol), ATP (78 mg, 0.16 mmol) and UTP (74 mg, 0.16 mmol) were dissolved in water in a 50 mL centrifuge tube containing TrisHCl buffer (100 mmol, pH 7.5) and MgCl2 (20 mmol). After the addition of appropriate amount of EcGalK (2.2 mg), BLUSP (3.0 mg) and NmLgtC (2.2 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 6 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H2O/HOAc, 8:3:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by Bio-Gel P-2 column (eluted with H2O) to provide trisaccharide 7 (65 mg, 94%) as a white solid. [a]27 D ¼ þ61.2 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.96 (d, J ¼ 4.0 Hz, 1H, H-100 ), 4.51 (d, J ¼ 12.3 Hz, 1H, H-10 ), 4.50 (d, J ¼ 12.5 Hz, 1H, H-1), 4.37 (t, J ¼ 6.6 Hz, 1H, H-500 ), 4.05e3.57 (m, 18H), 3.47 (t, J ¼ 6.6 Hz, 2H, CH2N3), 3.32 (t, J ¼ 8.1 Hz, 1H, H-2), 1.92 (p, J ¼ 6.6 Hz, 2H, CH2CH2N3); 13C NMR (150 MHz, D2O) d 103.26 (C10 ), 102.09 (C-1), 100.32 (C-100 ), 78.67 (C-4), 77.37 (C-40 ), 75.42 (C30 ), 74.80 (C-50 ), 74.43 (C-3), 72.91 (C-2), 72.17 (C-5), 70.92 (C-20 ), 70.82 (C-500 ), 69.14 (C-300 ), 68.97 (C-400 ), 68.58 (C-200 ), 67.36 (OCH2CH2), 60.55 (C-600 ), 60.39 (C-6), 60.07 (C-60 ), 47.90 (CH2N3), 28.27 (CH2CH2N3); HRMS (ESI) m/z calcd for C21H37N3NaO16 [MþNa]þ 610.2072, found 610.2073. 1.3. Gala1e4Galb1e4GlcNAcbProN3 (8) LacNAcbProN3 6 5 (37 mg, 0.08 mmol), Galactose (Gal, 20 mg, 0.10 mmol), adenosine 5'-triphosphate (ATP, 56 mg, 0.11 mmol) and uridine 5'-triphosphate (UTP, 53 mg, 0.11 mmol) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mmol, pH 7.5) and MgCl2 (20 mmol). After the addition of appropriate amount of EcGalK (1.5 mg), BLUSP (2.0 mg) and NmLgtC (1.5 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 8 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H2O/HOAc, 8:3:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by Bio-Gel P-2 column (eluted
with H2O) to provide trisaccharide 8 (46 mg, 92%) as a white solid. [a]27 D ¼ þ47.3 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.95 (d, J ¼ 4.2 Hz, 1H, H-100 ), 4.53 (d, J ¼ 7.8 Hz, 2H, H-1 and H-10 ), 4.36 (t, J ¼ 6.3 Hz, 1H, H-500 ), 4.04 (d, J ¼ 6.0 Hz, 1H, H-40 ), 4.03 (d, J ¼ 6.0 Hz, 1H, H-300 ), 4.02e3.56 (m, 17H), 3.40e3.34 (m, 2H), 2.05 (s, 3H, NHCOCH3), 1.84 (p, J ¼ 6.3 Hz, 2H, CH2CH2N3); 13C NMR (150 MHz, D2O) d 174.38 (NHCOCH3), 103.24 (C-10 ), 101.05 (C-1), 100.26 (C-100 ), 78.75 (C-4), 77.26 (C-40 ), 75.40 (C-30 ), 74.77 (C-50 ), 72.39 (C-3), 72.12 (C-5), 70.88 (C-20 ), 70.77 (C-500 ), 69.11 (C-400 ), 68.90 (C-300 ), 68.52 (C200 ), 67.10 (OCH2CH2), 60.47 (C-600 ), 60.32 (C-60 ), 60.00 (C-6), 55.23 (C-2), 47.75 (CH2N3), 28.08 (CH2CH2N3), 22.14 (NHCOCH3); HRMS (ESI) m/z calcd for C23H40N4NaO16 [MþNa]þ651.2337, found 651.2341. 