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NMR Chemical Shifts in Structural Biology of Glycosaminoglycans Vitor H. Pomin* Program of Glycobiology, Institute of Medical Biochemistry, and University Hospital Clementino Fraga Filho, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-913, Brazil

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CONTENTS

NMR Chemical Shifts in Glycobiology Glycosaminoglycans: Structure and Function Chondroitin Sulfate and Dermatan Sulfate Heparin and Heparan Sulfate Hyaluronic Acid Keratan Sulfate NMR Chemical Shifts in Structural Analysis of GAGs Chondroitin Sulfate and Dermatan Sulfate Heparin and Heparan Sulfate Hyaluronic Acid Keratan Sulfate Pros and Cons of Using 1H-, 13C-, and 15NIsotopes in Solution NMR Structural Analysis of GAGs Use of Chemical Shifts in Detection of Contaminants in GAG-Based Therapeutic Preparations NMR Chemical Shifts in Functional Studies of GAGs Chemical Shift Prediction of GAGs Conclusions Author Information Corresponding Author Notes Biography Acknowledgments References

In addition to that, a vast collection of experimental methods, in terms of pulse sequence, is available for biomolecular NMR. They comprise uni- or multidimensional as well as homo- and heteronuclear experiments. These experiments enable assessment of a large number of physicochemical aspects from the samples aimed to investigation by NMR. This large collection of methods might be virtually unlimited. The number and combinations of pulse sequences, transferring magnetization through different isotopes (usually 1H, 13C, and 15N), give rise to several permutations of experiments available to address structural interrogations. Furthermore, NMR spectroscopy has a multitude of parameters available for measurements. A brief list containing the principal observables will certainly include (i) chemical shifts at the first place, which reflect the local chemical environment of a given nuclei; (ii) scalar and dipolar couplings, both depend on averaged conformational states; (iii) NOE resonances, which result from through-space internuclear contacts perceived by mechanisms of cross-relaxation of the dipole−dipole interactions; (iv) transferred NOE signals, which enable observation of the biding surface or physical contacts in intermolecular complexes; (v) latitudinal and longitudinal relaxation times that reflect, within different NMR time scales (usually in the range of nanosecond to second), the overall or specific dynamic aspects of the molecules, internal parts of motions, or how fast the molecules can move or tumble around in solution (Brownian motion). The relaxation phenomena occur in conformation- and molecular weight (MW)-dependent manner and thus serve as useful parameters to assess these characteristics when unknown. Among all these parameters, here, I will discuss only the role of NMR chemical shifts in structural and functional studies of glycosaminoglycans (GAGs), a physiologically and medically relevant class of carbohydrates which is deeply studied worldwide. Among all types of biologically active glycans, GAGs have shown a special position in glycobiology strikingly because of their efficient clinical properties together with the multiple key roles they can play in the body. GAGs are polysaccharides ubiquitously distributed in vertebrates, but they can also be found in certain invertebrates,24−26 although in these latter organisms within the propensity of showing uncommon structures when compared to the regular mammal GAGs. Regardless the origin or structural behavior, NMR spectroscopy has widely been employed in the majority of the studies about GAGs, whenever their structural properties might be a matter of interest, question, or concern. Although various

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uclear magnetic resonance (NMR) spectroscopy has been considered so far the utmost advanced and information-rich technique for structural analyses of biomolecules, including complex carbohydrates.1−5 Its application is very broad and covers the subjects of chemistry, biology, biochemistry, physics, biophysics, medicine, material, and polymer sciences.6 The wealth of information that can be generated and supplied by NMR spectroscopy is unsurpassed by any other analytical technique or method.6 The information type may comprise (i) structural characteristics of molecules of several sorts such as organic,7 inorganic,8 and biological molecules;1,9,10 (ii) properties of the intermolecular complexes such as kinetics of the on/off rates, affinity constants, and structural mapping of the binding surfaces and atoms involved in the contact;11−13 (iii) molecular dynamics,14,15 and conformational states;13,16 and (iv) imaging of biological tissues for medical diagnosis17 or in vivo experiments. Solutionstate,1−5,9−16,18 gel-state19 (partial alignment by oriented media),20,21 and solid-state22,23 exist in NMR spectroscopy primarily for different analytical purposes but also to overcome solubility limitations of the molecules subjected to analysis. © 2013 American Chemical Society

Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2014 Received: June 15, 2013 Published: August 2, 2013 65

dx.doi.org/10.1021/ac401791h | Anal. Chem. 2014, 86, 65−94

Analytical Chemistry

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and after a series of international meetings and publications discussing this matter, novel reports documenting residual amounts of GAG species different from those used as the main ingredient in biomedical formulations have still been appearing.47,54 In synthesis, these two above-mentioned aspects are time relevant. The advent of the glycomics projects, including NMR structural biology of GAGs together with the still current warnings about contamination of therapeutic GAG supplies, comprise two topics of high demand and priority for discussion nowadays with the international scientific community.

works using solution NMR in structure, function, and dynamic analyses of GAGs, or derivatives, do exist, few are those that explain, in a basic and systematic way, how to interpret and use at the deepest the complex NMR data generated therein. Here I am concerned to fulfill this mission through this document. I have chosen chemical shifts because this type of observable is the most accurate, informative, and readily available parameter for analysis of molecules via NMR spectroscopy. In addition, chemical shifts are also the mostly used NMR observable. They reflect with great reliability the overall or specific structural features of the molecules, under a multitude of experimental and sample conditions. Its utility is widely broad and meaningful regarding the fields of structural biology and analytical chemistry. In order to accomplish this mission, I will be providing here (i) the theoretical basis of NMR chemical shifts, (ii) background on the chemistry and biology of each GAG type, and (iii) comprehensive discussion about the application and proper interpretation of NMR chemical shifts in structural analysis of GAGs, based on the three magnetically sensitive nuclei presented in those glycans (1H, 13C, and 15N). This is primarily done with the aim of offering to the public a systematic way of investigating and interpreting the chemical, biochemical, and even functional aspects of each GAG type by chemical shifts from solution NMR. This document additionally provides critical discussion concerning the following topics: (i) the benefits and disadvantages in using each magnetically sensitive isotope in structural analyses of GAGs, (ii) the utility of NMR chemical shifts in the detection and recognition of GAG species other than those used as the main ingredient to formulate biomedical preparations, (iii) the study of functional aspects of GAGs based on migration of NMR chemical shifts, and finally (iv) the possibility of chemical shift prediction of GAGs. Overall, this document might ultimately serve as general reference in the field of analytical chemistry, glycobiology, and biomolecular NMR. It can be adopted as a useful manual destined to guide any study involving GAG molecules, in which NMR chemical shifts, their respective assignments, and changes of values according to structural or functional aspects of GAGs, might be a matter of question. Two main aspects justify and strongly support an up-to-date review in the field regarding the utility of NMR chemical shifts in structural biology of GAGs. First, NMR spectroscopy has been playing in the past few years a pivotal role to the progress of the currently ongoing glycome age. Its contribution concerns both structure and function determination of many biologically relevant glycans,5 including GAGs.27,28 Hence, this role of NMR must be highlighted and explained in a document through a basic and systematic way, as here intended using its major observable, the chemical shifts. Second, the international scientific community has agreed that chemical shifts from NMR spectroscopy analysis must be used as the preferential analytical method, or parameter, for assessment of possible contaminants in biomedical preparations of GAGs.29 The concerns about contamination of GAG-based supplies destined to health care has initiated with the global warning named “Heparin Crisis” which has happened in 2007−2008.30−32 Since that time, many reports concerning contamination in GAG-based biomedical supplies, or describing ways to avoid or to detect possible contaminants, have been appeared in the literature.29−53 Most of these reports also discuss the applicability and usefulness of NMR spectroscopy for monitoring the quality of these GAG preparations. Even after this worldwide alarming notification

