The complex of Maclura pomifera agglutinin with the T-antigen disaccharide (-D-Gal-(133)-␣-D-GalNAc-(13O)-Me) was investigated by NMR spectroscopy in aqueous solution. Intramolecular transferred nuclear Overhauser enhancement (NOE) effects between the monosaccharide moieties were used to derive the ligand conformation in the lectin-bound state. Ligand protons in contact with the protein were identified by saturation transfer difference experiments and intermolecular transferred NOE effects. It is demonstrated that structural differences exist for the ligand-lectin complex in aqueous solution as compared with the previously published crystal structure (Lee, X., Thompson, A., Zhiming, Z., Ton-that, H., Biesterfeldt, J., Ogata, C., Xu, L., Johnston, R. A. Z., and Young, N. M. (1998) J. Biol. Chem. 273, 6312-6318). In order to accommodate the O-methyl group of the disaccharide, the amino acid side chain of Tyr-122 has to rotate from its position in the crystal. The NMR data are in accord with two conformational families at the -(133)glycosidic linkage in the solution complex with interglycosidic angles / ؍ 45/؊65°and ؊65/ ؊18°. These differ from the bound conformation of the ligand in the crystal ( / ؍ 39/؊8°) and are not highly populated by the ligand in the free state. The reason for the structural differences at the -(133)glycosidic linkage are hydrogen bonds that stabilize the relative orientation of the monosaccharide units in the crystal. Our results demonstrate that the crystallization of a protein-carbohydrate complex can interfere with the delicate process of carbohydrate recognition in solution.The core 1 disaccharide 1, -D-Gal-(133)-␣-D-GalNAc, Scheme 1, is O-glycosidically linked to serine or threonine, a major component of anti-freeze glycoproteins (2) and mucins (3). Associated with carcinoma, it is one of the best characterized carbohydrate tumor markers (4) called Thomson-Friedenreich or T-antigen (5, 6). Some plant lectins with a high specificity toward the T-antigen structure have the potential to be used as specific diagnostic probes for carcinoma detection. These are peanut agglutinin (7), Amaranthus caudatus agglutinin (8), and two lectins from the plant family of Moraceae, jacalin (9) and Maclura pomifera agglutinin (MPA). 1 The crystal structure of the reducing T-antigen disaccharide 2 in complex with MPA was recently solved to a resolution of 2.2 Å (1) and displays an interesting binding mode of the disaccharide. The reducing GalNAc moiety resides in the binding site of the lectin, closely surrounded by four aromatic amino acids that form a hydrophobic pocket around the ligand. The N-acetyl group as well as the Gal unit are not in direct contact with the protein. With the exception of one Asp side chain, the H-bonds to the ligand all involve atoms of the peptide backbone or water molecules. This recognition of the reducing sugar unit seems to be a specific feature of the lectins from the Moraceae family since the closely related jacalin binds methyl-␣-D-galactopyranoside in the same ...