1.4. GlcNAcb1e3Galb1e4GlcbProN3 (9) LactosebProN3 5 (80 mg, 0.19 mmol), N-acetylglucosamine (GlcNAc, 54 mg, 0.20 mmol), ATP (124 mg, 0.25 mmol) and UTP (118 mg, 0.25 mmol) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mmol, pH 8.0) and MgCl2 (20 mmol). After the addition of appropriate amount of NahK-GlmU (1.2 mg) and HpLgtA (0.6 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 16 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/ MeOH/H2O/HOAc, 4:2:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by silica gel flash column chromatography (EtOAc/MeOH/H2O, 7:2:1,v/v) and Bio-Gel P-2 column (eluted with H2O) to provide trisaccharide 9 (105 mg, 89%) as a white solid. [a]27 D ¼ þ3.4 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.70 (d, J ¼ 8.4 Hz, 1H, H-100 ), 4.50 (d, J ¼ 7.8 Hz, 1H, H-1), 4.45 (d, J ¼ 7.8 Hz, 1H, H-10 ), 4.16 (d, J ¼ 3.0 Hz, 1H, H-40 ), 4.03e3.46 (m, 20H), 3.32 (t, J ¼ 8.4 Hz, 1H, H-2), 2.05 (s, 3H, NHCOCH3), 1.93 (p, J ¼ 6.6 Hz, 2H, CH2CH2N3); 13C NMR (151 MHz, D2O) d 174.95 (NHCOCH3), 102.92 (C-10 ), 102.84 (C-100 ), 102.10 (C-1), 81.96 (C-30 ), 78.34 (C-4), 75.64 (C-500 ), 74.88 (C-50 ), 74.76 (C-5), 74.35 (C-300 ), 73.55 (C-3), 72.79 (C-2), 70.00 (C-20 ), 69.68 (C-400 ), 68.33 (C-40 ), 67.36 (OCH2CH2), 60.96 (C-10 ), 60.47 (C-600 ), 60.06 (C6), 55.65 (C-200 ), 47.87 (CH2N3), 28.23 (CH2CH2N3), 22.19 (NHCOCH3); HRMS (ESI) m/zcalcd for C23H40N4NaO16 [MþNa]þ 651.2337, found 651.2346. 1.5. Galb1e4GlcNAcb1e3Galb1e4GlcbProN3 (10) Trisaccharide 9 (80 mg, 0.13 mmol), galactose (Gal, 30 mg, 0.17 mmol), ATP (84 mg, 0.17 mmol) and UTP (80 mg, 0.17 mmol) were dissolved in water in a 50 mL centrifuge tube containing TrisHCl buffer (100 mmol, pH 7.5) and MgCl2 (20 mmol). After the addition of appropriate amount of EcGalK (2.5 mg), BLUSP (3.0 mg) and NmLgtB (1.3 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 12 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H2O/HOAc, 4:2:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by silica gel flash column chromatography (EtOAc/MeOH/H2O, 6:2:1, v/v) and Bio-Gel P-2 column (eluted with H2O) to provide tetrasaccharide 10 (94 mg, 93%) as a white solid. [a]27 D ¼ þ1.5 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.73 (d, J ¼ 8.4 Hz, 1H, H-100 ), 4.50 (d, J ¼ 7.8 Hz, 1H, H-1), 4.49 (d, J ¼ 7.8 Hz, 1H, H-1000 ), 4.46 (d, J ¼ 8.4 Hz, 1H, H-10 ), 4.17 (d, J ¼ 3.6 Hz, 1H, H-40 ), 4.03e3.96 (m, 3H), 3.94 (d, J ¼ 3.0 Hz,
D. Tsering et al. / Carbohydrate Research 438 (2017) 39e43
1H, H-4000 ), 3.87 (dd, J ¼ 4.8, 12.6 Hz, 1H, H-6b), 3.84e3.54 (m, 19H), 3.48 (t, J ¼ 6.6 Hz, 2H, CH2N3), 3.33 (t, J ¼ 8.4 Hz, 1H, H-2), 2.05 (s, 3H, NHCOCH3), 1.93 (p, J ¼ 6.6 Hz, 2H, CH2CH2N3); 13C NMR (151 MHz, D2O) d 174.89 (NCOCH3), 102.92 (C-10 ), 102.84 (C-1000 ), 102.73 (C-100 ), 102.09 (C-1), 82.05 (C-30 ), 78.35 (C-4), 78.14 (C-400 ), 75.33 (C-5000 ), 74.87 (C-50 ), 74.75 (C-5), 74.53 (C-500 ), 74.34 (C-3), 72.79 (C-2), 72.49 (C-3000 ), 72.16 (C-300 ), 70.96 (C-2000 ), 69.95 (C-20 ), 68.55 (C-4000 ), 68.31 (C-40 ), 67.36 (OCH2CH2), 61.04 (C-6000 ), 60.96 (C60 ), 60.06 (C-600 ), 59.85 (C-6), 55.18 (C-200 ), 47.87 (CH2N3), 28.24 (CH2CH2N3), 22.22 (NHCOCH3); HRMS (ESI) m/zcalcd for C29H50N4NaO21 [MþNa]þ 813.2865, found 813.2886.
