Tailored Multivalent Neo-Glycoproteins: Synthesis, Evaluation, and

Oct 4, 2017 - Tailored Multivalent Neo-Glycoproteins: Synthesis, Evaluation, and Application of a Library of Galectin-3-Binding Glycan Ligands. Domini...
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Tailored Multivalent Neo-glycoproteins: Synthesis, Evaluation and Application of a Library of Galectin-3-binding Glycan Ligands Dominic Laaf, Pavla Bojarova, Helena Pelantova, Vladimir Kren, and Lothar Elling Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00520 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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Bioconjugate Chemistry

Tailored Multivalent Neo-glycoproteins: Synthesis, Evaluation and Application of a Library of Galectin-3-binding Glycan Ligands Dominic Laaf,† Pavla Bojarová,‡ Helena Pelantová,‡ Vladimír Křen,*,‡ and Lothar Elling*,† †

Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstrasse 20, 52074 Aachen, Germany, email: [email protected], phone: +49 241 80 28350 ‡

Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 14220 Prague, Czech Republic, email: [email protected], phone: +420 296 442 510

ABSTRACT: Galectin-3 (Gal-3), a member of the β-galactoside binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc, LacNAc type 2) based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N’,N’’-diacetyllactosamine (GalNAcβ1,4GlcNAc, LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich ELISA format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer related biomedical research.

The glycans displayed on cellular surfaces encode physiologically relevant information, which is deciphered by lectins. The glycan-ligand specificity and affinity are determined by the sequence, configuration and conformation of monosaccharide building blocks. Multivalent glycan presentation strongly enhances lectin binding strength and essentially influences processes such as cell adhesion, cell-cell communication and signaling events.1-7 Galectin-3 (Gal-3) is one of the most extensively studied β-galactoside binding lectins as it is involved in tumor progression and metastasis.8-11 Due to its systemic appearance in blood serum, Gal-3 has attracted special attention and is considered as a cancer biomarker.12-15 Diagnosing high-risk patients by means of circulating Gal-3 in blood serum is therefore an attractive goal in human medicine. Strategies for the synthesis of potent and specific Gal-3 glycan ligands have been followed in order to inhibit tumor angiogenesis, migration and invasion. Binding of Gal-3 was triggered by chemical derivatization of known ligands such as galactose, lactose and N-acetyllactosamine (Galβ1,4GlcNAc, LacNAc)16-23 or using glycans isolated from natural sources, e.g. modified citrus pectin.24 The N’,N’’-diacetyllactosamine (LacdiNAc, GalNAcβ1,4GlcNAc) motif was recently confirmed as a highly

specific glycan epitope for binding Gal-3.25 Here, we present the synthesis and evaluation of bovine serum albumin (BSA) based multivalent neo-glycoproteins loaded with a variety of glycan species. The enzymatic synthesis of the corresponding monovalent oligosaccharides carrying a tBoc-protected linker was accomplished by cascade reactions of Leloir-glycosyltransferases and chemoenzymatic coupling, outlined in Scheme 1. We obtained (poly-)LacNAc oligosaccharides of increasing length, which carried five different terminal glycan epitopes. Squarate linker chemistry enabled a tunable and defined conjugation of the functionalized oligosaccharides to BSA used as a scaffold for multivalent glycan presentation (Scheme 2). Evaluation in galectin binding assays revealed new multivalent neo-glycoproteins as specific ligands and potent inhibitors for human Gal-3. Owing to its outstandingly high affinity to the LacdiNAc presenting BSA neoglycoconjugate, Gal-3 could be trapped from human blood serum. Furthermore, we validated a neoglycoprotein sandwich ELISA for an antibody-free Gal-3 detection. Our findings suggest that the tailored neoglycoproteins prepared in this study are promising glycotools for further investigations in terms of imaging and targeting of Gal-3.

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Scheme 1. (Chemo-)enzymatic synthesis of functionalized poly-LacNAc derivatives.