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NMR CHEMICAL SHIFTS IN GLYCOBIOLOGY Chemical shifts are perhaps the most important and the mostly employed measurable parameter available in the field of NMR spectroscopy. They have been explored not only in the currently used Fourier-transformed NMR pulsed method but also in the early periods of NMR, during which the continuouswave method of sweeping magnetic fields of different strengths was the used way to distinguish the resonant frequencies of different nuclei of the same isotope within the same molecule. Chemical shifts can be considered the first explored observation in the history of NMR and possibly the most valuable piece of information available so far to investigate the structural properties of molecules subjected to this spectroscopy technique. Even with the existence of other relevant NMR observables like (i) spin-couplings, (ii) NOE resonances, and (iii) cross-peaks in multidimensional spectra, assignments of chemical shifts are still the main requirement to allow interpretation and exploration of these other observables. More recently in the NMR timeline, possibly since the early 80s when biomolecular NMR has started to emerge, chemical shifts had proved to be of extreme utility in structural and functional analysis of molecules of biological significance. At that time, protein and nucleic acids were the main molecular classes under NMR investigation. Consequently, during the development of biomolecular NMR, all the methods and parameters have primarily been designed to address chemical and biological questions of these molecular types, especially proteins.5,10 Nowadays, however, NMR spectroscopy has been turned quite useful to uncover the molecular aspects and functionalities of carbohydrates as well,1−5,18 including glycans of complex structures or of high-order degrees of flexibility.55 The relatively recent association of NMR with glycobiology results mostly from (i) the recent boom of the glycomics,56 (ii) the progress and sophistication of NMR methods together with instrumentation development, allowing the extraction of worthy information from the more flexible molecules like glycans in solution,27,28,55 and finally (iii) the recent discoveries about essential roles of carbohydrates as either key players in physiology,56 as efficient biomedical agents or ingredients,39,54 or as potential drug candidates.57 It is worth mentioning that carbohydrates, not so long time ago, have mostly been considered just a mere class of molecules of either energetic or structural function. However, this conception has considerably changed nowadays, and the contribution of sugars to biology and biochemistry has become practically equal as those of proteins and nucleic acids. Theoretically speaking, chemical shift (δ) could be defined as the resonant frequency of a given nucleus related to a specific standard frequency, both under the application of the same external magnetic field (B0). The chemical shift values of a molecule noted by the individual lines of its resultant NMR 66



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spectrum are B0-independent because they are always referenced to a standard. Also, the difference between the frequencies of the measurable nuclei and the specific standard changes equally proportional to different B0 intensities (magnetic strengths). Conversely and fortunately, chemical shifts are directly dependent on the chemical environment of the measurable nuclei within the molecules. This aspect is extremely important since it will ultimately lead to multiple lines in the recorded NMR spectrum. The chemical environment may be assumed as a final product of many contributing factors that surround the observable nuclei. The principal structural aspects are (i) the local interactions such as throughbond and through-space contacts with other atoms located nearby and (ii) the local fluctuations such as oscillations caused by dynamics or upon conversable conformational changes experienced by the molecules during the NMR experiment. This information explains why chemical shifts are so sensitive to temperature, pH, and counterions. These conditions physically impact the above-mentioned aspects of the atomic nuclei of the molecules when observed by NMR chemical shifts. Since the atomic nuclei within a given molecule are always surrounded by either through-bond or through-space electrons of the adjacent atoms and because electrons also have magnetic moments, an effective magnetic field (Beff) is what is being actually experienced by each observable nuclei under the application of the external magnetic field B0. The Beff, which is slightly but quantitatively different from the laboratory magnetic field B0, gives rise to different precession velocities (frequencies) of the composing nuclei of the molecules. The Beff of each individual nucleus varies accordingly to their particular surrounds. This ultimately leads to different resonant frequencies for the several chemically distinct nuclei of the molecule, in terms of a single isotope type. This explains why multiple resonance lines, even from the same isotope type, 1H for instance, can be produced from a single molecule type when subjected to 1H NMR analysis. The shielding constant (σ) is a dimensionless quantity that varies from nucleus to nucleus and reflects their particular chemical differences of each individual nucleus. It mathematically represents the local contributions seen in the individual chemical environment of the observed nucleus when placed under external magnetization (B0), followed by the radiofrequency pulse(s) during the NMR experiment. The shielding constant is an important factor that translates the abovementioned physicochemical properties or chemical environment of the several composing atomic nucleus in a mathematical language. The multitude of chemically different nuclei of a given molecule, with their particular σ values, is the basic explanation for the multiple lines seen in the resultant NMR spectrum. These lines result from different resonant frequencies (measured in Hz) of each chemically distinct nuclei, consequently leading to different chemical shift values. The unit of chemical shifts is measured in parts per million (ppm), not in the frequency magnitude (Hz). The conventional use of NMR chemical shift values rather than nuclear precessional frequencies is because of the different magnetic fields (B0) available across the laboratories in the world. If the magnetic fields are different, even just slightly (just in order of magnitude of few Hz), it will lead to different resonant frequencies from the same nucleus of the same molecule, even under the same sample and experimental conditions. To avoid this variation, chemical shifts, which are set relatively to a

standard resonance, are adopted instead of just resonant frequencies as a function of the magnetic field. This procedure guarantees the same values (in parts per million) of a certain nucleus of a given molecule, even submitted to different magnetic strengths. One could easily realize that the chemical differences of composing nuclei, mathematically represented by their individual shielding constants, are very small since the resonant frequencies are perceived in the order of magnitude of the sixth power. In order to (i) mathematically picture the chemical shift values (δ, in ppm) measured in relation to a reference frequency of a standard and to (ii) understand the real contribution of the different Beff, which is proportionally related to each individual nuclear σ of the molecules and ultimately seen by multiple lines in the NMR spectrum, the two following equations must be assumed: (i) δ(ppm) = 106 × (υobs − υref)/υref and (ii) Beff = B0(1 − σ). The abbreviations υobs and υref stand, respectively, for the precessional frequencies of the observed nucleus and the referece nucleus. The reference frequency comes from the use of certain chosen standards, usually tetramethylsilane for 1H, methanol for 13C, and liquid ammonia for 15N. These standards have resonances highly shielded as further explained in more detail. As a consequence, their resonances are located far upfield in the spectrum. Usually they serve to mark the starting point in the chemical shift scale (0 ppm). They are used to precisely calibrate the chemical shift scales independent of the variations of the magnetic fields. They are used worldwide and adopted as international standards for any NMR work. In summary, chemical shift values (or resonant frequencies), observed by positionally different lines in the NMR spectra, are important pieces of information to the diagnostics of the local chemical properties that surround measurable atomic nucleus of a given molecule subjected to NMR analysis. Only specific types of nuclear isotopes can be screened by NMR spectroscopy because of their quantum spin-numbers (one half) associated with technical properties of the NMR instruments. The magnetically sensitive nuclei of the most biomolecules are 1 H, 13C, and 15N. Like proteins and nucleic acids, GAGs are also polymers that bear amino groups, although amino groups are not constituent groups in most carbohydrates as in the former molecular classes. The presence of amino sugars in GAGs makes chemical shifts of all three magnetically sensitive isotopes (1H, 13C, and 15N) suitable for NMR experimentation and analysis. Below, I will be discussing the structural properties of GAGs through the perspective of NMR chemical shifts based on these three isotope types. Since we are treating this document as a comprehensive material about NMR and GAGs, we must provide at this moment of the document, right after explaining the basics of NMR chemical shifts, a general view about the structural features of each GAG type. Likewise, given that the biological actions of GAGs are extremely relevant for many different subjects such as pharmacology, biochemistry, and cell physiology; and because these actions are strictly dependent on the structural features of GAGs, a minimum discussion regarding the biology of GAGs must be offered as well. This will make a comprehensive connection between NMR chemical shifts of GAGs and their structural biology. Moreover, some biological actions of GAGs can be even concluded on the basis of measuring the changes of NMR chemical shift values. An event of cell signaling-related GAG−protein interaction 67