[6] [7] [8]
[9]
[10]
1.6. Gala1e4Galb1e4GlcNAcb1e3Galb1e4GlcbProN3 (4) Tetrasaccharide 10 (79 mg, 0.10 mmol), Gal (23 mg, 0.13 mmol), ATP (66 mg, 0.13 mmol) and UTP (63 mg, 0.13 mmol) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mmol, pH 7.5) and MgCl2 (20 mmol). After the addition of appropriate amount of EcGalK (1.5 mg), BLUSP (2.0 mg) and NmLgtC (2.0 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 12 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H2O/HOAc, 4:2:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by Bio-Gel P-2 column (eluted with H2O) to provide pentasaccharide 4 (52 mg, 55%) as a white solid. [a]27 D ¼ þ28.7 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.96 (d, J ¼ 4.2 Hz, 1H, H-10000 ), 4.72 (d, J ¼ 8.4 Hz, 1H, H-100 ), 4.55 (d, J ¼ 7.8 Hz, 1H, H-1000 ), 4.50 (d, J ¼ 7.8 Hz, 1H, H-1), 4.45 (d, J ¼ 7.8 Hz, 1H, H-10 ), 4.37 (t, J ¼ 6.6 Hz, 1H, H-5000 '), 4.17 (d, J ¼ 3.0 Hz, 1H, H-40 ), 4.03e3.57 (m, 29H), 3.47 (t, J ¼ 6.9 Hz, 2H, CH2N3), 3.32 (t, J ¼ 8.7 Hz, 1H, H-2), 2.05 (s, 3H, NHCOCH3), 1.92 (p, J ¼ 6.6 Hz, 2H, CH2CH2N3); 13C NMR (150 MHz, D2O) d 174.91 (NCOCH3), 103.21 (C10 ), 102.92 (C-1000 ), 102.70 (C-100 ), 102.10 (C-1), 100.27 (C-10000 ), 82.03 (C-30 ), 78.43 (C-4), 78.34 (C-400 ), 77.30 (C-4000 ), 75.41, 74.87, 74.76, 74.57, 74.34, 72.78, 72.19, 72.13, 70.89, 70.79, 69.96, 69.12, 68.92, 68.53, 68.32 (C-40 ), 67.36 (OCH2CH2), 60.94 (C-60 ), 60.49 (C-60000 ), 60.36 (C-6000 ), 60.04 (C-600 ), 59.81 (C-6), 55.33 (C-200 ), 47.86 (CH2N3), 28.22 (CH2CH2N3), 22.18 (NHCOCH3); HRMS (ESI) m/z calcd for C35H60N4NaO26 [MþNa]þ 975.3393, found 975.3416.
[11] [12]
[13] [14]
[15] [16]
[17]
[18] [19] [20] [21]
Acknowledgments [22]
This work was financially supported by National Natural Science Foundation of China (Grant Nos. 21372145, 21672128), State Key Laboratory of Microbial Technology (M2016-06) and Department of Science and Technology of Shandong Province (2016GSF121002).
[23] [24]
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.carres.2016.11.019. [25]
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Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Enzymatic synthesis of human blood group P1 pentasaccharide antigen Dawa Tsering a, 1, Congcong Chen a, 1, Jinfeng Ye a, Zhipeng Han a, Bai-qian Jing b, **, Xian-wei Liu a, Xi Chen c, Fengshan Wang a, d, Peixue Ling a, e, Hongzhi Cao a, f, * a
National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, China Department of Pharmacy, Qilu Hospital, Shandong University, Jinan, 250012, China c Department of Chemistry, University of California, One Shields Avenue, Davis, CA, 95616, USA d Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drugs, Shandong University, Jinan, 250012, China e Shandong Academy of Pharmaceutical Science, Jinan, 250101, China f State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 July 2016 Received in revised form 30 November 2016 Accepted 30 November 2016 Available online 5 December 2016
The enzymatic synthesis of biologically important and structurally unique human P1PK blood group type P1 pentasaccharide antigen is described. This synthesis features a three-step sequential one-pot multienzyme (OPME) glycosylation for the stepwise enzymatic chain elongation of readily available lactoside acceptor with cheap and commercially available galactose and N-acetylglucosamine as donor precursors. This enzymatic synthesis provides an operationally simple approach to access P1 pentasaccharide and its structurally related Gb3 and P1 trisaccharide epitopes. © 2016 Elsevier Ltd. All rights reserved.
Keywords: P1 antigen Human blood group Glycosylation One-pot multienzyme Glycosyltransferase
The P1 antigen was discovered by Landsteiner and Levine [1] in 1927, and for a long time it was the only antigen belonging to previous human P blood group system. The P1 antigen determinant was identified as the trisaccharide 1 by Morgan and Watkins [2] in 1964, which shares the same terminal globobiose unit (Gala1e4Gal) as Pk trisaccharide antigen 2 (also known as Gb3 or CD77 belongs to previous human blood group GLOB system), and the P1 antigen was eventually elucidated as a pentasaccharide glycosphingolipid 3 (R' ¼ ceramide) by Naiki and Marcus [3] in 1974 (Fig. 1). Different hypotheses were proposed on the puzzling biosynthetic pathway of P1 antigen for many years, while, the results of recent studies showed that the P1/Pk synthase encoded by
* Corresponding author. National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, China. ** Corresponding author. E-mail addresses: [email protected] (B.-q. Jing), [email protected] (H. Cao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.carres.2016.11.019 0008-6215/© 2016 Elsevier Ltd. All rights reserved.
A4GALT gene is the only a1e4-galactosyltransferase responsible for the synthesis of terminal Gala1e4-linkage for both P1 and Pk antigens [4]. Based on their genetic contribution, the P1 and Pk antigens have been reassigned to the same blood group system (ISBT no. 003), and the system name has also been changed from P to the P1PK by the International Society of Blood Transfusion (ISBT) in 2011 [5]. Similar to other human blood group carbohydrate antigens, the P1 antigen mainly presents on red blood cells which may cause acute intravascular hemolytic transfusion reactions and recurrent spontaneous abortions [5b,6]. The P1 antigen was also identified on B lymphocytes, granulocytes and monocytes [7]. Most recently, Jacob et al. disclosed that the P1 antigen also over expressed on ovarian cancer cells and involved in cancer migration, thus it could be used as a new biomarker for ovarian cancer diagnosis [8]. The P1 and Pk antigens can also act as cellular receptors for several pathogens and toxins [9] such as Shigella dysenteriae, Escherichia coli O157, E. coli O104, Streptococcus suis, Pseudomonas aeruginosa, or HIV virus [10] and B19 parvovirus [11]. Every year,
40
D. Tsering et al. / Carbohydrate Research 438 (2017) 39e43
Fig. 1. Structure of human P1PK blood group system type Pk (P1PK3) and P1 (P1PK1) antigens (R' ¼ ceramide).