In (alkyl)di(aryl)(8-dim ethylamino-naphth-l-yl)phosphonium salts and in the parent phos phines, there is no experimental evidence of dative N -^P interactions. Conclusions to the contrary are based on the choice of improper standards of comparison.In tro d u c tio n /?e/7 -D isubstituted naphthalenes continue to be cherished objects o f research for the investigation of donor/acceptor (D ->A) interactions. This includes com pounds w ith donor substituents D o f only m od erate nucleophilicity and "acceptor" substituents A w hose electrophilicity is, at best, rather w eak or even doubtful. E .g ., a /?m -dim ethylam ino group or even a m ethoxy group has frequently been cho sen as the donor substituent [2 ], though 1-dim ethylam ino-naphthalene and 1 -m ethoxy-naphthalene are o f the dim ethylaniline and anisole type, respec tively.A m ong tertiary am ines, dim ethylaniline is a fairly w eak nucleophile. In 8-substituted 1-dim ethylam ino-naphthalenes, sterical hindrance in the peri space forces the (C H^^N group out of the Cio plane (see form ula 1) [3] so that efficient resonance o f the lone pair at N and the arom atic system is precluded; however, the N atom, bound to tw o sp3-carbons and one sp2 -carbon atom , neverthe less is a m uch poorer nucleophile than in aliphatic tertiary am ines (for com parisonmutatis mutan dis -note that 1-dim ethylam ino-naphthalene is less basic and N ,N ,2,6-tetram ethyl-aniline (2) only very slightly more basic than dim ethylaniline w hereas all three o f them are less basic than trim ethylam ine by more than five pow ers o f ten in the K B scale [4]). In 1 -m ethoxy-naphthalenes, sterical inhibition of res onance is m uch sm aller even in the presence of 8-substituents, and the cr-donicity of the anisole-type oxygen atom is, indeed, very poor.Am ong the "acceptor" substituents, am ide [2], silyl [5] and phosphino groups [6 -9] have been used w hich range, at best, at the bottom end of any electrophilicity scale. Such choice is surprising be cause the geom etry o f the fairly rigid naphthalene skeleton puts the peri substituents at a "natural" dis tance o f ca. 250 pm [10,11], much too long for a conventional bonding interaction [12]. The form a tion o f a dative bond D^A [13] of norm al length would require a considerable distortion of the Cio skeleton w hose energetic bill could be paid only by a strong p eri bond (such as a C-C or an am ide N-CO bond [14]). For the bonding interaction between a weak nucleophile and a p o o r electrophile, the naph thalene skeleton w ould act as a spacer which pre vents the substituents from com ing into bonding distance.Though still very m oderately so, a phosphonium phosphorus atom is a better electrophile than P in phosphines [15]. Earlier, we had investi gated the possibility o f a dative (C H 3)2N M P + in teraction in (alkyl)(8-dim ethylam ino-naphth-l-yl)-[=D A N ]di(phenyl)phosphonium salts [16]. Such in teraction could be described either as an isomerism (3A ^ 3B) or as a bond/no bond resonance (3A <->• 3B) [16]. In the form er case...
We report an improved synthesis of the T‐antigen disaccharide [β‐D‐Gal‐(1→3)‐α‐D‐GalNAc‐OMe], incorporating recycling of the undesired β‐glycosyl acceptor [methyl 2‐azido‐4,6‐benzylidene‐2‐deoxy‐β‐D‐galactopyranoside (9β)] through anomerization by treatment with FeCl3. The conformational analysis of the disaccharide made use of high quality NOE data in combination with extensive Metropolis Monte‐Carlo (MMC) and molecular dynamic (MD) simulations. To sample the conformational space sufficiently, 9.5·106 Monte‐Carlo steps were collected for the MMC simulations, while the fully solvated MD simulations were performed for 10 ns for comparison. In general, the MMC and MD simulations agreed very well. Comparison of theoretical NOE curves from both MMC and MD simulations with the experimental curves showed that the disaccharide populates two regions of conformational space, with a population of about 95% for the global minimum energy region and about 5% for a local minimum energy region.
We report an improved synthesis of the T-antigen disaccharide [β-D-Gal-(1Ǟ3)-α-D-GalNAc-OMe], incorporating recycling of the undesired β-glycosyl acceptor [methyl 2-azido-4,6-benzylidene-2-deoxy-β-D-galactopyranoside (9β)] through anomerization by treatment with FeCl 3 . The conformational analysis of the disaccharide made use of high quality NOE data in combination with extensive Metropolis Monte-Carlo (MMC) and molecular dynamic (MD) simulations. To sample the conformational space sufficiently, 9.5·10 6[a]
Synthesis and Conformational Analysis of the T-Antigen Disaccharide (β-D-Gal-(1→3)-α-D-GalNAc-OMe).--(BUKOWSKI, RALPH; MORRIS, LAURA M.; WOODS, ROBERT J.; WEIMAR, THOMAS; Eur. J.
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