Nitrous Acid Depolymerization Research Articles

Regiospecific Grafting of Chitosan Oligomers Brushes onto Silicon Wafers.

The functionalization of surfaces using chitosan oligomers is of great interest for a wide range of applications in biomaterial and biomedical fields, as chitosan oligomers can provide various functional properties including biocompatibility, wetting, adhesion, and antibacterial activity. In this study, an innovative process for the regiospecific chemical grafting of reducing-end-modified chitosan oligomers brushes onto silicon wafers is described. Chitosan oligomers (COS) with well-defined structural parameters (average DP ~19 and DA ~0%) and bearing a 2,5-anhydro-d-mannofuranose (amf) unit at the reducing end were obtained via nitrous acid depolymerization of chitosan. After a silanization step where silicon wafers were modified with aromatic amine derivatives, grafting conditions were studied to optimize the reductive amination between aldehydes of amf-terminated COS and aromatic amines of silicon wafers. Functionalized surfaces were fully characterized by AFM, ATR-FTIR, ellipsometry, contact angle measurement, and ToF-SIMS techniques. Smooth surfaces were obtained with a COS layer about 3 nm thick and contact angle values between 72° and 76°. Furthermore, it was shown that the addition of the reducing agent NaBH3CN could positively improve the COS grafting density and/or led to a better stability of the covalent grafting to hydrolysis. Finally, this study also showed that this grafting process is also efficient for chitosan oligomers of higher DA (i.e., ~21%).

Molecular Weight Characteristics and Anticoagulant Activity of Low-Molecular-Weight Heparin Obtained By Nitrous Acid Depolymerization

Molecular Weight Characteristics and Anticoagulant Activity of Low-Molecular-Weight Heparin Obtained By Nitrous Acid Depolymerization

2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides.

Diblock oligosaccharides based on renewable resources allow for a range of new but, so far, little explored biomaterials. Coupling of blocks through their reducing ends ensures retention of many of their intrinsic properties that otherwise are perturbed in classical lateral modifications. Chitin is an abundant, biodegradable, bioactive, and self-assembling polysaccharide. However, most coupling protocols relevant for chitin blocks have shortcomings. Here we exploit the highly reactive 2,5-anhydro-d-mannose residue at the reducing end of chitin oligomers obtained by nitrous acid depolymerization. Subsequent activation by dihydrazides or dioxyamines provides precursors for chitin-based diblock oligosaccharides. These reactions are much faster than for other carbohydrates, and only acyclic imines (hydrazones or oximes) are formed (no cyclic N-glycosides). α-Picoline borane and cyanoborohydride are effective reductants of imines, but in contrast to most other carbohydrates, they are not selective for the imines in the present case. This could be circumvented by a simple two-step procedure. Attachment of a second block to hydrazide- or aminooxy-functionalized chitin oligomers turned out to be even faster than the attachment of the first block. The study provides simple protocols for the preparation of chitin-b-chitin and chitin-b-dextran diblock oligosaccharides without involving protection/deprotection strategies.

Reducing-End Functionalization of 2,5-Anhydro-d-mannofuranose-Linked Chitooligosaccharides by Dioxyamine: Synthesis and Characterization.

The nitrous acid depolymerization of chitosan enables the synthesis of singular chitosan oligosaccharides (COS) since their reducing-end unit is composed of 2,5-anhydro-d-mannofuranose (amf). In the present study, we describe a chemical method for the reducing-end conjugation of COS-amf by the commercially available dioxyamine O,O′-1,3-propanediylbishydroxylamine in high mass yields. The chemical structure of resulting dioxyamine-linked COS-amf synthesized by both oximation and reductive amination ways were fully characterized by 1H- and 13C-NMR spectroscopies and MALDI-TOF mass spectrometry. The coupling of chemically attractive linkers such as dioxyamines at the reducing end of COS-amf forms a relevant strategy for the development of advanced functional COS-based conjugates.