millions of people lose their lives caused by Shiga toxin-producing E. coli (STEC) infection. The Shiga toxins 1 and 2 (Stx1 and Stx2) are the major virulence of STEC and the leading cause of acute kidney failure resulting from life threatening hemolytic uremic syndrome (HUS) [9a,9c]. Currently no treatment is available for STEC infections, as conventional antibiotic treatment is reported to enhance toxin production and progression of HUS [12]. Accumulating evidence confirmed that the terminal Gala1e4Gal moiety of glycolipid is the functional toxin receptor on mammalian cells, which makes it an attractive target for the development of Shiga toxin neutralizer as an alternative approach for the treatment of STEC infections [9a-c,13]. The past few decades have seen tremendous achievements in the design and synthesis of various globobiose and Pk trisaccharide based inhibitors, especially the multivalent Pk trisaccharide based glycoconjugates [13,14]. One of the most successful multivalent inhibitor is a Pk trisaccharide based pentavalent ligand developed by Bundle and coworkers, which can neutralize Shiga toxin in vitro up to 10 million times more potently than monovalent ligand [15]. Apart from previous efforts on the development of Shiga toxin inhibitors mainly focused on the Pk trisaccharide, the P1 trisaccharide has been recognized as a better inhibitor than its counterpart in early 1990s [16], which has been used for selective affinity capture and purification of Shiga-like toxin 1 [17] and uropathogenic E. coli [18]. Most recently, Cherian et al. showed that multivalent P1 trisaccharide-decorated mucin-type protein bound with high avidity to Shiga toxin [19]. Gallegos et al. demonstrated that the P1 trisaccharide was 7-fold more potent binding to Shiga toxin 1 than Pk trisaccharide, and P1 pentasaccharide was over 30 times more potent than Pk trisaccharide [20]. Taking together, these results indicate that the P1 pentasaccharide can be utilized as a better lead compound for the development of more potent and selective ligands for the neutralization, detection or purification of Shiga toxins. Despite the potential utility of P1 pentasaccharide antigen, a highly efficient synthetic approach is still missing, as previous chemical [21] or whole cell synthesis [22] were only focused on the P1 trisaccharide. Herein, we report an operationally simple approach for the enzymatic synthesis of the P1 pentasaccharide 4 which was attached to a 3-azidopropyl aglycone as chemical handle for future application. As depicted in Fig. 2, our approach relies on
the one-pot multienzyme (OPME) [23] based sequential glycosylation of readily available lactoside 5. Therefore, the P1 pentasaccharide antigen 4 can be produced in 3 steps from 5 after b1e3N-acetyl-glucosaminylation, b1e4-galactosylation and a1e4galactosylation. Our synthesis commenced with the enzymatic synthesis of P1 trisaccharide determinant 8 from disaccharide 6 (Scheme 1). Previously, an a1e4-galactosyltransferase from Neisseria meningitidis (NmLgtC) has been successfully applied for the enzymatic synthesis of Pk trisaccharide and its various derivatives [24], and it also has been used for the preparation of P1 trisaccharide from recombinant E. coli cells [22]. To confirm whether NmLgtC is suitable for in vitro synthesis of non-reducing end a1e4-linked galactose unit of P1 antigen, the small scale pilot reaction (50 mL) in Tris-HCl buffer (100 mM, pH 7.5) was initially examined using LacNAc glycoside 6 as acceptor and UDP-Gal as donor, and the lactoside 5 was used as acceptor for positive control (Scheme 1a). These pilot reactions demonstrated that lactoside 5 was a better substrate as its reaction went faster than LacNAc glycoside 6 for NmLgtC-catalyzed a1e4-galactosylation, but both reactions could be completed in 1 h in over 80% yields as determined by thin-layer chromatography (TLC). This single enzyme (NmLgtC) catalyzed glycosylation provides an easy access to P1 trisaccharide determinant by using expensive UDP-Gal (1020 US$ per 100 mg from Sigma) which is not practical for large scale synthesis. For the large scale synthesis of P1 trisaccharide 8, a one-pot three-enzyme system (OPME 1) was developed to lower the cost which coupled two enzymes EcGalK [24a] and BLUSP [25]) for in situ generation of UDP-Gal directly from cheap galactose (36.8 US$ per 100 g from Sigma). As shown in Scheme 1b, the galactose was first converted to galactose-1-phosphate (Gal-1-P) by an E. coli galactokinase (EcGalK) [24a] in Tris-HCl buffer (100 mM, pH 7.5) in the presence of adenosine 5'-triphosphate (ATP, 1.3 equiv) and MgCl2 (20 mM). The resulting Gal-1-P was utilized by a Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP) [25] in the presence of uridine 5'-triphosphate (UTP, 1.3 equiv) to form the UDP-Gal, and the in situ generated UDP-Gal was taken by NmLgtC as a donor to provide the P1 trisaccharide 8 (46 mg) in 92% yield after purification. Following the same approach, the Pk trisaccharide 7 (65 mg) was also prepared in one-pot in 94% yield after purification using lactoside 5 as acceptor. The formation of a1e4linked galactoside of P1 trisaccharide 8 was confirmed by 1H, 13C, and 2D NMR spectroscopies (see ESIy for details). Taking advantage of the substrate promiscuity of NmLgtC, the one-pot three-enzyme a1e4-galactosylation (OPME 1) was demonstrated as a practical and highly efficient approach for the preparative-scale synthesis of P1 tirsaccharide 8 and Pk trisaccharide 7 from readily available disaccharide acceptors and cheap galactose in one pot. Encouraged by these results, the total synthesis of P1 pentasaccharide 4 from lactoside 5 was explored through a sequential three-step OPME chain elongation as depicted in Scheme 2. A one-pot three-enzyme system (OPME 2) was
Fig. 2. Retrosynthetic analysis of P1 pentasaccharide 4.