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Figure 1. (A) Representative structure of the repeating disaccharide unit of chondroitin sulfate (CS) and dermatan sulfate (DS). CSs are composed of alternating 3-linked N-acetyl α-galactosamine (GalNAc) units and 4-linked β-D-glucuronic acid (GlcA) or 4-linked α-L-iduronic acid (IdoA) in the case of DS. (B) Representative structure of the repeating disaccharide unit of heparan sulfate (HS) and heparin (Hp). Both are composed of alternating 4-linked uronic acid and 4-linked α-glucosamine (GlcNAc) units. HS has β-D-GlcA as its major uronic acid type component, whereas Hp has α-L-iduronic acid (IdoA). HS is frequently N-sulfated at the glucosamine unit, with less extension of N-acetylation and just rare amounts of Nfree (NH2 group). On the other hand, Hp is predominantly composed of 2-sulfated IdoA together with N,6-disulfated glucosamine units. Although the 3-O-sulfation at the GlcNS6S unit occurs more often in Hp than in HS, it still happens quite rarely in Hp. This chemical group is highlighted by the dashed ellipse in the middle unit of the structure in panel C, designated as A*. It is an essential structural feature for the anticoagulant activity, which results in the antithrombin high-affinity Hp pentasaccharide, whose structure is represented in panel C. (C) This pentasaccharide has already been chemically synthesized by the pharmaceutical company GlaxoSmithKline under the trademark name Arixtra, also largely known as fondaparinux. The letters between parentheses are used as labels for further assignments in the 1D 1H NMR spectrum (Figure 6E). They are the following: A for the nonreducing terminal N,6-disulfated glucosamine; U for nonsulfated glucuronic acid; A* for internal N,3,6-trisulfated glucosamine; I for 2-sulfated iduronic acid; and AM for reducing terminal 1-α-methylated N,6-disulfated glucosamine. (D) Representative structure of the repeating disaccharide unit of hyaluronic acid (HA). It is composed of alternating 4-linked β-GlcA and 3-linked α-GlcNAc units. HA, also known as hyaluronan, is the only nonsulfated GAG. (E) Representative structure of the repeating disaccharide unit of keratan sulfate (KS). It is composed of alternating 3-linked β-galactose and 4-linked α-glucosamine units. KS can bear sulfation at the 6-position of any unit, although sulfation at GlcNAc occurs usually more often. In all panels, the glycosidic bonds and the methyl substitution at the reducing end residue (panel C) are indicated by continuous-lined ellipses, whereas monosaccharide types are indicated by rectangles. Chemical variations of each disaccharide are represented below each structure.

between GAG families, among cell types, tissues, organisms, pathological/physiological conditions, species, and likely environmental and abiotic conditions that may influence the balance between phenotype and genotype. This structural heterogeneity in GAGs, especially those observed by different sequences within a single chain of a single GAG type, permits in turn a big beneficial variability of molecular interactions with functional proteins.64−66 The GAG−protein interactions regulate diverse key processes of the body including cell−cell interactions and cellular signaling events.64−69 The physiological consequences of these interactions are vast. Some examples are modulations in organogenesis/growth control,58,70−72 morphogenesis,73 cell growth/adhesion,74,75 coagulation/thrombosis,76,77 regeneration/wound healing,78,79 tumorigenesis/metatasis,78,80 angiogenesis,81 neural development/regeneration,82,83 and inflammation,84 besides mediating

analyzed by chemical shift migration will be taken here as an illustrative example.

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GLYCOSAMINOGLYCANS: STRUCTURE AND FUNCTION GAGs are mostly found at the extracellular matrixes (ECM),58 attached to a protein core forming the well-known proteoglycans. Proteoglycans are mostly membrane-associated glycoconjugates, but released proteoglycans can also be found in the ECM.58−61 Minor amounts of proteoglycans might be intracellularly found as well.60 Heparin (Hp) is an unusual GAG type since it essentially occurs inside granules of mast cells, and like hyaluronan,62 it is not naturally found linked to a polypeptide chain,63 as proteoglycans. The structures of GAGs can vary enormously: among disaccharide units of the same polymer, among polymeric chains of the same GAG type, 68



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infections by viral85 and micropathogens,86 and prion internalization.87−89 With this great variety of functions, structural studies of GAGs may represent an essential step for generating the minimal background about the structural determinants involved in those biological functions.55,64−66,90 Actually, the fact of revealing and understanding the principal chemical features involved in those biological actions of GAGs comprise a sine-qua-non mission of great priority not only in glycosaminoglycanomics studies but also in glycomics. As mentioned before, NMR spectroscopy has been pursuing the protagonist role in this regard across the last 2 decades. Generally speaking, as indicated by the name glycosaminoglycan, GAGs are structurally composed of disaccharide repeating units of alternating N-acetyl hexosamines (glycosamino) and another monosaccharide (glycan). The hexosamines can be glucose-based, N-acetyl glucosamine (GlcNAc), or galactose-based, N-acetyl galactosamine (GalNAc). On the other hand, the adjacent monosaccharide can be either an uronic acid type, glucuronic acid (GlcA), or its C5-epimerized form, the iduronic acid (IdoA), or the neutral sugar galactose (Gal).60,64 These monosaccharide types composed basically the backbone of GAGs, but they are modified accordingly to each GAG (sub)-family. GAG polymers made by GlcNAc units can be further processed at their N-sites during the chain modification period of biosynthesis. The processing can be either a deacetylation followed by a sulfation reaction that results in a sulfonamide group (NHSO3−) or just the less often deacetylation reaction that ends up in producing glucosamine (GlcNH) residues composed of N-free amino groups (NH2). The polymers made by GalNAc units are also known as galactosaminoglycans (GalGs). They are kept unprocessed at the N-position, although heavily modified by O-linked sulfation substitutions that can occur at any monosaccharide unit. The O-sulfonation also occurs at the GlcNAc-composed polymers. In fact, they happen very extensively in this type of GAGs. Sulfate esters may occur at (de)acetylated hexosamines as well as at (un)epimerized uronic acids. However, they occur more often at the N-deacetylated N-sulfated hexosamines, followed by the epimerized uronic acid, IdoA. The O-sulfonation occurs just rarely at the unepimerized GlcA unit. The number of structures resultant from all these reactions is big, but common characteristics are kept among GAG families and subfamilies. For example, GAG polymers have their backbones composed of either GlcNAc or GalNAc units. This characteristic will help to define the classification of the major GAG families. Further structural modifications, such as epimerization and additional sulfation, will result in subfamilies. More important than classification is that different structures will give to each GAG type, distinct physicochemical and biological properties.64,67,68,91 Below we will separately take each GAG family and subfamilies in order to present and explain these structural variations and show the underlying consequences of both these physicochemical and biological aspects for each GAG type. Discussion concerning NMR chemical shifts of each GAG family is given right thereafter. Chondroitin Sulfate and Dermatan Sulfate. Subfamilies of chondroitin sulfates (CSs), the only GalGs, are classified accordingly to structural variations depicted at Figure 1A. These variations comprise (i) different extensions and (ii) positions of O-linked sulfate groups, which can occur at any monosaccharide type (C2 and/or C3 in GlcA/IdoA units and C4 and/or C6 in GalNAc units), and (iii) epimerization levels

of the uronic acids, which always convert GlcA units into IdoA units (Figure 1A). Five types of naturally occurring CS subfamilies are generated as a consequence of these structural variations. They are CS-A, CS-B also known as dermatan sulfate (DS), CS-C, CS-D, and CS-E. CS-A is mostly 4-sulfated at the GalNAc units, while CS-C is predominantly 6-sulfated at the same unit type. CS-B, widely known by its own name DS, has α-IdoA rather than β-GlcA units. The IdoA units in DS may undergo 2-O-sulfonation, while the GalNAc units are mostly 4sulfated. CS-D has sulfation at both GlcA and GalNAc units, respectively, at their 2- and 6-positions. CS-E is majorly 2,4disulfated at the composing GalNAc units. An uncommon CS type is the oversulfated chondroitin sulfate (OSCS), which is highly sulfated at all available hydroxyl sites, such as C2 and C3 positions of the GlcA together with C4- and C6-positions of the GalNAc units. This latter CS type derives only from chemical synthesis. Obviously, all the structurally distinct CSs, or the microsequences presented throughout their chains, give rise to differential levels of biological responses as well as to different NMR spectral profiles, as discussed further. CSs are the most abundant GAG in the body. Regardless the subfamily, naturally occurring CSs are responsible for a multitude of functions. Besides the key roles that CSs can play at connective tissues as structural components of the ECM,59 these GalGs can also participate in neural development and regeneration,92−94 wound healing,95 infection,96 growth factors signaling,97,98 morphogenesis, and cell division.94,98,99 They are also receptors for various pathogens, including those of high threat to human health.96 Because of their important contribution in cartilages and other connective tissues, CS polymers can be explored as nutraceutical.54 They are utilized as either therapeutic agents or as active ingredients in food supplements normally found in diets of athletes, dancers, football players, or any of those who are frequently submitted to activities that results in physical impacts of their knees or susceptible for abrasion conditions of their joints’ cartilage. Despite the ubiquitous nature and vital functions of CSs, their structural characterization has been challenging. Also NMR spectroscopy has helped to clarify some of the related questions. The biological functions of CS are directly related to their sulfation patterns.100 For example, OSCS, which is heavily sulfated, can cause kallikrein-dependent hypotension,30,33 and this was the main clinical reason for the number of lethal cases that have happened during the “Heparin Crisis” in late 2007 and early 2008.30,31 Besides the commonest CS-A, CS-B, and CS-C polymers, unusual structures may also be found in nature. They comprise the CS-D (mostly composed of 2-sulfated GlcA together with 6-sulfated GalNAc units),101 CS-E (essentially composed of di4,6-sulfated GalNAc units),102,103 and the rarest CS-K, which contains significant amounts of 3-sulfated GlcNAc units.104 These types of structures are more abundant in marine invertebrates,102−105 in which, like the regular mammal CSs, they participate primarily as structural components of the ECM of specific tissues.102 Although in very lower abundance in terrestrial mammals, these CS types of rarer structures are still responsible for relevant biological roles such as those involving interactions with heparin-binding factors including midkine,106 L- and P-selectins,107 CD44, and chemokines;107 in neurite elongation;108 in bone formation and biomineralization;109 and in blocking HSV invasion, even when found at substantially lower concentrations.110 The binding with CD44 seems to be 69