Oligosaccharides mapping of nitrous acid degraded heparin through UHPLC-HILIC/WAX-MS

Building blocks characterization is a significant approach for understanding the molecular structure of heparin and its derivatives. Nitrous acid (HONO) depolymerization of heparin generates oligosaccharides that maintain the epimerization conformation on C5 of the uronic acids, reflecting the authentic structure of the parental chain. HONO treatment at pH 1.5 selectively cleaves the bond between N-sulfated glucosamine and hexuronic acid, resulting mainly disaccharides, as well as tetra-, tri-, and mono-saccharides. The tetrasaccharides are derived from the structure of N-acetylated domains while tri-, and mono-saccharides are derived from the reducing or the non-reducing end of the heparin chain. The resulted oligosaccharides were separated and analyzed using a UHPLC-HILIC/WAX-MS method. We succeeded in the identification of 19 tetrasaccharides, 19 trisaccharides and 4 monosaccharides species, majority of which is structurally characterized. By comparing the theoretical possibilities and actual occurrence of the well-characterized tetrasaccharides, we demonstrated that the biosynthesis of heparin is a systematic process.

Reducing-end “clickable” functionalizations of chitosan oligomers for the synthesis of chitosan-based diblock copolymers

Chitooligosaccharides (COS) produced by nitrous acid depolymerization of chitosan are unique chitosan oligomers due to the presence of the 2,5-anhydro-d-mannofuranose (amf) unit at their reducing end. In this work, we focused on the reductive amination and the oximation of the amf aldehyde group towards various functionalized anilines, hydrazides and O-hydroxylamines. The aim of this work was to synthesize new COS-based building blocks functionalized at their reducing end by different “clickable” chemical groups such as alkene, alkyne, azide, hydrazide and thiol. Targeted functionalized COS were synthesized in excellent mass yields and fully characterized by NMR spectroscopy and MALDI-TOF mass spectrometry. Our results showed these functionalizations are quantitative, versatile and can be easily performed in mild reaction conditions. Finally, these COS-based building blocks could be useful intermediates for the development of advanced functional COS-based conjugates, as illustrated in this work by the synthesis of new COS-poly(ethylene glycol) (PEG) diblock copolymers.

Hydrophilic interaction chromatography-multiple reaction monitoring mass spectrometry method for basic building block analysis of low molecular weight heparins prepared through nitrous acid depolymerization

Low molecular weight heparins (LMWHs) are important anticoagulant drugs that are prepared through depolymerization of unfractionated heparin. Based on the types of processing reactions and the structures of the products, LMWHs can be divided into different classifications. Enoxaparin is prepared by benzyl esterification and alkaline depolymerization, while dalteparin and nadroparin are prepared through nitrous acid depolymerization followed by borohydride reduction. Compositional analysis of their basic building blocks is an effective way to provide structural information on heparin and LMWHs. However, most current compositional analysis methods have been limited to heparin and enoxaparin. A sensitive and comprehensive approach is needed for detailed investigation of the structure of LMWHs prepared through nitrous acid depolymerization, especially their characteristic saturated non-reducing end (NRE) and 2,5-anhydro-d-mannitol reducing end (RE). A maltose modified hydrophilic interaction column offers improved separation of complicated mixtures of acidic disaccharides and oligosaccharides. A total of 36 basic building blocks were unambiguously identified by high-resolution tandem mass spectrometry (MS). Multiple reaction monitoring (MRM) MS/MS quantification was developed and validated in the analysis of dalteparin and nadroparin samples. Each group of building blocks revealed different aspects of the properties of LMWHs, such as functional motifs required for anticoagulant activity, the structure of heparin starting materials, cleavage sites in the depolymerization reaction, and undesired structural modifications resulting from side reactions.

Octanoic Hydrazide-Linked Chitooligosaccharides-2,5-Anhydro-d-Mannofuranose

Chitooligosaccharide with one 2,5-anhydro-d-mannofuranose unit at the reducing end (COSamf) was prepared by nitrous acid depolymerization of chitosan. The reducing-end functionalization of COSamf by reductive amination with octanoic hydrazide in the presence of NaBH3CN was achieved in high yield. The chemical structure of the targeted octanoic hydrazide-linked COSamf was fully characterized by NMR spectroscopy and MALDI-TOF mass spectrometry. This synthesis opens the way to a new generation of COSamf derivatives with potential amphiphilic properties.