RESULTS AND DISCUSSION Preparation, isolation and characterization of functionalized oligosaccharides. Linker-modified LacNAc type 2 oligosaccharides composed of up to eight sugar units (3a-c, 4a-c) were obtained in a one-pot synthesis with human β4-galactosyltransferase (β4GalT) and H. pylori β3-N-acetylglucosaminyltransferase (β3GlcNAcT) (Scheme 1) as published previously.26 Isolation of pure compounds was achieved by hydrophilic interaction chromatography (HILIC) with an average overall yield of 95 %. The diversity of oligosaccharide ligands was expanded by specific glycosylation with three additional glycosyltransferases or by chemo-enzymatic biotinylation. GlcNAc-terminated LacNAc oligomers 3a-c were either elongated by enzyme variant of β4GalT (β4GalTY284L)27 or E. coli β3galactosyltransferase (WbgO, β3GalT),28, 29 generating either LacdiNAc (5a-c) or LacNAc type 1 (6a-c) as the terminal glycosylation epitope, respectively (Scheme 1 A, B). Moreover, galactose oxidase treatment30, 31 of Galterminated glycans 4a-c followed by reductive amination afforded biotinylated products 7a-c (Scheme 1 C). Alternatively, murine α3-galactosyltransferase (α3GalT)32, 33 was employed to synthesize the Galili epitope (Galα3Galβ4GlcNAc, 8a-c, Scheme 1 D). After solid-phase extraction or preparative reversed-phase HPLC, products 7a-c were isolated in overall yields of 74-98 % (Table S3). Integrity, purity and structure of isolated products were confirmed by HPLC-ESI-MS and NMR. Enzyme characterization. Enzyme activities of β4GalTY284L, β3GalT and α3GalT were determined with various LacNAc oligosaccharide acceptors (Table S2, Figure S3). Acceptors of moderate length (3a, 4a) were generally favored by β4GalTY284L, β3GalT and α3GalT. Reduced yields in the case of products 6b (31 %) and 6c

(42 %) were due to the low activity of β3GalT with the more extended LacNAc oligosaccharides 3b and 3c and their incomplete conversion. Thus, we concluded that 3a and 4a offer ideal distances between the linker-Boc protecting group and the non-reducing end for an optimal enzymatic performance. Neo-glycoprotein synthesis and analysis. The homobifunctional diethyl squarate (3,4-diethoxy-3cyclobutene-1,2-dione) is a well suited agent to generate multivalent neo-glycoproteins from non-glycosylated protein scaffolds.34, 35 While the direct conjugation of glycans and proteins by reductive amination results in sugar ring opening at the reducing end,36 application of diethyl squarate enables the conjugation of intact glycans. According to our previous studies,25, 37 two sequential amidation reactions, tunable by pH control, were required to generate neo-glycoproteins with a varying glycan density (Scheme 2). According to this procedure, the deprotected primary amines of compounds 4b-8b were conjugated with diethyl squarate under neutral conditions (pH 7.0). The resulting squarate monoamide esters 9-13 were isolated by preparative HPLC in high yields (83-92 %). The reduced yield of 46 % in the case of product 12 was caused by the formation of a diamide side product (Table S4). Integrity of isolated products was confirmed by HPLC-ESI-MS (Supporting Information). In the second amidation reaction, the available lysine residues of BSA reacted with compounds 9-13 under slightly alkaline conditions (pH 9.0), outlined in Scheme 2. The ratio of reactants (compounds 9-13 to lysine residues of BSA) was adjusted to 0.375. Since the remaining ethyl ester reactive site is hydrolyzed under reaction conditions with a half-life of four days (Figure S5), we chose a batchwise addition of squarate monoamide esters 9-13, which improved the reaction yield by 20 % compared to our previous reports.25

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OH OH

OH OH

OH O

R OH

OH O

O

O HO

O OH

NH

O

O HO

H N

NH

O

O

H N

N H

S

O

O

OHOH O R=

HO

OH

4b-8b

H

OH

S

O

O HO

O

NH H N H

NH O O

4b, 9 (83%), 14 (n=17) (77%)

HN i, ii

O

HN OH NH

OH O

R=

HO OH

OH OH

OH OH

OH O

R OH

O

O HO

O OH

NH

O

OH O

O

O

O HO

H N

NH

O

H N

HO

O

N H

OH O

R=

S

O

HO OH

O

NH O

O NH O

7b, 12 (46%), 17 (n=19) (72%)