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sulfation pattern-independent.107 CD44 binds to hyaluronic acid, which is the only nonsulfated GAG type. Heparin and Heparan Sulfate. Hp is perhaps the worldwide most studied GAG type. This is because of its great anticoagulant potential which is widely explored in the global pharmaceutical market.111,112 Hp is the second mostly used therapeutic biomolecule, placed just behind the hormone insulin which is used for treatment of diabetes type I. Hp occurs essentially inside granules of mast cells and is believed to be released and involved in inflammatory events.113 Hp molecules have surely the most heterogeneous structure among all GAGs (Figure 1B). Diverse microsequences can be found throughout its polymeric chain,114 and they are quite important in mediating the differential interactions with the various proteins in order to trigger diverse biological responses.115 Hp is majorly composed of alternating 4-linked α-iduronic acid (>70%) or β-glucuronic acid and 4-linked αglucosamine units with varying substitutions (Figure 1B).39,63,112 During the biosynthesis of Hp, this molecule undergoes more extensive sulfation than any other GAG type. This makes Hp not only the most sulfated GAG type but also the most negatively charged polymer naturally found on earth. Sulfation can occur at the 2-O-position of the IdoA as well as at the 6-, 3-, and N-positions of the glucosamines (Figure 1B).116 The 3- and N-sulfations are very significant in terms of biological functions.117−119 The 3-O-sulfonation happens just occasionally within the Hp chain but is an essential feature for the high-affinity interaction with antithrombin (AT), which results in the anticoagulant effect.61 The N-sulfation content that happens in detriment of the N-acetylation content is the major contributor for forming the N-sulfated clusters (NS domains) of the Hp chains. This NS domain shows better interactions with basic fibroblast growth factor (bFGF or FGF2),120 for instance. The degrees of N-acetylation/N-sulfation represented by extensions of the NA/NS domains are also responsible for creating a clear distinction between Hp and the structurally close-related GAG type, the heparan sulfate (HS). Although both GAG types share roughly the same principal disaccharide unit (Figure 1B), the amounts of substitutions and their distributions are quite different.63,121 During the biosynthesis of HS, for example, this polymer undergoes much less chain modifications than Hp. This will result in a series of structural distinctions between these two GAG subfamilies such as (i) HS has around a 0.8−1.8 sulfate/hexosamine ratio rather than a 1.8−2.6 ratio for Hp, this not including the additional 2O-sulfonation that occurs in IdoA units of Hp; (ii) sulfamate groups generated by the N-deacetylation/N-sulfation reaction happen around 40−60% in HS rather than ≥80% in Hp; (iii) the epimerization degree measured directly by the IdoA content is around 30−50% in HS as opposed to over 70% in Hp;72 (iv) the additional 3-O-sulfonation (the last modification during the Hp/HS biosynthesis) occurs in 0−0.3% in HS rather than ∼30% in Hp. This specific and rare 3-sulfation at the glucosamines is strictly involved in mediating and stabilizing the AT−Hp complex118,122 through the specific pentasaccharide sequence of high affinity for AT (Figure 1C). The distinct 3-Osulfonation amounts between Hp and HS explains their differential actions or levels of response in anticoagulation.63 Hp is clinically explored in the production of anticoagulants destined to the pharmaceutical market, especially through the use of low-molecular weight heparin (LMWH) derivatives,111 which have less side effects, more efficient activity, and bioavailability.111 The HS−AT complex, which occurs physio-

logically, happens much more occasionally and, as a result, makes the anticoagulant action of HS much lower.76 The AT affinity for HS must be lower in order to keep the physiological balance between clotting and anticlotting in the body, as opposed to the therapeutic potential of Hp, whose affinity for AT must be much higher in order to effectively guarantee the stoppage and the prevention of the blood clotting. Aside from the lower interactions of HS with AT to just keep the hemostatic balance,123 proteoglycans of HS additionally interacts with several other important proteins or peptides in other physiological systems such as (i) cytokines/chemokines and selectins in inflammation;124 (ii) prion and virus particles during infections;88,125,126 (iii) growth factors such as bFGF, 121,127 and vascular endothelial growth factor (VEGF),81,128 both involved in angiogenesis,81 growth control,129,130 morphogenesis,131 and tumor neovascularization during cancer progression and invasion;81 and finally (iv) ECM proteins such as collagen and integrins, both directly involved in adhesion, migration, and differentiation of cells.132 These latter cases of molecular interactions are responsible to keep the ECM structurally and functionally working. Most of the abovementioned biological actions seem to be related to specific structural domains (NA, NS, or NA/NS) or their distributions in HS proteoglycans at the cell surfaces.75 Hyaluronic Acid. Hyaluronic acid (HA) or hyaluronan, widely distributed in connective, epithelial, and neural tissues, is the only GAG type that is not modified by sulfation or epimerization at the GlcA moiety during the chain polymerization nor linked to a protein core. HA is composed of the repeating disaccharide structure of [-GlcA-β(1 → 3)-GlcNAcβ(1 → 4)-]n (Figure 1D) and possesses the highest degree of polymerization among all GAGs, perhaps in the range of 104 in terms of disaccharide units.62 Despite the lack of sulfation and less structural heterogeneity, HA still retains enough capability in interactions with other molecules, especially functional proteins133 like growth factors, cytokines, and adhesive proteins involved in angiogenesis, tumor proliferation, migration, and invasion.134 Nevertheless, the main biological role of HA is to assemble the ECM, especially in soft connective tissues such as vitreous humor, synovial fluid, umbilical cord, and the intercellular space of the epidermis.135 In all these tissues, HA can be found in relatively high concentrations, something close to 15−20 g as the total HA amount in the adult human body.62 The large molecular domains of HA result in viscoelasticity that flows at high concentrations besides filling empty spaces effectively.136 HA is also a scaffolding molecule onto which proteoglycans are noncovalently bound to form large molecular aggregates (aggrecan) whose high-negatively charged densities serve to provide turgor pressure in cartilage tissues by drawing in water and ions.60 This serves for both structure and function of cartilages. Moreover, since HA together with CS are the major structure-forming components in the globe of the eye and cornea,137 these GAGs can be used together to compose ophthalmic solutions for treatments and prophylaxis.138 The most famous physicochemical property of HA is its capacity of forming gels in solution.139,140 This property can be pharmaceutically and industrially useful as either a vehicle to make specific media or biologically active sera.141 For instance, HA-based medium can be employed in cosmetics to soften and smooth skin owing to its regenerative and hydrating properties besides being an important component of the ECM of this particular tissue. In addition, HA-based serum can be used as a 70



Review

Figure 2. Fully assigned 1D 1H NMR spectra (expansions from δH 5.5 to 1.5 ppm, which cover all signals) of the mostly studied and relevant CS types: (A) CS-A sodium salt from bovine trachea (Sigma-Aldrich), (B) CS-B (also known as DS) sodium salt from porcine intestinal mucosa (Sigma-Aldrich), (C) CS-C sodium salt from shark cartilage (Sigma-Aldrich), and (D) OSCS produced in our laboratory. All spectra were recorded at pH 7.0, 298 K, in a Varian 800 MHz NMR instrument, with samples dissolved in 99.9% D2O. The composing GalNAc, GlcA, and IdoA units are labeled as A, U, and I, respectively. The numbers that follow these letters represent the position of the 1H within the sugar ring. A4-SO3−, A6-SO3−, and CH3 represent, respectively, the 1H-signals of the 4- and 6-sulfated sites and the methyl protons of the acetyl groups. Peaks labeled with asterisks denote contaminants from solvent or other non-GAG materials, while peaks labeled with question mark denote unassigned signals due to structural heterogeneity arising from minor components within the polymeric CS chain. OSCS has less sulfated components of upfield resonances.148 HOD means the signal from residual proton-containing water, although suppression of the water signal was set bythe presaturation hard pulse. All spectra were acquired and assigned by the author based on literature values of chemical shifts.