Quantification of aldehyde terminated heparin by SEC-MALLS–UV for the surface functionalization of polycaprolactone biomaterials

A straight forward strategy of heparin surface grafting employs a terminal reactive-aldehyde group introduced through nitrous acid depolymerization. An advanced method that allows simultaneously monitoring of both heparin molar mass and monomer/aldehyde ratio by size exclusion chromatography, multi-angle laser light scattering and UV-absorbance (SEC-MALLS–UV) has been developed to improve upon heparin surface grafting. Advancements over older methods allow quantitative characterization by direct (aldehyde absorbance) and indirect (Schiff-based absorbance) evaluation of terminal functional aldehydes. The indirect quantitation of functional aldehydes through labeling with aniline (and the formation of a Schiff-base) allows independent quantitation of both polymer mass and terminal functional groups with the applicable UV mass extinction coefficients determined. The protocol was subsequently used to synthesize an optimized heparin-aldehyde that had minimal polydispersity (PDI<2) and high reaction yields (yield >60% by mass). The 8kDa weight averaged molar mass heparin-aldehyde was then grafted on polycaprolactone (PCL), a common implant material. This optimized heparin-aldehyde retained its antithrombin activity, assessed in freshly drawn blood or surface immobilized on PCL films. Anticoagulant activity was equal to or better than the 24kDa unmodified heparin it was fragmented from.

Structure and anticoagulant activity of fucosylated glycosaminoglycan degraded by deaminative cleavage

Fucosylated glycosaminoglycans (FGs) are complex glycosaminoglycans that exhibit potent anticoagulant activity. To study the relationship between molecular size and biological activity, oligosaccharides with (2,5)-anhydro-d-talose units at new reducing ends were prepared by hydrazine deacetylation and nitrous acid depolymerization. The product chemical structures were analyzed by one- and two-dimensional NMR methods. Additionally, anticoagulant activities were evaluated by clotting assay and chromogenic substrate cleavage. The results demonstrated that under mild deacetylation and deaminative cleavage conditions, both products were relatively homogeneous and sulfated fucose branch types and sulfate substituents remained stable. These depolymerized FGs with different molecular sizes had potent intrinsic anticoagulant activities, which were similar to those that were obtained by free-radical depolymerization with similar molecular weights. Decreasing molecular weight may weaken activity but not significantly affect factor Xase and heparin cofactor II (HCII)-mediated thrombin inhibition.

Disaccharide Analysis of Glycosaminoglycans Using Hydrophilic Interaction Chromatography and Mass Spectrometry

Heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) participate in many important biological processes. Quantitative disaccharide analysis of HS and CS/DS is essential for the characterization of GAGs and enables modeling of the GAG domain structure. Methods involving enzymatic digestion and chemical depolymerization have been developed to determine the type and location of sulfation/acetylation modifications as well as uronic acid epimerization. Enzymatic digestion generates disaccharides with Δ-4,5-unsaturation at the nonreducing end. Chemical depolymerization with nitrous acid retains the uronic acid epimerization. This work shows the use of hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) for quantification of both enzyme-derived and nitrous acid depolymerization products for structural analysis of HS and CS/DS. This method enables biomedical researchers to determine complete disaccharide profiles on GAG samples using a single LC-MS platform.

Mass Spectrometric Method for Determining the Uronic Acid Epimerization in Heparan Sulfate Disaccharides Generated Using Nitrous Acid

Heparan sulfate (HS) glycosaminoglycans (GAGs) regulate a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS, which requires identification of the sulfation pattern as well as the uronic acid epimerization. In order to model HS structure, it is necessary to quantitatively profile depolymerization products. To date, liquid chromatography-mass spectrometry (LC-MS) methods for profiling heparin lyase decomposition products have been shown. These enzymes, however, destroy information about uronic acid epimerization. Deaminative cleavage using nitrous acid (HONO) is a classic method for GAG depolymerization that retains uronic acid epimerization. Several chromatographic methods have been used for analysis of deaminative cleavage products. The chromatographic methods have the disadvantage that there is no direct readout on the structures producing the observed peaks. This report demonstrates a porous graphitized carbon (PGC)-MS method for the quantification of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Here, we demonstrate the separation and identification of uronic acid epimers as well as geometric sulfation isomers. The results are comparable to those expected for benchmark HS and heparin samples. The data demonstrate the utility of PGC-MS for quantification of HS nitrous acid depolymerization products for structural analysis of HS and heparin.

