Recognition of septanose carbohydrates by concanavalin A

Castro, Steve; Duff, Michael R.; Snyder, Nicole L.; Morton, Martha D.; Kumar, Challa V.; Peczuh, Mark W.

doi:10.1039/b509243d

Cited by 43 publications

(31 citation statements)

References 37 publications

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“…However, the addition of 5 to the solution did result in a small but measurable reduction in the fraction of ConA 2 bound to 6. Following the procedures outlined above, the intrinsic K a for the ConA 2 -5 interaction was determined to be (1.0 Ϯ 0.4) ϫ 10 4 M Ϫ1 , which is in reasonable agreement with the ITC-derived value of (7.9 Ϯ 1.0) ϫ 10 3 M Ϫ1 [18], Table 1.…”

Section: Cona-monosaccharide Bindingsupporting

confidence: 66%

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Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

El-Hawiet

Kitova

Liu

et al. 2010

J. Am. Soc. Mass Spectrom.

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A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying proteinligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (L ref ), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein-ligand complex in the gas phase, is added to the solution. The fraction of P bound to L ref , which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. The assay is based on the direct detection and quantification of the abundance (Ab) of ligand-bound and unbound protein ions in the gas phase, e.g., PL nϩ and P nϩ , respectively. A key assumption is that the measured abundance ratio (R) is equivalent to the equilibrium concentration ratio of ligand-bound and free protein in solution, eq 1: The direct ES-MS assay has been used to measure affinities for a range of protein-ligand complexes, including antibody-antigen, lectin-carbohydrate, enzymesubstrate/inhibitor complexes and, in many instances, the K a values agree well with constants obtained by other analytical methods, including isothermal titration calorimetry (ITC), surface plasmon resonance, and frontal affinity chromatography MS [4 -10]. However, there have also been reports of protein-ligand complexes that could not be detected by ES-MS or, if detected, the relative abundance of ligand-bound and unbound protein ions did not match the distribution expected in solution, with less binding observed in the gas phase [11][12][13][14]. These anomalous results are often due the occurrence of in-source dissociation, whereby the gaseous complexes undergo partial or complete dissociation during ES-MS analysis. If the gas-phase PL ions are kinetically labile and undergo dissociation during analysis, the magnitude of the measured R value and, correspondingly, the K a value will be artificially low. In the extreme case, where no PL ions survive, in-source dissociation will result in a false negative. Recently, it was shown that solution or gas-phase additives can, in some instances, protect complexes from in-source dissociation [12,15]. However, this approach does have its limitations and the detection of very labile gas-phase complexes, which rapidly dissociate at ambient temperature, by ES-MS remains problematic. Here, we describe an indirect ES-MS approach to quantify protein-ligand interactions that are highly Address reprint requests to Dr.

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Section: Cona-monosaccharide Bindingsupporting

confidence: 66%

“…Each subunit possesses a single carbohydrate binding site with specificity for the ␣-pyranose forms of Glc or Man. The affinity of dimeric ConA for the monosaccharide 5 was determined to be (7.9 Ϯ 1.0) ϫ 10 3 M Ϫ1 at pH 5.2 and 25°C by ITC [18].…”

Section: Determination Of Protein-ligand Affinitiesmentioning

confidence: 99%

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

El-Hawiet

Kitova

Liu

et al. 2010

J. Am. Soc. Mass Spectrom.

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“…Septanoses have attracted some attention recently as non-natural analogues of pyranoses which are expected to display significantly different conformational behavior. [47] To this end, the vinyl magnesium chloride used in the sequence outlined above for the protected l-rhodinals was simply replaced by allyl magnesium bromide, under otherwise identical conditions. The resulting alcohol 6 was protected as TBS ether 7, which gave, under tandem RCM-isomerization conditions, a ring expanded l-rhodinal analogue 8 (Scheme 2).…”

Section: Resultsmentioning

confidence: 99%

The Tandem Ring‐Closing Metathesis–Isomerization Approach to 6‐Deoxyglycals

Schmidt

Biernat

2008

Chemistry A European J

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Protected 3,6-dideoxyglycals have been synthesized de novo as single isomers starting from ethyl lactate by using the tandem RCM-isomerization reaction as the key step. Different relative configurations become accessible by addition of vinyl- or allyl-metal compounds to protected lactaldehydes under Cram-chelate or Felkin-Anh control. The concept is exemplified for glycals of L-rhodinose and L-amicetose, as well as for ring-expanded non-natural analogues thereof. This novel approach to glycals is also applicable to the synthesis of disaccharide glycals via a reiterative strategy, as exemplified for the dimer of L-rhodinose and its non-natural ring expanded analogue.

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“…Although limited, the biological and biophysical activities of the reported septanose carbohydrates indicate that these structures may be useful probes for glycobiology. 2,3 Structurally related polyhydroxyazepanes are known to be inhibitors of glycosidase and protease enzymes. 4 In each of these cases, the low energy conformations of the specific seven-membered rings were used to explain the reported activities.…”

Section: Introductionmentioning

confidence: 99%

Conformational analysis of methyl 5-O-methyl septanosides: effect of glycosylation on conformer populations

DeMatteo

Song

Fenton

et al. 2006

Carbohydrate Research

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Recognition of septanose carbohydrates by concanavalin A

Abstract: The ability of the jack bean lectin concanavalin A (ConA) to bind seven membered ring (septanose) monosaccharides has been investigated by isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR spectroscopy.

Cited by 43 publications

References 37 publications

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

The Tandem Ring‐Closing Metathesis–Isomerization Approach to 6‐Deoxyglycals

Conformational analysis of methyl 5-O-methyl septanosides: effect of glycosylation on conformer populations

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