lower concentrations,144 and HA is not covalently attached at this big molecular assembly. The KS/CS proteoglycans are held at the HA chain by protein linkers. Some vertebrates, like rats and other rodents, do not have KS as a structural component of their aggrecans as opposed to those normally found in humans and cows.61 In the cornea, KS proteoglycans maintain the even spacing of type I collagen fibrils in this tissue in such a way that allows the light beams to pass through without scattering.145 This phenomenon is regulated by the structural integrity of KS, since defects in sulfation or chain formation will lead to macular corneal dystrophy and keratoconus, due to distortions in the fibril organization and cornea opacity.146 Like HA and CS, KS can be also explored as an active ingredient of ophthalmic solutions destined to repair abnormal conditions of the cornea,147 even though KS is a minor component of this tissue.

vehicle to other functional ingredients since HA has the capacity of forming gel rich of skin-absorptive properties.135,142 This will help to both carry the active ingredients and release them below the epithelial layer. Keratan Sulfate. Keratan sulfate (KS) has some particularities that make this GAG type different from the others. First, the oligosaccharide linker responsible to initiate the synthesis of KS is different from the regular tetrasaccharide sequence of xylose-galactose-galactose-glucuronic acid (XylGal-Gal-GlcA) units observed in CS/DS and HS/Hp and resembles those of membrane-associated glycoproteins. There are basically two types of KS (KSI and KS II), both distinguished by their oligosaccharide linkage region attached to the protein core. Second, the structure of KS has no acidic unit like IdoA or GlcA as normally found in the other GAGs but rather the neutral sugar Gal. The KS structure comprises a sulfated poly-N-acetyllactosamine chain made up of [-Gal-β(1 → 4)-GlcNAc-β-(1 → 3)-] disaccharide units (Figure 1E). KS can bear sulfation at the 6-position of any unit, although sulfation at GlcNAc occurs usually more often. The KS structure resembles those found on conventional glycoproteins and mucins, except the fact that these latter ones are not sulfated.61,143 In terms of biological functions, KS is widely considered a functional structural component of the ECM of specific tissues such as cornea and connective tissues that contains aggrecan.144 The proteoglycan aggrecan is a large molecular assembly composed of both KS/CS and HA, although KS are found at

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NMR CHEMICAL SHIFTS IN STRUCTURAL ANALYSIS OF GAGs From now on we will be treating the role of NMR chemical shifts on the structural analyses of GAGs. Since these polymers are highly sulfated, as discussed above, and because of sulfate groups and their patterns of distributions are utilized to define GAG subfamilies, and above all, are one of the main structural determinants for the biological activities of GAGs, it seems very important at this point of the document, an explanation about the chemical effects of sulfate esters on the local environment of the atomic nuclei of GAGs placed nearby to these chemical groups. This will help the understandings about chemical shift 71



Review

Figure 3. Partially assigned 1H/13C-gHSQC spectra (expansions from δH 7.0 to 3.0 ppm and δC 120.0 to 40.0 ppm, panels A−C or from δH 6.0 to 3.2 ppm and δC 112.0 to 50.0 ppm, panels D and E, which cover all signals except those from acetyl methyl groups) of the most common CS disaccharides in their unreduced forms: (A) ΔC0S, (B) ΔC6S, (C) ΔC4S; and two reduced CS hexasaccharides of different sulfation patterns obtained from chondroitinase digestions: (D) ΔC444S-ol and (E) ΔC664S-ol. All spectra were recorded at pH 7.0, 298 K in a Varian 800 MHz NMR instrument, with samples dissolved in 99.9% D2O. Only cross-peaks denoting sulfation sites are indicated or assigned in the panels. The asterisks in plot D represent signals from a CS contaminant. The structures are the following: (A) ΔC0S as ΔUA-(β1 → 3)-GalNAc-α,β; (B) ΔC6S as ΔUA-(β1 → 3)-GalNAc-6S-α,β; (C) ΔC4S as ΔUA-(β1 → 3)-GalNAc-4S-α,β; (D) ΔC444S-ol as ΔUA-(β1 → 3)-GalNAc-4S-(β1 → 4)-GlcA(β1 → 3)-GalNAc-4S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-4S-ol; and (E) ΔC664S-ol as ΔUA-(β1 → 3)-GalNAc-6S-(β1 → 4)-GlcA-(β1 → 3)GalNAc-6S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-4S-ol, where ΔUA = Δ4,5unsaturated uronic acid; GalNAc = N-acetyl galactosamine; GlcA = glucuronic acid; S = sulfation group, in which the numbers before “S” represent the ring position; -ol stands for reduced sugars (open rings at the reducing-end termini).28,90 In the panels, nre-6S, mid-6S, nre-4S, mid-4S, and 4S-ol belong, respectively, to the assignments of the 1H/13C pairs of 6sulfated and 4-sulfated GalNAc from the nonreducing, middle, and reduced disaccharide units within the CS hexasaccharide chains. Modified with permission from ref 90. Copyright 2012 Oxford University Press.

values and variations in GAG atomic nuclei as a function of closely located sulfate groups. The resonances in the NMR spectrum will consequently change accordingly to the levels and positions of sulfates as well as to nonsulfated or desulfated sites. The high electronic density of sulfate groups will provoke a deshielding effect of the neighboring atomic nuclei such as 13 C, 15N, 13C-bound 1H, or 15N-bound 1H. As a consequence, when these GAG atomic nuclei are placed close to sulfate esters, they will suffer an electronic deshielding process induced by sulfation. This will end up in displacing their respective NMR chemical shifts toward a spectral region of resonances of higher frequencies (high δ values). Atoms have resonances of higher frequencies when their nuclei are more deshielded. Moreover, their shielding constants are thus of lower values. In this condition, the resonant frequencies are higher because the atomic nuclear accessibility for magnetism and experimental manipulation is enhanced due to the movement of the electroncloud (electronic shield) away from these atoms. The movement of the electronic shielding away is occasional by chemical groups of high-electronic density like sulfates that significantly attract the electrons closer to them. In the NMR jargon, the spectral region of resonances of higher frequencies is conventionally known as the downfield region, which is placed at the left-hand side of the horizontal chemical shift scale (higher values of ppm). The opposite side is known as the

upfield region. In summary, sulfation in GAGs will usually cause a migration of the neighboring atomic nuclei to the downfield region of the NMR spectrum as opposed to desulfation that will lead to an upfield displacement of the nearby nuclear resonances, regardless of the isotope type chosen for experimentation and analysis. Sometimes, however, β-effects may be influential, and this will lead to an upfield resonance shift when close to the sulfate groups. This is seen more in a few 13C-atoms of GAGs. Sulfation was taken here just as an introductory example to explain the influence of a specific structural feature of GAGs on the chemical environment of their composing nuclei, the respective influences on shielding constants, and the resultant chemical shift values obtained from the NMR spectrum. It is worthy saying at this point that this case of chemical determinants provoked by sulfation was assumed here as just an isolated and individual factor. In fact, the final result (shielding or deshielding contributions) will be seen as a consequence of many surrounding chemical determinants which act together at the same time. However, we can surely take one single component at each time, like sulfation, to understand its individual effect on the local environment of the atomic nuclei of GAGs. Other structural features of GAGs such as α/β-anomericity, glycosylation sites, N-acetylation/N-sulfation, C5-epimerization of uronic acids, and adjacent residue type will also lead to either deshielding or 72



Review

Figure 4. Fully assigned 1H/15N-gHSQC spectra (expansions from δH 8.15 to 7.75 ppm and δN 124.6 to 118.0 ppm, which cover all signals) of the most common native CS polymers: (A) CS-A, (B) CS-C, (C) OSCS, and (D) CS-B (also known as DS). All spectra were recorded at pH 4.5, 298 K, in a Varian 800 MHz NMR instrument, with samples dissolved in 85/15% H2O/D2O. These standards were the same used to make Figure 2. Note that all resonances show at least one distinct chemical shift. Modified from ref 28. Copyright 2010 American Chemical Society.