Further Studies on the Isolation and Characterization of Heparin Contaminants Isolated From Recalled Batches of Unfractionated Heparin

The main contaminant from the recalled batches of unfractionated heparin (UFH) during the heparin crisis in 2008–2009 was reported to be oversulfated chondroitin sulfate (OSCS). Numerous reports have suggested the presence of other contaminants such as the oversulfated dermatan sulfate, oversulfated heparan sulfate and other non‐heparin GAGs. To further investigate the composition of non‐heparin GAG contaminants, a nitrous acid depolymerization method with stepwise methanolic precipitation was employed. The isolated non‐heparin GAGs from different batches were analyzed for the molecular and structural profile and biologic activities including anticoagulant, antiprotease and protease activation properties. Additional studies were carried out on the antithrombotic, bleeding and hemodynamic effects of these contaminants The interaction of the contaminants with heparin binding proteins were studied using surface enhanced laser desorption ionization methods. Differences in the molecular profile and structural composition were noted. Based on the structural analysis, non‐OSCS GAGs were also found to be present. In most instances OSCS was the main contaminant, however other non‐heparin GAGs were also found to be present in some of the batches. Additional characterization of the contaminants in the recalled batches of UFH is warranted to fully understand the composition of these contaminants.

Inhibition of allergic airway responses by heparin derived oligosaccharides: identification of a tetrasaccharide sequence

BackgroundPrevious studies showed that heparin's anti-allergic activity is molecular weight dependent and resides in oligosaccharide fractions of <2500 daltons.ObjectiveTo investigate the structural sequence of heparin's anti-allergic domain, we used nitrous acid depolymerization of porcine heparin to prepare an oligosaccharide, and then fractionated it into disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide fractions. The anti-allergic activity of each oligosaccharide fraction was tested in allergic sheep.MethodsAllergic sheep without (acute responder) and with late airway responses (LAR; dual responder) were challenged with Ascaris suum antigen with and without inhaled oligosaccharide pretreatment and the effects on specific lung resistance and airway hyperresponsiveness (AHR) to carbachol determined. Additional inflammatory cell recruitment studies were performed in immunized ovalbumin-challenged BALB/C mice with and without treatment.ResultsThe inhaled tetrasaccharide fraction was the minimal effective chain length to show anti-allergic activity. This fraction showed activity in both groups of sheep; it was also effective in inhibiting LAR and AHR, when administered after the antigen challenge. Tetrasaccharide failed to modify the bronchoconstrictor responses to airway smooth muscle agonists (histamine, carbachol and LTD4), and had no effect on antigen-induced histamine release in bronchoalveolar lavage fluid in sheep. In mice, inhaled tetrasaccharide also attenuated the ovalbumin-induced peribronchial inflammatory response and eosinophil influx in the bronchoalveolar lavage fluid. Chemical analysis identified the active structure to be a pentasulfated tetrasaccharide ([IdoU2S (1→4)GlcNS6S (1→4) IdoU2S (1→4) AMan-6S]) which lacked anti-coagulant activity.ConclusionsThese results demonstrate that heparin tetrasaccharide possesses potent anti-allergic and anti-inflammatory properties, and that the domains responsible for anti-allergic and anti-coagulant activity are distinctly different.

Study of chitosan and its derivatives as preservatives for field natural rubber latex

In this study, the chitosan and its derivatives were tested for their preservative activities for field natural rubber (NR) latex. A series of chitosans with different molecular weights were obtained by nitrous acid depolymerization. The chemically modified chitosans, N-carboxymethyl chitosan (NCMCh), N-sulfated chitosan (NSCh), and N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (NHTACh), were prepared from high and low-molecular weight chitosans. Preservative activities for field NR latex of these chitosans were investigated based on the measurement of volatile fatty acids (VFA) number of the treated latex. The preservative activities of chitosan increased with decreasing molecular weights. The low- molecular weight NSCh and NHTACh exhibited good preservative activity for the latex. By the use of low-molecular weight NHTACh in combination with octylphenol poly (ethyleneglycolether) (Nonidet P40), the latex was successfully preserved for more than 1 month in the low-ammonia condition. The results showed an attractive feature to develop the preservative system, which was possible to reduce the concentrations of ammonia and carcinogenic nitrosamine in the NR latex. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Online Reverse Phase-High-Performance Liquid Chromatography-Fluorescence Detection-Electrospray Ionization-Mass Spectrometry Separation and Characterization of Heparan Sulfate, Heparin, and Low-Molecular Weight-Heparin Disaccharides Derivatized with 2-Aminoacridone

A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ~15-21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of the commercial products prior to enzymatic digestion or prepurification steps to eliminate any excess of AMAC reagent or interference from proteins, peptides, and other sample impurities before RP-HPLC-Fd-ESI-MS injection were necessary. This method has applicability for the rapid differentiation of pharmaceutical Heps and LMW-Heps prepared by means of different depolymerization processes and for compositional analysis of small amounts of samples derived from biological sources by using the highly sensitive fluorescence detector.

