Characterization of Phosphoranes Obtained by the Spontaneous Carbonyl-Catalyzed Phosphorylation of Monosaccharides and Polyols in Aqueous Media.

Phosphorylation is a very important biochemical process in metabolism and biochemical marking. The mechanism for the biophosphorylation of substrates and the hydrolysis/transesterification of RNA has been suggested to proceed through phosphorane intermediates. Although the phosphorane intermediate/transition state has long been a subject of many theoretical models and studies, it has neither been isolated nor characterized, with most information derived from the hydrolysis and radiolabeling of cyclic phosphotriesters. We herein present the first report of the spontaneous phosphorylation of sugars and polyols in the absence of enzymes. That is, aldehydes and ketones combine with inorganic phosphates to form activated phosphates that phosphorylate alcohols without the requirement of any enzyme or additional activating agent. This phosphorylation is particularly favored in polyhydroxycarbonyls that can form internal cyclic acetals to give rise to the corresponding acetal phosphoranes. We have further characterized these phosphoranes and demonstrated their dehydration to the corresponding phosphates by using high-resolution mass spectroscopy. The phosphorylation of adenosine and uridine to form the corresponding phosphoranes was also achieved.

Structural elucidation of fucosylated chondroitin sulfates from sea cucumber using FTICR-MS/MS.

Fucosylated chondroitin sulfates are complex polysaccharides extracted from sea cucumber. They have been extensively studied for their anticoagulant properties and have been implicated in other biological activities. While nuclear magnetic resonance spectroscopy has been used to extensively characterize fucosylated chondroitin sulfate oligomers, we herein report the first detailed mass characterization of fucosylated chondroitin sulfate using high-resolution Fourier transform ion cyclotron resonance mass spectrometry. The two species of fucosylated chondroitin sulfates considered for this work include Pearsonothuria graeffei (FCS-Pg) and Isostichopus badionotus (FCS-Ib). Fucosylated chondroitin sulfate oligosaccharides were prepared by N-deacetylation-deaminative cleavage of the two fucosylated chondroitin sulfates and purified by repeated gel filtration. Accurate mass measurements obtained from electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry measurements confirmed the oligomeric nature of these two fucosylated chondroitin sulfate oligosaccharides with each trisaccharide repeating unit averaging four sulfates per trisaccharide. Collision-induced dissociation of efficiently deprotonated molecular ions through Na/H+ exchange proved useful in providing structurally relevant glycosidic and cross-ring product ions, capable of assigning the sulfate modifications on the fucosylated chondroitin sulfate oligomers. Careful examination of the tandem mass spectrometry of both species deferring in the positions of sulfate groups on the fucose residue (FCS-Pg-3,4- OS) and (FCS-Ib-2,4- OS) revealed cross-ring products 0,2Aαf and 2,4X2αf which were diagnostic for (FCS-Pg-3,4- OS) and 0,2X2αf diagnostic for (FCS-Ib-2,4- OS). Mass spectrometry and tandem mass spectrometry data acquired for both species varying in oligomer length (dp3-dp15) are presented.

Single Stage Tandem Mass Spectrometry Assignment of the C-5 Uronic Acid Stereochemistry in Heparan Sulfate Tetrasaccharides using Electron Detachment Dissociation.

The analysis of heparan sulfate (HS) glycosaminoglycans presents many challenges, due to the high degree of structural heterogeneity arising from their non-template biosynthesis. Complete structural elucidation of glycosaminoglycans necessitates the unambiguous assignments of sulfo modifications and the C-5 uronic acid stereochemistry. Efforts to develop tandem mass spectrometric-based methods for the structural analysis of glycosaminoglycans have focused on the assignment of sulfo positions. The present work focuses on the assignment of the C-5 stereochemistry of the uronic acid that lies closest to the reducing end. Prior work with electron-based tandem mass spectrometry methods, specifically electron detachment dissociation (EDD), have shown great promise in providing stereo-specific product ions, such as the B3´ -CO2, which has been found to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) in some HS tetrasaccharides. The previously observed diagnostic ions are generally not observed with 2-O-sulfo uronic acids or for more highly sulfated heparan sulfate tetrasaccharides. A recent study using electron detachment dissociation and principal component analysis revealed a series of ions that correlate with GlcA versus IdoA for a set of 2-O-sulfo HS tetrasaccharide standards. The present work comprehensively investigates the efficacy of these ions for assigning the C-5 stereochemistry of the reducing end uronic acid in 33 HS tetrasaccharides. A diagnostic ratio can be computed from the sum of the ions that correlate to GlcA to those that correlate to IdoA. Graphical Abstract ᅟ.

Structural Analysis of Heparin-Derived 3-O-Sulfated Tetrasaccharides: Antithrombin Binding Site Variants.

Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for heparin's anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a heparin with heparin lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in heparin and low-molecular-weight heparin products is described.

Assignment Of Hexuronic Acid Stereochemistry In Synthetic Heparan Sulfate Tetrasaccharides With 2-O-Sulfo Uronic Acids Using Electron Detachment Dissociation.

The present work focuses on the assignment of uronic acid stereochemistry in heparan sulfate (HS) oligomers. The structural elucidation of HS glycosaminoglycans is the subject of considerable importance due to the biological and biomedical significance of this class of carbohydrates. They are highly heterogeneous due to their non-template biosynthesis. Advances in tandem mass spectrometry activation methods, particularly electron detachment dissociation (EDD), has led to the development of methods to assign sites of sulfo modification in glycosaminoglycan oligomers, but there are few reports of the assignment of uronic acid stereochemistry, necessary to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA). Whereas preceding studies focused on uronic acid epimers with no sulfo modification, the current work extends the assignment of the hexuronic acid stereochemistry to 2-O-sulfo uronic acid epimeric tetrasaccharides. The presence of a 2-O-sulfo group on the central uronic acid was found to greatly influence the formation of B3, C2, Z2, and Y1 ions in glucuronic acid and Y2 and 1,5X2 for iduronic acid. The intensity of these peaks can be combined to yield a diagnostic ratios (DR), which can be used to confidently assign the uronic acid stereochemistry.

The Interaction of Heparin Tetrasaccharides with Chemokine CCL5 Is Modulated by Sulfation Pattern and pH.

Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding (44)RKNR(47) "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both (44)RKNR(47) motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.