Figure 5. Fully assigned 1H/15N-gHSQC spectra (expansions from δH 8.3 to 7.5 ppm and δN 123.1 to 119.1 ppm, which cover all signals) of reduced CS hexasaccharides with different sulfation patterns. They have been obtained from either chondroitnase (panels A, B, and D) or hyaluronidase (C) digestions. All spectra were recorded at pH 4.5, 298 K, in a Varian 800 MHz NMR instrument, with samples dissolved in 85/15% H2O/D2O. The structures are the following: (A) ΔC666S-ol as ΔUA-(β1 → 3)-GalNAc-6S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-6S-(β1 → 4)-GlcA-(β1 → 3)GalNAc-6S-ol; (B) ΔC664S-ol as ΔUA-(β1 → 3)-GalNAc-6S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-6S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-4S-ol; (C) C644S-ol as GlcA-(β1 → 3)-GalNAc-6S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-4S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-4S-ol; and (D) ΔC444S-ol as ΔUA-(β1 → 3)-GalNAc-4S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-4S-(β1 → 4)-GlcA-(β1 → 3)-GalNAc-4S-ol, where ΔUA = Δ4,5unsaturated uronic acid; GalNAc = N-acetyl galactosamine; GlcA = glucuronic acid; S = sulfation group, in which the numbers before “S” represent the ring position; -ol stands for reduced sugars (open rings at the reducing-end termini).28,90 In the panels, 6S nonred, 6S mid, 6S-ol, 4S nonred, 4S mid, and 4S-ol belong respectively to the assignments of the amide 1H/15N pairs of 6-sulfated and 4-sulfated GalNAc from the nonreducing, middle, and reduced disaccharide units in the CS hexasaccharides. The peaks with 15N-chemical shifts around 120.6 and 121.6 ppm are respectively from 4- and 6-sulfated GalNAc units. Data modified with permission from ref 27 (Copyright 2013 Springer) and data modified from ref 28 (Copyright 2010 American Chemical Society).

(Figure 3), and 1H/15N-gHSQC (Figures 4 and 5), recorded for native CS polymers (Figures 2 and 4) as well as for some CS-derived oligosaccharides like disaccharides (Figure 3A−C) and hexasaccharides (Figures 3D,E and 5). These CS oligosaccharide taken herein have well-defined chemical structures and were obtained from digestions of the native polymers with lyase or hydrolase enzymes, followed by fractionation procedures in size-exclusion and strong-anion chromatographies.90 1D 1H NMR is the most used and quickest NMR method considering experimental time and sensitivity. It is also the most readily available NMR experimental type for collection of chemical shift-based data and the most reliable experiment when integration and nuclear amounts are under investigation. 1D 1H NMR spectrum of biomolecules, including complex carbohydrates such as GAGs or derivatives, gives rise to signals

shielding effects and thus downfield or upfield chemical shifts. These chemical features and their resultant influences on the NMR chemical shifts of certain atomic nuclei of GAGs will be individually explained below. Always at the first moment, a related discussion appears in the text. This systematic way of explanation will be helpful to learn from the use of NMR chemical shifts in structural interpretation of GAGs following thus the natural flow of information presented throughout the text regarding each GAG type and its specific structural features analyzed by NMR. Chondroitin Sulfate and Dermatan Sulfate. In order to offer a clear procedure for the proper recognition of the CS types (Figure 1A) and their main structural characteristics by the use of NMR chemical shifts, we first must present some illustrative NMR spectra, 1D 1H (Figure 2), 2D 1H/13Cgradient heteronuclear single quantum coherence (gHSQC) 73



Review

rings will cause the deshielding effects on the neighboring atoms. This explains why the 4-sulfated carbon-linked 1H usually is placed at a further downfield region of the NMR spectrum than the 1H-signal related to 6-sulfation. Nevertheless, both the 4- and 6-sulfation-related signals can be fairly used for the recognition of the CS types. Their integrals are indicative of the proper amounts of sulfation types within the CS sample. These amounts are determinants for subfamily classification, spectral profile, and biological responses. Other well-resolved resonances exist along with the ones from the sulfated sites. They are the anomeric signals of both GalNAc (noted as signal A1 with δH at ∼4.55 ppm) and GlcA (noted as signal U1 with δH at ∼4.48 ppm) units in panels A and C of Figure 2 as well as few additional components like U2, U3, and methyl groups, with δH, respectively, at 3.36, 3.58, and 2.0 ppm. These signals are easily distinguishable and readily assignable. However, in the CS-B (DS) spectrum (Figure 2B), chemical shift dispersion is lower than CS-A and CS-B. The anomeric I1 starts to superimpose with the residual water proton signal and the U3 signal starts to overlap with the I2. Even though resonances from GlcA are minor, they are still diagnostic of the residual unepimerized uronic acid, noted GlcA units, and labeled as U in the spectrum. They must be minor in CS-B species, based on the data of Figure 1A. This can be clearly proved by the dominance of IdoA signals in the 1D 1H NMR spectrum of DS. Low intensities of peaks U2 and U3 are observed comparatively to high-intense signals I2 and I3 (Figure 2B). Despite the lower chemical shift dispersion, the 1D 1H NMR spectrum of DS is fairly resolved and very diagnostic for structural assignments based solely on 1Hchemical shifts, besides being also diagnostic of this CS type. As discussed previously about chemical influences of sulfation, this chemical group will cause a downfield chemical shift displacement of the surrounding nuclei when compared to nonsulfated sites. This resonance shift is relatively proportional to the amounts of the nearby sulfate groups and their positions within the sugar ring. This can be clearly seen by the 1D 1H NMR spectrum of the OSCS (Figure 2D). In this spectrum, the majority of the 1H-signals have suffered a downfield chemical shift displacement in comparison to the signals from the CS-C, the original CS source used for oversulfation reaction; see comparatively panels D and C of Figure 2. This downfield displacement of most of the 1H-signals is particularly due to the oversulfated state of OSCS, in which most of the protons of the GalNAc and GlcA units are suffering long- and/or short-range electronic deshielding effects from oversulfation. Although most of the signals are not clearly resolved in the OSCS spectrum (Figure 2D), both anomeric 1H-signals (A1 at δH of ∼4.81 and U1 at δH of ∼4.92 ppm) and some additional ring protons (overlapped U2 and U4 at δH of 4.50 ppm) are otherwise readily recognizable and assignable. Aside from low chemical shift dispersion, the 1D 1H spectrum of OSCS is characteristic of this CS type due to downfield shifts of the major resonances. Usually, the 1D 1H NMR spectral profiles from the less common CS types such as CS-D, CS-E, and CS-K, seen more like that one of the OSCS, are poorly resolved. Just a few minor components are readily available for assignments. See the 1D 1 H NMR spectrum of a CS-E molecule in ref 150 as an illustrative example of a CS type of rarer structure. Conversely, CS derivatives of low-MW and well-defined chemical structures such as disaccharides, tetrasaccharides, and hexasaccharides, regardless of the CS-origins, have a great tendency of showing 1D 1H NMR spectra profiles of much better resolution. This is

coming only from the nonexchangeable protons when the samples are dissolved in deuterium oxide (D2O), the most common solvent used in NMR experiments to avoid residual signal from the water. The nonexchangeable 1H-signals arise from hydrogens directly bound to carbons in the GAG molecules. Hydrogens attached to highly electronegative atoms such as oxygens, nitrogens, and sulfurs, respectively, in hydroxyl, amide/amine, and sulfhydryl groups exchange rapidly with the solvent. These are groups of high electronegativity character. They attract the electron-clouds close to them and thus facilitate the detachment and release of the adjacent protons to exchange with the solvent. The fast exchanging rates of these protons are pH- and temperature-dependent, and vary within different NMR time scales. Consequently the exchangeable protons in the 1H-related NMR experiments performed under the regular experimental conditions, especially those involving physiological pH (7.3) and/or room (25 °C) or body temperatures (37 °C), are ultimately undetected by this approach. This provides some beneficial contributions: it decreases signal density and resonance overlap tendency. These contributions facilitate peak identification and structural determination. This is a similar rational for engineering deuterated proteins for NMR analysis.149 On the basis of 1D 1H NMR spectra recorded for the major polymeric CS types (Figure 2), distinguished profiles can be seen, characteristically for each CS subfamily. This serves as a useful signature to quick identify the CS type subjected to analysis. The routinely employed 1D 1H NMR experiment of CSs also serves to assess possible contaminants (signals marked with asterisks on panel D, Figure 2) as well as to assess minor components (signals labeled with question mark at panel D, Figure 2). Although not that extensive, chemical shift degeneracy can still be noted in the 1D 1H NMR spectra of CSs, and it occurs within different degrees depending on the subfamily. The low signal dispersion is typical of complex carbohydrates whose NMR resonances are essentially condensed in a very limited spectral range, usually from 6.0 to 3.0 ppm for 1H-chemical shifts.1,27 Few exceptions arise from side chain chemical groups attached to the sugar rings such as methyl protons of acetyl groups which have 1H-chemical shifts at ∼2.0 ppm. The low chemical shift dispersion of the major carbon-bound 1H resonances in carbohydrates, including GAGs, sometimes makes difficult to achieve complete assignments via 1D 1H NMR. This is because that resonance overlap is easy to occur (see, for instance, signals noted as U5 and A3 with δH at ∼3.8 ppm in panels A, C, and D of Figure 2). However, well-resolved peaks still exist as discussed next, and they are valuable for spin and structural assignments as well as for the rapid recognition of the CS subfamilies. Fortunately, nonexchangeable protons attached to sulfated carbons are among the resolved signals in CS-A (mostly 4sulfated) and CS-C (predominantly 6-sulfated) spectra. The resonance of the proton attached to the 4-sulfated carbon is usually placed 1.0 ppm more downfield (δH around 5.2 ppm) than the 6-sulfated one (δH of around 4.2 ppm). See comparatively plots A and C of Figure 2. The 6-sulfation makes 1H-chemical shifts of the closer protons at the more upfield region of the spectrum than the 4-sulfation. This occurs likely due to its location outside of the sugar ring and thus less susceptibility to short-range chemical influences from the most hydroxyl oxygens of the ring, especially the O5 heteroatom. The presence of hydroxyl oxygens of high electronegative density attached at the C2, C3, and C4 carbons of the sugar 74