D. Tsering et al. / Carbohydrate Research 438 (2017) 39e43
Scheme 1. a) Small scale pilot reaction to confirm the feasibility of synthesis of P1 trisaccharide using a1e4-galactosyltransferase NmLgtC; b) One-pot three-enzyme preparative scale synthesis of P1 trisaccharide 8 and Pk trisaccharide 7. NmLgtC, N. meningitides a1e4-galactosyltransferase; EcGalK, E. coli galactokinase; BLUSP, B. longum UDP-sugar pyrophosphorylase; Reagents and conditions: a) The total volume of small scale reaction is 50 mL which contains UDP-Gal (13 mM), Tris-HCl buffer (100 mM, pH 7.5), MgCl2 (20 mM), NmLgtC (5 mg), disaccharide 5 (10 mM, 1 equiv), 37 C, disaccharide 6 (10 mM, 1 equiv), 1 h, over 80% for 7 and 8 based on TLC estimation, respectively; b) Galactose (20 mg, 1.3 equiv), disaccharide 6 (37 mg, 0.08 mM), ATP (1.3 equiv), UTP (1.3 equiv), Tris-HCl buffer (100 mM, pH 7.5), MgCl2 (20 mM), EcGalK (1.5 mg), BLUSP (2.0 mg), NmLgtC (1.5 mg), 37 C, 8 h, 92% for 8 (46 mg) in onepot; 94% for 7 (65 mg) in one-pot from disaccharide 5 (50 mg).
employed for the synthesis of trisaccharide 9 from lactoside 5 (Scheme 2). In this system, N-acetylglucosamine (GlcNAc) was converted to uridine 5'-diphosphate-N-acetylglucosamine (UDPGlcNAc) in Tris-HCl buffer (100 mM, pH 8.0) in the presence of ATP (1.3 equiv), UTP (1.3 equiv), MgCl2 (20 mM) and a fusion enzyme, NahK-GlmU [26] constructed from B. longum N-acetylhexosamine1-kinase (NahK) and E. coli N-acetylglucosamineuridylyltransferase (GlmU). The in situ generated UDP-GlcNAc was utilized by a H. pylori b1e3-N-acetylglucosaminyltransferase (HpLgtA) [27] as a donor to furnish the trisaccharide 9 (105 mg) in 89% yield. The b1e4-galactosylation of trisaccharide 9 was performed by using a one-pot three-enzyme b1e4-galactosylation system (OPME 3). The OPME 3 shares same two enzymes (EcGalK and BLUSP) in OPME 1 for in situ generation of UDP-Gal from galactose but using a different galactosyltransferase from N. meningitides (NmLgtB) [28] to form the desired b1e4-linked galactose unit of 10 (Scheme 2). The tetrasaccharide 10 (94 mg) was obtained in 93% yield after
41
Scheme 2. Sequential three-step OPME synthesis of P1 pentasaccharide 4. NahKGlmU, B. longum N-acetylhexosamine-1-kinase (NahK) and E. coli N-acetylglucosamineuridylyltransferase (GlmU) fusion enzyme; HpLgtA, Helicobacter pylori b1e3-Nacetylglucosaminyltransferase; NmLgtB, N. meningitides b1e4-galactosyltransferase; Reagents and conditions: OPME 2, GlcNAc (54 mg, 1.3 equiv), 5 (80 mg, 0.19 mM, 1.0 euiv), ATP (1.3 equiv), UTP (1.3 equiv), Tris-HCl buffer (100 mM, pH 8.0), MgCl2 (20 mM), NahK-GlmU (1.2 mg), HpLgtA (0.6 mg), 37 C, 16 h, 89% for 9 (105 mg) in onepot; OPME 3, Gal (30 mg, 1.3 equiv), 9 (80 mg, 0.13 mM, 1.0 euiv), ATP (1.3 equiv), UTP (1.3 equiv), Tris-HCl buffer (100 mM, pH 7.5), MgCl2 (20 mM), EcGalK (2.5 mg), BLUSP (3.0 mg), NmLgtB (1.3 mg), 37 C, 12 h, 93% for 10 (94 mg) in one-pot; OPME 1, Gal (23 mg, 1.3 equiv), 10 (79 mg, 0.1 mM, 1.0 euiv), ATP (1.3 equiv), UTP (1.3 equiv), TrisHCl buffer (100 mM, pH 7.5), MgCl2 (20 mM), EcGalK (1.5 mg), BLUSP (2.0 mg), NmLgtC (2.0 mg), 37 C, 12 h, 55% for 4 (52 mg) in one-pot.
