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1.
Angew Chem Int Ed Engl ; 60(49): 25922-25932, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34523784

RESUMO

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life. EPO carries three N-glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline-assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor.


Assuntos
Eritropoetina/metabolismo , Ácido N-Acetilneuramínico/biossíntese , Ácido N-Acetilneuramínico/síntese química , Sialiltransferases/metabolismo , Eritropoetina/química , Glicosilação , Humanos , Estrutura Molecular , Ácido N-Acetilneuramínico/química , Photobacterium/enzimologia , beta-D-Galactosídeo alfa 2-6-Sialiltransferase
2.
Angew Chem Int Ed Engl ; 60(24): 13380-13387, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33756033

RESUMO

A library of glycoforms of human interleukin 6 (IL-6) comprising complex and mannosidic N-glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two-step refolding. The central glycopeptide segments were assembled by pseudoproline-assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N-glycans. Nine IL-6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non-glycosylated recombinant IL-6 from E. coli. Each IL-6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N-glycan. The clearance rates were atypical, since the 2,6-sialylated glycoforms of IL-6 cleared faster than the corresponding asialo IL-6 with terminal galactoses. Compared to non-glycosylated IL-6 the plasma clearance of IL-6 glycoforms was delayed in the presence of larger and multibranched N-glycans in most cases.


Assuntos
Glicopeptídeos/metabolismo , Interleucina-6/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Galactose/metabolismo , Glicopeptídeos/sangue , Glicopeptídeos/genética , Glicosilação , Humanos , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-6/farmacologia , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Espectrometria de Massas por Ionização por Electrospray
3.
Angew Chem Int Ed Engl ; 57(44): 14543-14549, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30144245

RESUMO

The occurrence of α1,6-linked core fucose on the N-glycans of mammalian glycoproteins is involved in tumor progression and reduces the bioactivity of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC). Since core-fucosylated N-glycans are difficult to isolate from natural sources, only chemical or enzymatic synthesis can provide the desired compounds for biological studies. A general drawback of chemical α-fucosylation is that the chemical assembly of α1,6-linked fucosides is not stereospecific. A robust and general method for the α-selective fucosylation of acceptors with primary hydroxy groups in α/ß ratios exceeding 99:1 was developed. The high selectivities result from the interplay of an optimized protecting group pattern of the fucosyl donors in combination with the activation principle and the reaction conditions. Selective deprotection yielded versatile azides of all mammalian complex-type core-fucosylated N-glycans with 2-4 antennae and optional bisecting GlcNAc.


Assuntos
Acetilglucosamina/química , Fucose/química , Polissacarídeos/química , Animais
4.
Angew Chem Int Ed Engl ; 57(32): 10178-10181, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29956878

RESUMO

The mini fungal lectin PhoSL was recombinantly produced and characterized. Despite a length of only 40 amino acids, PhoSL exclusively recognizes N-glycans with α1,6-linked fucose. Core fucosylation influences the intrinsic properties and bioactivities of mammalian N-glycoproteins and its level is linked to various cancers. Thus, PhoSL serves as a promising tool for glycoprofiling. Without structural precedence, the crystal structure was solved using the zinc anomalous signal, and revealed an interlaced trimer creating a novel protein fold termed ß-prism III. Three biantennary core-fucosylated N-glycan azides of 8 to 12 sugars were cocrystallized with PhoSL. The resulting highly resolved structures gave a detailed view on how the exclusive recognition of α1,6-fucosylated N-glycans by such a small protein occurs. This work also provided a protein consensus motif for the observed specificity as well as a glimpse into N-glycan flexibility upon binding.


Assuntos
Fucose/síntese química , Lectinas/química , Polissacarídeos/química , Configuração de Carboidratos , Sequência de Carboidratos , Fucose/análogos & derivados , Fucose/química , Modelos Moleculares
5.
Angew Chem Int Ed Engl ; 56(47): 14987-14991, 2017 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-28991403

RESUMO

The biological recognition of complex-type N-glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N-glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra-antennary N-glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N-acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N-glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N-acetyllactosamine-binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).

6.
Angew Chem Int Ed Engl ; 56(19): 5252-5257, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28378443

RESUMO

The main glycoforms of the hydrophobic lysosomal glycoprotein saposin D (SapD) were synthesized by native chemical ligation. An approach for the challenging solid-phase synthesis of the fragments was developed. Three SapD glycoforms were obtained following a general and robust refolding and purification protocol. A crystal structure of one glycoform confirmed its native structure and disulfide pattern. Functional assays revealed that the lipid-binding properties of three SapD glycoforms are highly affected by the single sugar moiety of SapD showing a dependency of the size and the type of N-glycan.


Assuntos
Carboidratos/química , Saposinas/síntese química , Saposinas/metabolismo , Configuração de Carboidratos , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Saposinas/química
7.
ACS Chem Biol ; 12(4): 1104-1112, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28192664

RESUMO

Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate-protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore their importance for the development of biomedical applications.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Oligossacarídeos/química , Polissacarídeos/química , Sítios de Ligação , Calorimetria , Configuração de Carboidratos , Difração de Raios X
8.
Angew Chem Int Ed Engl ; 55(35): 10487-92, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27443163

RESUMO

The occurrence of N-glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N-glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N-glycans of the complex type by employing modular TFAc-protected donors for all antennae. The TFAc-protected N-glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N-glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one-pot double glycosylation. With this new method, rare N-glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.

9.
Chem Commun (Camb) ; 47(37): 10485-7, 2011 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-21850361

RESUMO

When using benzyl ethers as permanent protecting groups in oligosaccharide synthesis selective oxidative debenzylation with NaBrO(3) + Na(2)S(2)O(4) under biphasic conditions is efficient and compatible with anomeric azides and many other functions.


Assuntos
Azidas/química , Benzeno/química , Monossacarídeos/química , Oligossacarídeos/química , Ácidos de Lewis/química , Oxirredução , Especificidade por Substrato
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