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Table 1. 1H- and 13C-Chemical Shifts (ppm) of Cross-Peaks in 13C-gHSQC Spectra Indicating Sulfation Positions in GalNAc Units from Unsaturated Reduced CS Hexasaccharides (Figure 3D,E)a

seen by the presence of well-resolved peaks of considerably narrower lines.101−104 This implies that chemical shifts in oligosaccharides are easier for assignments rather than in native CS polymers whose NMR resonances are inclined to showing enhanced line broadening. This happens most likely because of the large rotational correlation times (τc) typically found at high-MW molecules such as native CS polymers. This will result in longer relaxation times and thus broader lines in their NMR spectrum. Larger linewidths result in the propensity of enhancing signal overlap. Therefore, it is harder to accomplish chemical shift and structural assignments via solely 1D 1H NMR spectra of some GAG polymers, including native CSs of uncommon structures.150 Multidimensional NMR spectra can definitely help to overcome this issue of resonance overlap in native GAG polymers with the spread of peaks by the additional dimension. This can be seen in Figures 3−5, in which heteronuclear 13C- or 15N-chemical shifts are additionally detected via through-bond scalar-coupling (1JHX, where X denotes the heteroatom 13C or 15N). This NMR method allows correlation of the heteronuclear chemical shifts, in these cases carbon-13, with the 1H-resonances in the direct dimension. Actually, the paradigm of better resolved spectra of intermediate-sized oligosaccharides versus lower spectral resolution of native polymers can be seen for the major GAG types, as stated below for derivatives of the other families. As expected, on the basis of the physical foundations of NMR, especially relaxation time as a function of MW, GAG oligosaccharides have to result in sharper peaks and thus give more resolved NMR spectra. This is a particular phenomenon that is relevant is terms of the utility of NMR chemical shifts for structural interpretation of GAG molecules and derivatives. A more detailed explanation will be given in the next section concerning NMR chemical shifts of Hp and its low-MW derivatives. Nonetheless, the 13C-gHSQC method can serve as a useful NMR approach for the recognition of different sulfation patterns (non-, 2-, 4-, 6-sulfation) in CS samples. This can be pictured from the differentially sulfated CS disaccharides shown in Figure 3A−C. In plot A of this figure, the typical positions of the 1H/13C pairs of each sulfation type are indicated by dashed circles. Since the CS disaccharide of Figure 3A is nonsulfated, no signals are noted at the highlighted regions, as opposed to the presence of a 4-sulfation-related 1H/13C cross-peak in the spectra of the 4-sulfated CS disaccharide (Figure 3B) and to the presence of a 6-sulfation-related 1H/13C cross-peak in the spectra of the 6-sulfated CS disaccharide (Figure 3C). This diagnostic potential based on 1H/13C correlations is also useful for CS polymers, although heterogeneity may arise in these cases. In these latter cases, structural complexity makes more laborious chemical shift assignments. In plots B and C of Figure 3, doublet signals can be seen for the 4- and 6-sulfation-related 1 H/13C pairs. These doublets do not arise from scalar couplingrelated splitting but, in fact, from the α- and β-configurations of the reducing-end GalNAc due to the anomeric mutarotation in solution. From the 1H/13C-gHSQC spectra of the differentially sulfated CS disaccharides (Figure 3B,C), we can note a typical behavior of downfield 1H- and 13C-chemical shifts for the 4sulfated sites (δH/C = ∼4.7/80.0 ppm) in comparison to those from the 6-sulfated sites (δH/C = ∼4.4/70.0 ppm). This chemical shift behavior also occurs in longer well-defined CS oligosaccharides such as tetrasaccharides,101,102 hexasaccharides (Figure 3D,E and Table 1) and for the native CS polymers. The

position of sulfationb

1

H-/13C-chemical shifts

1

H4/13C4 of 4-sulfated GalNAc units

4S-ol mid-4S nre-4S 6S-ol mid-6S nre-6S

4.47/81.98 4.73/79.94 4.78/79.91 1 H6/13C6 of 6-sulfated GalNAc units 4.19/70.55 4.17/70.55 4.21/70.57

a

Data reproduced with permission from ref 90. Copyright 2012 Oxford University Press. bThe units labeled with S-ol, mid-, and nreindicate the reduced form of the reducing end, the middle, and the nonreducing end GalNAc residues along the oligomeric chain.

reason for this sulfation type-specific chemical shift position has been properly explained above. The most important at this point is that this differential tendency of 1H- and 13C-chemical shifts from 4- and 6-sulfation can be diagnostically useful for the assignment and determination of the sulfation distribution in intermediate-sized CS oligosaccharides in a sequence-specific manner (Figure 3D,E and Table 1). In some CS octasaccharides, see ref 90, for example, and perhaps, not much longer oligosaccharides, the 1H/13C-pairs that are related to these differentially sulfated sites start to show a tendency to overlap. Hence, the diagnostic utility of these sulfation-related 1H/13C resonances starts to fail in terms to determine the position of the disaccharide units within the oligomeric chain. Therefore, the diagnostic quality of the sulfation patterns of CS derivatives based on 1H/13C pairs is limited to the oligomeric extension of the derivative subjected to analysis. In polymers, although qualitatively and quantitatively informative, the 1H/13C resonances related to sulfation types as well as to minor components serve ultimately just to obtain a rough snapshot of the overall structural composition. No information about sequencing can be raised therein. On the other hand, the combined 1H- and 13C-chemical shifts from cross-peaks of intermediate-sized CS oligosaccharides, as abovediscussed, can be of great diagnostic potential in a sequencespecific manner. This was seen by the complete sequencing of unsaturated reduced CS oligosaccharides (Figure 3D,E). The assignments of the sulfated GalNAc units can be accomplished solely on the means of comparing the 1H-/13C-chemical shift values, as represented in Table 1. Hence, in CS oligosaccharides, the chemical shifts of 1H-/13C cross-peaks are diagnostically useful both in terms of fast recognition of the sulfation types and in determining the right positions of the sulfated GalNAc units within the oligosaccharide chains. See plots D and E of Figure 3 in conjunction with the values in Table 1 for the two types of CS hexasaccharides. Just to give an impression of the applicability of this approach, 13 other CS hexasaccharides of well-defined chemical structures have successfully been characterized in ref 90, primarily on the basis of the 1H/13C-chemical shift values of the related sulfation sites of the composing disaccharides. This is a useful example to prove the utility of 1H-/13C-chemical shifts on the structural characterization of GAG derivative oligosaccharides, for instance, the CS-derived oligosaccharides. 75



Review

Table 2. Some Important Properties for NMR Detection and Sensitivity of the Commonest Nuclei of Biomolecules nuclide

spin

natural abundance

gyromagnetic ratio γ [107 rad T−1 s−1]

proton (1H) carbon-12 (12C) carbon-13 (13C) nitrogen-14 (14N) nitrogen-15 (15N)

1/2 0 1/2 1

99.985 98.9 1.108 99.63

26.7522

799.734 (1)

1.00

10 ppm

6.7283 1.9338

201.133 (1/3.976) 57.820 (1/13.831)