purification. With tetrasaccharide 10 in hand, the aforementioned one-pot three-enzyme a1e4-galactosylation (OPME 1) system for the synthesis of P1 trisaccharide 8 was adapted to finish the total synthesis of P1 pentasaccharide 4 (Scheme 2). Although the NmLgtCcatalyzed a1e4-galactosylation of tetrasaccharide 10 was much slower than using disaccharide 5 as acceptor, the tetrasaccharide 10 was still well tolerated by a1e4-galactosyltransferase NmLgtC to afford P1 pentasaccharide 4 (52 mg) in 55% yield. In summary, a highly efficient three-step sequential one-pot multienzyme glycosylation approach was developed for the enzymatic synthesis of human blood P1PK system P1 pentasaccharide antigen 4. The three OPME systems comprised four enzymes for the in situ generation of two UDP-sugar donors (two for UDP-GlcNAc and two for UDP-Gal, respectively), and three glycosyltransferases for the introducing three different glycosidic linkages, respectively. Taking advantage of the substrate promiscuity of all seven bacterial enzymes, the P1PK pentasaccharide antigen 4 was synthesized from readily available lactoside 5 along with cheap galactose and GlcNAc as donor precursors under operationally simple three steps with an overall yield of 46%. This biologically important pentasaccharide can be used as probes for bacterial infection detection and development of antigen-based multivalent inhibitors for neutralization of bacterial toxins.
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1. Experimental 1.1. General methods All chemicals were obtained from commercial suppliers and used without further purification unless noted. Thin layer chromatography (TLC) was performed on silica gel plates 60 F254 (Merck, Billerica MA). Plates were visualized under UV light and/or by treatment with 5% sulfuric acid in ethanol or p-anisaldehyde sugar stain followed by heating. Silica gel 60 (300e400 mesh, Haiyang, Qingdao, China) was used for flash column chromatography. Gel filtration chromatography was performed using a column (100 cm 2.5 cm) packed with Bio-Gel P-2 Fine resins (BioRad, Hercules, CA). 1H NMR (600 MHz) and 13C NMR (150 MHz) spectra were recorded on Bruker AVANCE-600 spectrometer at 25 C. NMR spectra were calibrated using solvent signals (1H: d 7.26 for CDCl3, d 3.34 for CD3OD or d 4.79 for D2O, 13C: d 77.0 for CDCl3). High resolution electrospray ionization (ESI) mass spectra were obtained at the National Glycoengineering Research Center and Drug Testing and Analysis Center in Shandong University. 1.2. Gala1e4Galb1e4GlcbProN3 (7) LactosebProN3 5 [29] (50 mg, 0.12 mmol), Gal (28 mg, 0.16 mmol), ATP (78 mg, 0.16 mmol) and UTP (74 mg, 0.16 mmol) were dissolved in water in a 50 mL centrifuge tube containing TrisHCl buffer (100 mmol, pH 7.5) and MgCl2 (20 mmol). After the addition of appropriate amount of EcGalK (2.2 mg), BLUSP (3.0 mg) and NmLgtC (2.2 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 6 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H2O/HOAc, 8:3:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by Bio-Gel P-2 column (eluted with H2O) to provide trisaccharide 7 (65 mg, 94%) as a white solid. [a]27 D ¼ þ61.2 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.96 (d, J ¼ 4.0 Hz, 1H, H-100 ), 4.51 (d, J ¼ 12.3 Hz, 1H, H-10 ), 4.50 (d, J ¼ 12.5 Hz, 1H, H-1), 4.37 (t, J ¼ 6.6 Hz, 1H, H-500 ), 4.05e3.57 (m, 18H), 3.47 (t, J ¼ 6.6 Hz, 2H, CH2N3), 3.32 (t, J ¼ 8.1 Hz, 1H, H-2), 1.92 (p, J ¼ 6.6 Hz, 2H, CH2CH2N3); 13C NMR (150 MHz, D2O) d 103.26 (C10 ), 102.09 (C-1), 100.32 (C-100 ), 78.67 (C-4), 77.37 (C-40 ), 75.42 (C30 ), 74.80 (C-50 ), 74.43 (C-3), 72.91 (C-2), 72.17 (C-5), 70.92 (C-20 ), 70.82 (C-500 ), 69.14 (C-300 ), 68.97 (C-400 ), 68.58 (C-200 ), 67.36 (OCH2CH2), 60.55 (C-600 ), 60.39 (C-6), 60.07 (C-60 ), 47.90 (CH2N3), 28.27 (CH2CH2N3); HRMS (ESI) m/z calcd for C21H37N3NaO16 [MþNa]þ 610.2072, found 610.2073. 