O

O HO

O

O HO

O NH O

5b, 10 (85%), 15 (n=16) (81%) 9-13 OH OH O R=

iii

HO OH O

OH

OH OH O OH OH

OH OH

OH O

R OH

O

O

O HO

O NH O

O

OH

OH

O

O HO

NH O

H N

H N S

R=

O BSA

N H

N H

HO

HO O

OH

OH OH

OH O OH

O O NH

O

O HO

O NH O

8b, 13 (90%), 18 (n=18) (83%)

O

6b, 11 (92%), 16 (n=17) (76%)

n=16-19 14-18

Scheme 2. Preparation of neo-glycoproteins 14-18. Reagents and conditions: i) 1 M HCl, 4 °C, 48 h, Dowex® 66 free base; ii) 50:50 (EtOH:H2O), 30 mM HEPES (pH 7.0), Et3N, diethyl squarate, r.t., 18 h, preparative reversed-phase HPLC; iii) BSA (1.16 mg, 60 µM), 50 mM Na2B4O7, r.t., 7 d, VivaSpin®500.

The average proportion of reacted squarate monoamide esters (coupling efficiency) was 78 %. The number of modified lysine residues was quantified by trinitrobenzene sulfonic acid (TNBSA) assay25 (Table S5) and the products were evaluated by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE, Figure 1). A shift towards higher molecular masses (Figure 1) indicated a successful glycan attachment; a slight smearing of bands suggested that the hydrophilic glycan chains prevented SDS from binding to the protein core as published.25, 37 In summary, five neo-glycoproteins (14-18) were obtained, each presenting between 16 and 19 epitope-modified LacNAc oligomers (Scheme 2).

Evaluation of neo-glycoproteins as galectin ligands. Human galectins were expressed in E. coli Rosetta (DE3) cells and purified by immobilized metal-ion affinity chromatography as described elsewhere.25, 37, 38 Immobilized neo-glycoproteins 14-18 were used as galectin ligands in ELISA-type assays with galectin-1 (Gal-1), Gal-3 and N-terminally truncated Gal-3 lacking the N-terminal amino acid residues 1-62 (Gal-3Δ), respectively (Figure S6S10). Non-modified BSA served as a negative control that did not interact with galectins. Binding efficiencies [µM-1] of galectins were calculated as the ratio of the maximal binding signal (Bmax) and the galectin concentration for half-maximal binding (apparent Kd value) (Table 1, Table S6 in Supporting Information). Strong binding of Gal-1 was found when LacNAc terminated and Galili epitope presenting neo-glycoproteins 14 and 18 were used respectively. The latter confirmed previous reports on specific binding of Gal-1 to α3-linked digalactosides.39 However, in comparison with Gal-3 and Gal-3Δ, Gal-1 binding to neo-glycoproteins 14-18 was significantly (p4GlcNAc sequence with in situ UDP-Gal regeneration, Glycoconj. J. 13, 687-692. 34. Kamath, V. P., Diedrich, P., and Hindsgaul, O. (1996) Use of diethyl squarate for the coupling of oligosaccharide amines to carrier proteins and characterization of the resulting neoglycoproteins by MALDI-TOF mass spectrometry, Glycoconj. J. 13, 315-319. 35. Tietze, L. F., Schroter, C., Gabius, S., Brinck, U., Goerlach-Graw, A., and Gabius, H. J. (1991) Conjugation of p-