Isolation and Characterization of Contaminants in Recalled Unfractionated Heparin and Low-Molecular-Weight Heparin

Recently, a contaminant was found in some clinically used unfractionated heparin (UFH) preparations. Administration of this UFH was associated with an increased risk of developing a wide range of adverse effects including death. To further investigate the chemical profile of the contaminant, contaminated batches of UFH were treated by exhaustive nitrous acid depolymerization followed by methanol precipitation to remove heparin oligosaccharides. Because contaminated heparins may have been used as starting material in the production of low-molecular-weight heparins (LMWHs), a similar procedure was carried out using an experimental batch of enoxaparin prepared from contaminated heparin. While high-pressure liquid chromatography (HPLC) analysis of contaminated heparin did not distinguish the presence of the contaminant, it could readily be observed as a high-molecular weight shoulder in the elution profile of contaminated enoxaparin. Digesting contaminated heparin with heparinase-I prior to HPLC analysis showed the presence of a nondigestible component (15%-30% of the mixture). This contaminant was also resistant to degradation by chondroitinases A, B, and C. Proton nuclear magnetic resonance (NMR) indicated that the contaminant was oversulfated chondroitin sulfate (OSCS). Size-exclusion chromatography indicated that the mean molecular weight of the OSCS was 16.8 kD, comparable to that of a synthetic porcine cartilage OSCS preparation that was used as a reference material (17.2 kD). While varying degrees of high-molecular weight dermatan sulfate and other minor impurities were detected, OSCS appeared to be the major contaminant in these preparations. The process involved in the production of enoxaparin does not significantly degrade OSCS.

Low molecular weight molluscan glycosaminoglycan from bivalve Katelysia opima (Gmelin)

Heparin, the glycosaminoglycan (GAG), was isolated from mollusk Katelysia opima (bivalve). The crude extract was partially purified by DEAE cellulose fractionation using two different molar concentrations of saline solution. Among the fractions F1, F2, F3 and F4, fraction F3 had highmolecular weight and better activity. F3 was further processed to get low-molecular weight heparin (LMWH) by nitrous acid depolymerization, and the total yield was 1.2 g/kg. The crude unfractionated heparin (UFH), fractionated heparin and depolymerized heparin samples were colorimetrically analyzed for the metachromatic shift using azure-A dye, and all the samples were found to be metachromatically active. Both UFH and LMWH showed an anticoagulant activity of 160 USP U/mg. Fractionated molluscan and LMWH were found to have peaks similar to that of standard heparin when assessed by the FTIR spectrum. Finally, molecular weight was determined by gradient polyacrylamide gel electrophoresis for UFH, fractionated F3 and LMWH, which were found to be 31,000, 30,500 and 6000 Da, respectively.

Why differentiate low molecular weight heparins for venous thromboembolism?

Why differentiate low molecular weight heparins for venous thromboembolism?

Dalteparin sodium in the management of thromboembolic disorders

Low molecular weight heparins have largely replaced unfractionated heparin for the prevention and treatment of venous and arterial thromboembolic disease since they are efficacious, safe and more convenient. Dalteparin sodium (Fragmin®, Pfizer Inc.) is a low molecular weight heparin produced by controlled nitrous acid depolymerization of unfractionated heparin. Although specific indications and usage vary across countries, dalteparin has received regulatory approval for use in preventing venous thromboembolism following major abdominal and orthopedic surgery, treating acute deep vein thrombosis, and preventing recurrent venous thromboembolism in cancer patients. It is also used for the treatment of unstable coronary arterial disease and prevention of clotting of the extracorporeal system during hemodialysis. More recent research efforts have examined the potential role of dalteparin as an antineoplastic agent. Dalteparin has significantly improved and simplified the management of venous thromboembolism an...