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1.
PLoS One ; 10(7): e0132848, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26200113

RESUMEN

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-ß-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Polisacáridos/química , Trastuzumab/metabolismo , Acetilglucosaminidasa/metabolismo , Anticuerpos Monoclonales/química , Citotoxicidad Celular Dependiente de Anticuerpos , Glicosilación , Humanos , Trastuzumab/química
2.
Carbohydr Res ; 407: 122-30, 2015 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-25753904

RESUMEN

Toward applications in synthetic carbohydrate chemistry, we converted our previous acid-resistant heavy fluorous tag [(Rf)3C-CH2-OH, 1] to allyl alcohol derivatives [(Rf)3C-CH2-O-(CH2)n-CH=CH-CH2-OH, 3 (n=1) or 4 (n=3)] by means of olefin cross metathesis. They were then subjected to ß-glycosylation reactions by using a series of glycosyl donors, including glycosyl bromide and trichloroacetimidates. The terminal OH group in 3 and 4 was found to be ß-glycosylated in moderate yield when 2,3,4,6-tetra-O-benzoyl-D-galactosyl trichloroacetimidate was used as the glycosyl donor. Upon a detachment reaction using Pd(PPh3)4, the initial heavy fluorous tag 1 was recovered in high yield (>90%) together with 1-hydroxy sugar, indicating that not only the allyl ether linkage in the glycosides but also the internal di-alkyl ether linkage in 4 be cleaved by the action of the Pd-catalyst enabling long-range olefin transmigration. Potential utility was demonstrated by using the tetra-O-benzoyl-ß-D-galactosylated derivative of 3 in a series of deprotection, protection and glycosylation reactions, which were conductible in high yields without using chromatographic purification process. These findings prompt us to propose a general scheme in which the acid-resistant heavy fluorous compound 1 is applied as a recyclable tag in synthetic carbohydrate chemistry.


Asunto(s)
Glicósidos/síntesis química , Propanoles/síntesis química , Conformación de Carbohidratos , Técnicas de Química Sintética/métodos , Glicósidos/química , Glicosilación
3.
Bioorg Med Chem Lett ; 22(2): 1251-4, 2012 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-22177082

RESUMEN

The lactoside with PEG-fluorous tag was introduced to BHK-21(C-13) cells to generate a GM3-type oligosaccharide (Siaα2-3Galß1-4Glc). The GM3-type oligosaccharide obtained was easily immobilized by spotting onto commercially available polytetrafluoroethylene (PTFE) filter through non-covalent fluorous affinity and simply assessed by dot blot method using the interaction of carbohydrate- with proteins which recognize sialic acid such as virus membrane proteins.


Asunto(s)
Virus de la Influenza A/química , Oligosacáridos/química , Politetrafluoroetileno/química , Animales , Línea Celular , Cricetinae , Membranas Artificiales , Polietilenglicoles/química
4.
Org Biomol Chem ; 9(10): 3780-6, 2011 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-21483978

RESUMEN

Tin hydride mediated radical carbonylation and cyclization reaction was investigated using a variety of ω-alkynyl amines as substrates. In this reaction α-methylene and α-stannylmethylene lactams having five to eight membered rings were obtained as principal products. In cases where the nitrogen has a substituent capable of giving stable radicals, such as an α-phenethyl group, the lactam ring formation again took place with extrusion of an α-phenethyl radical. Coupled with the subsequent protodestannylation procedure (TMSCl plus MeOH), these reactions provide a useful entry to α-methylene lactams with incorporation of CO as a lactam carbonyl group. In cases where the amines do not have a substituent acting as a radical leaving group, a reaction course involving a 1,4-H shift is chosen so as to liberate tin radicals ultimately. Thus the proposed mechanism involves (i) nucleophilic attack of amine nitrogen onto a carbonyl group of α,ß-unsaturated acyl radicals/α-ketenyl radicals via lone pair-π* interaction, which leads to zwitterionic radical species, (ii) the subsequent proton shift from N to O to give hydroxyallyl radicals, (iii) 1,4-hydrogen shift from O to C, and (iv) ß-scission to give lactams with liberation of tin radicals. DFT calculations reveal that the 1,4-hydrogen shifts, the key step of the reaction mechanism, can proceed under usual reaction conditions. On the other hand, an S(H)i type reaction to give lactams may be the result of the ß-scission of the similar zwitterionic radical intermediates. DFT calculations also predict that an S(H)i type reaction would result when the intermediate has a good (radical) leaving group such as a phenethyl group.

5.
J Am Chem Soc ; 128(24): 7712-3, 2006 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-16771465

RESUMEN

Acyl radicals generated by the addition of alkyl or vinyl radicals to carbon monoxide cyclized onto the C-N bonds of imines and oxazoline with perfect 6-endo selectivity, driven by a preference for attack at nitrogen.

6.
Chem Commun (Camb) ; (21): 2482-3, 2004 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-15514826

RESUMEN

Free-radical carbonylation of omega-alkynylamines with tributyltin hydride gives a mixture of alpha-methylene lactams and alpha-stannylmethylene lactams. Nucleophilic addition of an internal amino group to the carbonyl group of alpha-ketenyl radicals is proposed as the cyclization step. The subsequent unusual 1,4-H shift from the resulting 1-hydroxyallyl radical, followed by elimination of the beta-tributyltin radical leads to the formation of alpha-methylene lactams.


Asunto(s)
Aminas/síntesis química , Etilenos/química , Cetonas/química , Ciclización , Radicales Libres/química , Estructura Molecular , Compuestos de Trialquiltina/química
8.
Org Biomol Chem ; 1(23): 4262-7, 2003 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-14685329

RESUMEN

Free-radical mediated cyclizative carbonylations of azaenynes were carried out using TTMSS as a radical mediator to compare the efficiency and the stereochemistry with those using tributyltin hydride. Using a substrate concentration of 0.1 M, the reactions gave good yields of alpha-silylmethylene lactams having four to seven-membered rings. The observed E-diastereoselectivity of the resulting vinylsilane moiety is in sharp contrast to the Z-selectivity observed during the analogous carbonylation using tributyltin hydride. When hexanethiol was used as the radical mediator, alpha-thiomethylene lactams were formed with E-favoring stereoselectivity again. Ab initio and DFT molecular orbital calculations on the stability of E and Z products were carried out for a set of five-membered methylene lactams bearing SnH3, SiH3, and SMe groups. The distinct thermodynamic preference for the Z-isomer was only predicted for the Sn-bearing lactam. A steric effect due to the bulky (TMS)3Si group is proposed for the E-selectivity observed in the TTMSS-mediated reaction.

9.
J Am Chem Soc ; 125(19): 5632-3, 2003 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-12733892

RESUMEN

Free-radical mediated stannylcarbonylation of azaenynes provides a general [n + 1]-type annulation approach leading to alpha-stannylmethylene lactams. The cyclization is unusual in its breadth, covering 4-exo, 5-exo, 6-exo, 7-exo, and 8-exo modes.

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