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aminophenyl glycosides with squaric acid diester to a carrier protein and the use of neoglycoprotein in the histochemical detection of lectins, Bioconjugate Chem. 2, 148-153. 36. Gildersleeve, J. C., Oyelaran, O., Simpson, J. T., and Allred, B. (2008) Improved Procedure for Direct Coupling of Carbohydrates to Proteins via Reductive Amination, Bioconjugate Chem. 19, 1485-1490. 37. Laaf, D., Bojarová, P., Mikulová, B., Pelantová, H., Křen, V., and Elling, L. (2017) Two-Step Enzymatic Synthesis of β-D-N-Acetylgalactosamine-(1→4)-D-N-Acetylglucosamine (LacdiNAc) Chitooligomers for Deciphering Galectin Binding Behavior, Adv. Synth. Catal. 359, 2101-2108. 38. Kupper, C. E., Böcker, S., Liu, H., Adamzyk, C., van de Kamp, J., Recker, T., Lethaus, B., Jahnen-Dechent, W., Neuss, S., Müller-Newen, G., and Elling, L. (2013) Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology, Curr. Pharm. Des. 19, 5457-5467. 39. Miller, M. C., Ribeiro, J. P., Roldós, V., MartínSantamaría, S., Cañada, F. J., Nesmelova, I. A., André, S., Pang, M., Klyosov, A. A., Baum, L. G. et al. (2011) Structural aspects of binding of α-linked digalactosides to human galectin-1, Glycobiology 21, 1627-1641. 40. Song, X., Xia, B., Stowell, S. R., Lasanajak, Y., Smith, D. F., and Cummings, R. D. (2009) Novel Fluorescent Glycan Microarray Strategy Reveals Ligands for Galectins, Chem. Biol. 16, 36-47. 41. Cheng, D., Liang, B., and Li, Y. (2015) Serum Galectin-3 as a Potential Marker for Gastric Cancer, Med. Sci. Monit. 21, 755760. 42. Collins, P. M., Bum-Erdene, K., Yu, X., and Blanchard, H. (2014) Galectin-3 Interactions with Glycosphingolipids, J. Mol. Biol. 426, 1439-1451. 43. López-Lucendo, M. F., Solís, D., André, S., Hirabayashi, J., Kasai, K.-I., Kaltner, H., Gabius, H.-J., and Romero, A. (2004) Growth-regulatory Human Galectin-1: Crystallographic Characterisation of the Structural Changes Induced by Singlesite Mutations and their Impact on the Thermodynamics of Ligand Binding, J. Mol. Biol. 343, 957-970. 44. Ahmad, N., Gabius, H.-J., André, S., Kaltner, H., Sabesan, S., Roy, R., Liu, B., Macaluso, F., and Brewer, C. F. (2004) Galectin-3 Precipitates as a Pentamer with Synthetic Multivalent Carbohydrates and Forms Heterogeneous Crosslinked Complexes, J. Biol. Chem. 279, 10841-10847. 45. Lepur, A., Salomonsson, E., Nilsson, U. J., and Leffler, H. (2012) Ligand Induced Galectin-3 Protein Self-association, J. Biol. Chem. 287, 21751-21756. 46. Andrade, J. D., and Hlady, V. (1987) Plasma Protein Adsorption: The Big Twelvea, Ann. N. Y. Acad. Sci. 516, 158-172. 47. Cederfur, C., Salomonsson, E., Nilsson, J., Halim, A., Öberg, C. T., Larson, G., Nilsson, U. J., and Leffler, H. (2008) Different affinity of galectins for human serum glycoproteins: Galectin-3 binds many protease inhibitors and acute phase proteins, Glycobiology 18, 384-394. 48. Iurisci, I. D. A., Cumashi, A., Sherman, A. A., Tsvetkov, Y. E., Tinari, N., Piccolo, E., D'Egidio, M., Adamo, V., Natoli, C., Rabinovich, G. A. et al. (2009) Synthetic Inhibitors of Galectin-1 and -3 Selectively Modulate Homotypic Cell Aggregation and Tumor Cell Apoptosis, Anticancer Res. 29, 403-410. 49. Rabinovich, G. A., Cumashi, A., Bianco, G. A., Ciavardelli, D., Iurisci, I., D’Egidio, M., Piccolo, E., Tinari, N., Nifantiev, N., and Iacobelli, S. (2006) Synthetic lactulose amines: novel class of anticancer agents that induce tumor-cell apoptosis