1.3. Gala1e4Galb1e4GlcNAcbProN3 (8) LacNAcbProN3 6 5 (37 mg, 0.08 mmol), Galactose (Gal, 20 mg, 0.10 mmol), adenosine 5'-triphosphate (ATP, 56 mg, 0.11 mmol) and uridine 5'-triphosphate (UTP, 53 mg, 0.11 mmol) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mmol, pH 7.5) and MgCl2 (20 mmol). After the addition of appropriate amount of EcGalK (1.5 mg), BLUSP (2.0 mg) and NmLgtC (1.5 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 8 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H2O/HOAc, 8:3:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by Bio-Gel P-2 column (eluted
with H2O) to provide trisaccharide 8 (46 mg, 92%) as a white solid. [a]27 D ¼ þ47.3 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.95 (d, J ¼ 4.2 Hz, 1H, H-100 ), 4.53 (d, J ¼ 7.8 Hz, 2H, H-1 and H-10 ), 4.36 (t, J ¼ 6.3 Hz, 1H, H-500 ), 4.04 (d, J ¼ 6.0 Hz, 1H, H-40 ), 4.03 (d, J ¼ 6.0 Hz, 1H, H-300 ), 4.02e3.56 (m, 17H), 3.40e3.34 (m, 2H), 2.05 (s, 3H, NHCOCH3), 1.84 (p, J ¼ 6.3 Hz, 2H, CH2CH2N3); 13C NMR (150 MHz, D2O) d 174.38 (NHCOCH3), 103.24 (C-10 ), 101.05 (C-1), 100.26 (C-100 ), 78.75 (C-4), 77.26 (C-40 ), 75.40 (C-30 ), 74.77 (C-50 ), 72.39 (C-3), 72.12 (C-5), 70.88 (C-20 ), 70.77 (C-500 ), 69.11 (C-400 ), 68.90 (C-300 ), 68.52 (C200 ), 67.10 (OCH2CH2), 60.47 (C-600 ), 60.32 (C-60 ), 60.00 (C-6), 55.23 (C-2), 47.75 (CH2N3), 28.08 (CH2CH2N3), 22.14 (NHCOCH3); HRMS (ESI) m/z calcd for C23H40N4NaO16 [MþNa]þ651.2337, found 651.2341. 1.4. GlcNAcb1e3Galb1e4GlcbProN3 (9) LactosebProN3 5 (80 mg, 0.19 mmol), N-acetylglucosamine (GlcNAc, 54 mg, 0.20 mmol), ATP (124 mg, 0.25 mmol) and UTP (118 mg, 0.25 mmol) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mmol, pH 8.0) and MgCl2 (20 mmol). After the addition of appropriate amount of NahK-GlmU (1.2 mg) and HpLgtA (0.6 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 16 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/ MeOH/H2O/HOAc, 4:2:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by silica gel flash column chromatography (EtOAc/MeOH/H2O, 7:2:1,v/v) and Bio-Gel P-2 column (eluted with H2O) to provide trisaccharide 9 (105 mg, 89%) as a white solid. [a]27 D ¼ þ3.4 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.70 (d, J ¼ 8.4 Hz, 1H, H-100 ), 4.50 (d, J ¼ 7.8 Hz, 1H, H-1), 4.45 (d, J ¼ 7.8 Hz, 1H, H-10 ), 4.16 (d, J ¼ 3.0 Hz, 1H, H-40 ), 4.03e3.46 (m, 20H), 3.32 (t, J ¼ 8.4 Hz, 1H, H-2), 2.05 (s, 3H, NHCOCH3), 1.93 (p, J ¼ 6.6 Hz, 2H, CH2CH2N3); 13C NMR (151 MHz, D2O) d 174.95 (NHCOCH3), 102.92 (C-10 ), 102.84 (C-100 ), 102.10 (C-1), 81.96 (C-30 ), 78.34 (C-4), 75.64 (C-500 ), 74.88 (C-50 ), 74.76 (C-5), 74.35 (C-300 ), 73.55 (C-3), 72.79 (C-2), 70.00 (C-20 ), 69.68 (C-400 ), 68.33 (C-40 ), 67.36 (OCH2CH2), 60.96 (C-10 ), 60.47 (C-600 ), 60.06 (C6), 55.65 (C-200 ), 47.87 (CH2N3), 28.23 (CH2CH2N3), 22.19 (NHCOCH3); HRMS (ESI) m/zcalcd for C23H40N4NaO16 [MþNa]þ 651.2337, found 651.2346. 