The assessment of provenance of heparin is becoming a major concern for the pharmaceutical industry and its regulatory bodies. Batch-specific [carbon (δ¹³C), nitrogen (δ¹⁵N), oxygen (δ¹⁸O), sulfur (δ³⁴S), and hydrogen (δD)] stable isotopic compositions of five different animal-derived heparins were performed. Measurements readily allowed their differentiation into groups and/or subgroups based on their isotopic provenance. Principle component analysis showed that a bivariate plot of δ¹³C and δ¹⁸O is the best single, bivariate plot that results in the maximum discrimination ability when only two stable isotopes are used to describe the variation in the data set. Stable isotopic analyses revealed that (1) stable isotope measurements on these highly sulfated polysaccharide (molecular weight ∼15 kDa) natural products (“biologics”) were feasible; (2) in bivariate plots, the δ¹³C versus δ¹⁸O plot reveals a well-defined relationship for source differentiation of hogs raised in the United States from hogs raised in Europe and China; (3) the δD versus δ¹⁸O plot revealed the most well-defined relationship for source differentiation based on the hydrologic environmental isotopes of water (D/H and ¹⁸O/¹⁶O); and (4) the δ¹⁵N versus δ¹⁸O and δ³⁴S versus δ¹⁸O relationships are both very similar, possibly reflecting the food sources used by the different heparin producers. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:457–463, 2015

The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin's core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1375–1383, 2014

Although most pharmaceutical heparin used today is obtained from porcine intestine, heparin has historically been prepared from bovine lung and ovine intestine. There is some regulatory concern about establishing the species origin of heparin. This concern began with the outbreak of mad cow disease in the 1990s and was exacerbated during the heparin shortage in the 2000s and the heparin contamination crisis of 2007–2008. Three heparins from porcine, ovine, and bovine were characterized through state-of-the-art carbohydrate analysis methods with a view profiling their physicochemical properties. Differences in molecular weight, monosaccharide and disaccharide composition, oligosaccharide sequence, and antithrombin III-binding affinity were observed. These data provide some insight into the variability of heparins obtained from these three species and suggest some analytical approaches that may be useful in confirming the species origin of a heparin active pharmaceutical ingredient. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1447–1457, 2013

Testosteronan, an unusual glycosaminoglycan (GAG) first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on β-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the GAG, heparan sulfate. Studies using ³⁵S-labeled 3′-phosphoadenosine-5′-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from ³⁴S-labeled 6-O-sulfo-N-sulfotestosteroran using ³⁴S-labeled PAPS because testosteronan was resistant to all of the tested GAG-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of ³⁴S into ∼14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to ∼20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O-sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin (AT) III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to AT III. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 675–685, 2013.

In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and dermatan sulfate (DS) as an impurity. While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field ¹H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by ¹H-NMR to assess the utility of ¹H-NMR for screening heparin adulterants. Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and contaminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharmaceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities. Heparin prepared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field ¹H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5–10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4017–4026, 2009

α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide–alkyne Huisgen cycloaddition (“click chemistry”). Our approach is to generate non-natural N-glycosides of sialic acid that are resistant to neuraminidase-catalyzed hydrolysis as opposed to the natural O-glycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid derivatives has been synthesized in 71–89 % yield. A disaccharide mimic of sialic acid has also been prepared using the α-sialic acid azide 1 and a C-8 propargyl sialic acid acceptor in 68 % yield. A model sialic acid coated dendrimer was also synthesized from a perpropargylated pentaerythritol acceptor. These novel sialic acid derivatives were then evaluated as potential neuraminidase inhibitors using a 96-well plate fluorescence assay; micromolar IC₅₀ values wereobserved, comparable to the known sialidase inhibitorNeu5Ac2en.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

The cover picture shows the binding of an influenza virus to the surface of a host cell. The hemaglutinin (blue spikes) binds to the sialic acid (green hexagons) residues present on the non-reducing end of the surface glycoprotein to gain entry into the cell. Once the cell is infected, the viral neuraminidases (pink pinwheels) cleave the sialic acids to escape. The 1,2,3-triazole-linked sialic acid derivatives (green spheres) were designed to act as non-hydrolyzable inhibitors to block the virus. Details are discussed in the article by R. J. Linhardt et al. on p. 2611 ff.

The solution conformation behavior of a dermatan-derived tetrasaccharide—ΔHexA-(13)-GalNAc4S-β-(14)-IdoA-α-(13)-red-GalNAc4S (S is a sulfate group)—has been explored by means of NMR spectroscopy, especially by NOE-based conformational analysis. The tetrasaccharide was present as four species, two of which are chemically different in the anomeric orientation of the reducing 2-deoxy-2-acetamido-galactose (red-GalNAc) residue, while the other two are the result of different conformations of the iduronic acid (IdoA) unit. The two α–β-interconverting anomers were present in a 0.6:1 ratio. Ring conformations have been defined by analysis of ³J_H,H coupling constants and interresidual NOE contacts. Both 2-deoxy-2-acetamido-galactose (GalNAc) residues were found in the ⁴C₁ chair conformation, the unsaturated uronic acid (Δ-Hex A) adopts a strongly predominant half-chair ¹H₂ conformation, while the IdoA residue exists either in the ¹C₄ chair or in the ²S₀ skewed boat geometries, in a 4:1 ratio. There is a moderate flexibility of Φ and Ψ torsions as suggested by nuclear Overhauser effects (NOEs), molecular modeling (MM), and molecular dynamics (MD) studies. This was further investigated by residual dipolar couplings (RDCs). One-bond CH RDCs (¹D_C,H) and long-range H–H (³D_H,H) RDCs were measured for the tetrasaccharide in a phage solution and interpreted in combination with restrained MD simulation. The RDC-derived data substantially confirmed the validity of the conformer distribution resulting from the NOE-derived simulations, but allowed an improved definition of the conformational behavior of the oligosaccharides in solution. In summary, the data show a moderate flexibility of the four tetrasaccharide species at the central glycosidic linkage. Differences in the shapes of species with the IdoA in skew and in chair conformations and in the distribution of the sulfate groups have also been highlighted.