and inhibit galectin-mediated homotypic cell aggregation and endothelial cell morphogenesis, Glycobiology 16, 210-220. 50. Christenson, R. H., Duh, S.-H., Wu, A. H. B., Smith, A., Abel, G., deFilippi, C. R., Wang, S., Adourian, A., Adiletto, C., and Gardiner, P. (2010) Multi-center determination of galectin-3 assay performance characteristics:: Anatomy of a novel assay for use in heart failure, Clin. Biochem. 43, 683-690. 51. Gopal, D. M., Kommineni, M., Ayalon, N., Koelbl, C., Ayalon, R., Biolo, A., Dember, L. M., Downing, J., Siwik, D. A., Liang, C.-S. et al. (2012) Relationship of Plasma Galectin-3 to Renal Function in Patients With Heart Failure: Effects of Clinical Status, Pathophysiology of Heart Failure, and Presence or Absence of Heart Failure, J. Am. Heart Assoc. 1, e000760. 52. Tang, W. H. W., Shrestha, K., Shao, Z., Borowski, A. G., Troughton, R. W., Thomas, J. D., and Klein, A. L. (2011) Usefulness of Plasma Galectin-3 Levels in Systolic Heart Failure to Predict Renal Insufficiency and Survival, Am. J. Cardiol. 108, 385-390. 53. Nayor, M., Wang, N., Larson, M. G., Vasan, R. S., Levy, D., and Ho, J. E. (2015) Circulating Galectin-3 Is Associated With Cardiometabolic Disease in the Community, J. Am. Heart Assoc. 5. 54. Saussez, S., Lorfevre, F., Lequeux, T., Laurent, G., Chantrain, G., Vertongen, F., Toubeau, G., Decaestecker, C., and Kiss, R. (2008) The determination of the levels of circulating galectin-1 and -3 in HNSCC patients could be used to monitor tumor progression and/or responses to therapy, Oral Oncol. 44, 86-93. 55. Pieters, R. J. (2009) Maximising multivalency effects in protein-carbohydrate interactions, Org. Biomol. Chem. 7, 20132025. 56. Bonduelle, C., Oliveira, H., Gauche, C., Huang, J., Heise, A., and Lecommandoux, S. (2016) Multivalent effect of glycopolypeptide based nanoparticles for galectin binding, Chem. Commun. 52, 11251-11254. 57. André, S., Sansone, F., Kaltner, H., Casnati, A., Kopitz, J., Gabius, H.-J., and Ungaro, R. (2008) Calix[n]arene-Based Glycoclusters: Bioactivity of Thiourea-Linked Galactose/Lactose Moieties as Inhibitors of Binding of Medically Relevant Lectins to a Glycoprotein and Cell-Surface Glycoconjugates and Selectivity among Human Adhesion/Growth-Regulatory Galectins, ChemBioChem 9, 1649-1661. 58. Bernatchez, S., Szymanski, C. M., Ishiyama, N., Li, J., Jarrell, H. C., Lau, P. C., Berghuis, A. M., Young, N. M., and Wakarchuk, W. W. (2005) A Single Bifunctional UDPGlcNAc/Glc 4-Epimerase Supports the Synthesis of three Cell Surface Glycoconjugates in Campylobacter jejuni, J. Biol. Chem. 280, 4792-4802. 59. Logan, S. M., Altman, E., Mykytczuk, O., Brisson, J. R., Chandan, V., Schur, M. J., St Michael, F., Masson, A., Leclerc, S., Hiratsuka, K. et al. (2005) Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori, Glycobiology 15, 721-733. 60. Sauerzapfe, B., Namdjou, D. J., Schumacher, T., Linden, N., Křenek, K., Křen, V., and Elling, L. (2008) Characterization of recombinant fusion constructs of human β1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus, J. Mol. Catal. B: Enzym. 50, 128-140.

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Scheme 1. (Chemo-)enzymatic synthesis of functionalized poly-LacNAc derivatives. 382x201mm (300 x 300 DPI)

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Scheme 2. Preparation of neo-glycoproteins 14-18.   Reagents and conditions: i) 1 M HCl, 4 °C, 48 h, Dowex® 66 free base; ii) 50:50 (EtOH:H2O), 30 mM HEPES (pH 7.0), Et3N, diethyl squarate, r.t., 18 h, preparative reversed-phase HPLC; iii) BSA (1.16 mg, 60 µM), 50 mM Na2B4O7, r.t., 7 d, VivaSpin®500. 321x171mm (300 x 300 DPI)

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Figure 1. SDS-PAGE analysis of BSA neo-glycoproteins 14-18, non-modified BSA (C), and PageRuler Prestained Protein Ladder as a molecular weight standard (M). 141x135mm (300 x 300 DPI)

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Figure 2. BSA neo-glycoprotein 15 as specific agent for trap-ping Gal-3 from human blood serum using antiHis6 specific (A) or anti-Gal-3 specific detection (B). B, Gal-3 concentra-tion=60 nM, n=6, ** corresponds to a significant difference with a confidence interval of p