1.5. Galb1e4GlcNAcb1e3Galb1e4GlcbProN3 (10) Trisaccharide 9 (80 mg, 0.13 mmol), galactose (Gal, 30 mg, 0.17 mmol), ATP (84 mg, 0.17 mmol) and UTP (80 mg, 0.17 mmol) were dissolved in water in a 50 mL centrifuge tube containing TrisHCl buffer (100 mmol, pH 7.5) and MgCl2 (20 mmol). After the addition of appropriate amount of EcGalK (2.5 mg), BLUSP (3.0 mg) and NmLgtB (1.3 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 12 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H2O/HOAc, 4:2:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by silica gel flash column chromatography (EtOAc/MeOH/H2O, 6:2:1, v/v) and Bio-Gel P-2 column (eluted with H2O) to provide tetrasaccharide 10 (94 mg, 93%) as a white solid. [a]27 D ¼ þ1.5 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.73 (d, J ¼ 8.4 Hz, 1H, H-100 ), 4.50 (d, J ¼ 7.8 Hz, 1H, H-1), 4.49 (d, J ¼ 7.8 Hz, 1H, H-1000 ), 4.46 (d, J ¼ 8.4 Hz, 1H, H-10 ), 4.17 (d, J ¼ 3.6 Hz, 1H, H-40 ), 4.03e3.96 (m, 3H), 3.94 (d, J ¼ 3.0 Hz,
D. Tsering et al. / Carbohydrate Research 438 (2017) 39e43
1H, H-4000 ), 3.87 (dd, J ¼ 4.8, 12.6 Hz, 1H, H-6b), 3.84e3.54 (m, 19H), 3.48 (t, J ¼ 6.6 Hz, 2H, CH2N3), 3.33 (t, J ¼ 8.4 Hz, 1H, H-2), 2.05 (s, 3H, NHCOCH3), 1.93 (p, J ¼ 6.6 Hz, 2H, CH2CH2N3); 13C NMR (151 MHz, D2O) d 174.89 (NCOCH3), 102.92 (C-10 ), 102.84 (C-1000 ), 102.73 (C-100 ), 102.09 (C-1), 82.05 (C-30 ), 78.35 (C-4), 78.14 (C-400 ), 75.33 (C-5000 ), 74.87 (C-50 ), 74.75 (C-5), 74.53 (C-500 ), 74.34 (C-3), 72.79 (C-2), 72.49 (C-3000 ), 72.16 (C-300 ), 70.96 (C-2000 ), 69.95 (C-20 ), 68.55 (C-4000 ), 68.31 (C-40 ), 67.36 (OCH2CH2), 61.04 (C-6000 ), 60.96 (C60 ), 60.06 (C-600 ), 59.85 (C-6), 55.18 (C-200 ), 47.87 (CH2N3), 28.24 (CH2CH2N3), 22.22 (NHCOCH3); HRMS (ESI) m/zcalcd for C29H50N4NaO21 [MþNa]þ 813.2865, found 813.2886.
[6] [7] [8]
[9]
[10]
1.6. Gala1e4Galb1e4GlcNAcb1e3Galb1e4GlcbProN3 (4) Tetrasaccharide 10 (79 mg, 0.10 mmol), Gal (23 mg, 0.13 mmol), ATP (66 mg, 0.13 mmol) and UTP (63 mg, 0.13 mmol) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mmol, pH 7.5) and MgCl2 (20 mmol). After the addition of appropriate amount of EcGalK (1.5 mg), BLUSP (2.0 mg) and NmLgtC (2.0 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction mixture was incubated in a shaking incubator at 37 C for 12 h with agitation at 140 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H2O/HOAc, 4:2:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by Bio-Gel P-2 column (eluted with H2O) to provide pentasaccharide 4 (52 mg, 55%) as a white solid. [a]27 D ¼ þ28.7 (c 1.0, H2O); 1H NMR (600 MHz, D2O) d 4.96 (d, J ¼ 4.2 Hz, 1H, H-10000 ), 4.72 (d, J ¼ 8.4 Hz, 1H, H-100 ), 4.55 (d, J ¼ 7.8 Hz, 1H, H-1000 ), 4.50 (d, J ¼ 7.8 Hz, 1H, H-1), 4.45 (d, J ¼ 7.8 Hz, 1H, H-10 ), 4.37 (t, J ¼ 6.6 Hz, 1H, H-5000 '), 4.17 (d, J ¼ 3.0 Hz, 1H, H-40 ), 4.03e3.57 (m, 29H), 3.47 (t, J ¼ 6.9 Hz, 2H, CH2N3), 3.32 (t, J ¼ 8.7 Hz, 1H, H-2), 2.05 (s, 3H, NHCOCH3), 1.92 (p, J ¼ 6.6 Hz, 2H, CH2CH2N3); 13C NMR (150 MHz, D2O) d 174.91 (NCOCH3), 103.21 (C10 ), 102.92 (C-1000 ), 102.70 (C-100 ), 102.10 (C-1), 100.27 (C-10000 ), 82.03 (C-30 ), 78.43 (C-4), 78.34 (C-400 ), 77.30 (C-4000 ), 75.41, 74.87, 74.76, 74.57, 74.34, 72.78, 72.19, 72.13, 70.89, 70.79, 69.96, 69.12, 68.92, 68.53, 68.32 (C-40 ), 67.36 (OCH2CH2), 60.94 (C-60 ), 60.49 (C-60000 ), 60.36 (C-6000 ), 60.04 (C-600 ), 59.81 (C-6), 55.33 (C-200 ), 47.86 (CH2N3), 28.22 (CH2CH2N3), 22.18 (NHCOCH3); HRMS (ESI) m/z calcd for C35H60N4NaO26 [MþNa]þ 975.3393, found 975.3416.
[11] [12]
[13] [14]
[15] [16]
[17]
[18] [19] [20] [21]
Acknowledgments [22]
This work was financially supported by National Natural Science Foundation of China (Grant Nos. 21372145, 21672128), State Key Laboratory of Microbial Technology (M2016-06) and Department of Science and Technology of Shandong Province (2016GSF121002).
[23] [24]
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.carres.2016.11.019. [25]
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