1.76 × 10−4 1.00 × 10−3

220 ppm

1/2

0.37

−2.7126

81.093 (1/9.861)

3.85 × 10−6

300 ppm

NMR frequency at 18.8 T (ratio related to 1H)

relative receptivity

chemical shift range

Table 3. 1H- and 15N-Chemical Shifts (ppm) of Cross-Peaks from Amide Groups from 15N-Acetyl Hexosamines of Different GAGs GAG type

structure

CS-A/CS-C

Figure 1A

CS-B (DS) OSCS diΔC4Sb

Figure 1A Figure 1A legend of Figure 3

diΔC6S

legend of Figure 3

HS/Hp

Figure 1B

Arixtra

Figure 1C

figure or ref

amino sugar type β-GalNAc-4(SO3−) β-GalNAc-6(SO3−) β-GalNAc-4(SO3−)

Figure 4A,C

β-GalNAc-4,6-di(SO3−) β-GalNAc-4(SO3−) α-GalNAc-4(SO3−) β-GalNAc-6(SO3−) α-GalNAc-6(SO3−) GlcA-linked α-GlcNAc-6(SO3−) IdoA-linked α-GlcNAc-6(SO3−) α-GlcN(SO3−) α-GlcN,6-di(SO3−) α-GlcN,3,6-tri(SO3−) α-GlcNH2 α-GlcN(SO3−) (A) α-GlcN,3,6-tri(SO3−) (A*) α-GlcN,6-di(SO3−)-OMe (AM)

Figure 4D Figure 4B refs 27,28 refs 27,28 Figure 7A−C Figure 7A−C ref 114 ref 114 ref 114 ref 152 ref 152

H-/15N-chemical shifts (ppm)a

1

7.96/120.91 7.94/121.61 8.09/120.46 7.94/121.21 8.35/121.50 8.14/122.40 8.24/121.80 8.09/122.75 8.36/123.62 8.27/123.72 5.36/94.0 5.44/94.1 5.60/92.1 NAc/33.30 5.98/93.00 5.50/91.53 5.54/94.46

a Chemical shifts are relative to trimethylsilylpropionic acid at 0 ppm for 1H and liquid ammonia for 15N, obtained in experiments at 25 °C and pH 4.5. bThe CS disaccharides used were unreduced in order to keep the α/β-anomeric mutarotation. cNot assigned in the reference.

new avenue for increased sensitivity problems when analysis of sugars at natural isotopic abundance are undertaken.6 The advent of cryogenically cooled probes has also contributed in this field of sensitivity enhancement in glycobiology NMR. In addition, some works have also shown that cell cultures grown with glutamine with 15N-labeled side chains can produce GAGs considerably labeled with 15N-isotopes.28,161 Anyway, the 15Nrelated results generated from these improved NMR technologies and methods have recently brought not only novel insights into the structural biology of GAGs but also have considerably facilitated the structural analysis of such glycans, and their derivatives, based on their 15N-related chemical shifts.28 Some of these recent and novel results will be discussed below. The 15N-related resonances of GAGs belong necessarily to the amide nitrogen of their composing hexosamine units. Therefore, one would expect that just a few NMR signals would be generated from this type of NMR approach since the amides in GAGs are just a single structural component of the GAG disaccharide repeating units (Figure 1). As discussed further, although few in numbers, the resultant 15N-related signals with their respective chemical shifts are very useful for structural characterization of GAG polymers and derivatives. These signals are very sensitive to both long- and short-range structural variations of the composing disaccharide units of GAG families and subfamilies. These structural variations may include glycosidic bond types, anomericity, adjacent uronic acid types, sulfation patterns, and of course, N-substitutions. Even

Recently, a novel NMR approach for the structural characterization of GAGs and derivatives has emerged. This new scope of analysis is based on the much less sensitive and less explored 15N isotope.27,28 The main reason that has limited or impaired the use of this isotope in biomolecular NMR studies of GAGs in the past, is the low magnetic susceptibility of this nucleus, negative gyromagnetic ratio 10-fold lower than that of the proton and relativity receptivity, around 3.85 × 10−6 compared to the value of 1.0 set for 1H, associated with its low natural abundance (∼0.37%). See Table 2 for details and comparison among the main NMR used nuclides. Nowadays, however, technology and instrumentation has developed and NMR studies of GAGs based on 15N have not only proved possible27,28,114,136,151−162 but also turned into a more routine practice. This fact is mainly due to the progress and spread of modern NMR instruments of ultrahigh magnetic fields, cryoprobe technology, the recent development of isotopic GAG-labeling strategies, together with the development of novel combinations of 2D pulse sequences for detection of specific 15N-related magnetization transfer and coherences. Recently, ultrahigh magnetic field NMR spectrometers (900, 920 MHz and 1GMz) have become available for application in structural analysis of sugars.163,164 This instrumentation development has decreased the severe tendency for spectral overlapping mainly due to enhanced sensitivity, besides providing additional benefits in accessing the information from chemical exchange and relaxation. Small-volume NMR spectroscopy has also been implemented, and this may be a 76



Review

CS-A, since the former CS type is composed of over 95% Csulfation type and the balance being A-sulfation type (Figure 4B); while the latter one is, respectively, C- and A-type in the amounts of 65% and 35% (Figure 4A). This novel 15N NMR method works out not only for high-MW CS polymers but also for well-defined intermediate-sized CS oligosaccharides as described next. Like the ability of the 13C-HSQC method in determining the sulfation patterns of intermediate-sized CS oligosaccharides in a sequence-specific manner through the note of specific 1H-/13Cchemical shift values (Table 1), 1H-/15N-chemical shifts derived from 15N-HSQC spectra can also be used for the same analytical purpose. Note the possibility of rapid characterization of the sulfation patterns in CS hexasaccharides through the use of specific chemical shifts notation from their 15N-HSQC spectra (Figure 5). The 6-sulfation can be simply distinguished from 4-sulfated sites in all composing disaccharide units within the hexasaccharide chain, since the differential sulfation-related peaks are suitable for being sorted out in a relatively clear and easy way. The C-type sulfation in GalNAc units produce amide 1 H/15N pairs with more downfield 15N-chemical shifts (somewhere between δN 121.1 and 122.1 ppm) than those from A-sulfated hexosamines (somewhere between δN 120.1 and 121.2 ppm) (Figure 5). This assignment is quite similar to the differential 15N-chemical shifts of the A- and C-type sulfation seen in the 15N-HSQC spectra of native CS-A and CSC polymers (Figure 4A,B and Table 3). The differential 15Nchemical shifts of amides from 6- or 4-sulfated GalNAc units can be taken as a general rule since it is also applicable to CS disaccharide units, see Table 3 and refs 27 and 28. The upfield 15 N-chemical shift of 4-sulfation and the downfield 15Nchemical shift of 6-sulfation have been explained in the above paragraph, also in comparison with the opposite behavior of the 13 C-chemical shifts of each sulfation type. The 1H-/15Nchemical shift assignments in the CS hexasaccharides enables additional recognition of the position of the disaccharides within the polymeric chain (see the assignments labeled as nonred, mid, and -ol, in panels of Figure 5), besides the recognition of the anomeric configuration and ratio in disaccharides.27,28 This way of rapid chemical shift assignments for sequence-specific recognition of CS oligosaccharides resembles the one described above for the 13C-HSQC method, although this latter 13C-related method will always end up giving a more crowded spectral profile due to the presence of resonances from both sulfation-related and -unrelated carbonbound nonexchangeable 1 H (Figure 3D,E). This will consequently lead to a higher probability of resonance overlap as opposed to the 15N-related NMR method which relies solely on amide groups of GAGs. The fact of simpler spectral profiles from the 15N-based NMR methods associated with the considerable amounts of valuable structural information that can be generated therein represents a tremendous advantage in relationship to the more conventional 1H- and/or 13C-based NMR methods, even though the considerably lower magnetic sensitivity of the 15N isotope may be a limiting factor. This implies that ultrahigh magnetic field NMR instruments (especially those equal or above 800 MHz) must be chosen for this novel approach if we consider the following aspects: (i) feasible time for acquisition of the total experiment; (ii) spectral quality in which reliable peaks, way above the spectral floor (good signal-to-noise ratio) can be recorded; and (iii) use of isotopically unlabeled samples from which spectral resolution

sensitive to these features and thus informative for the structural studies of GAGs, or derivatives, the resultant 15Nrelated NMR peaks are indeed short in number. They never exceed more than two or three resonances per 1H/15N-HSQC spectrum. Nevertheless, this considerably smaller number of signals allows spin and structural assignments in a faster and easier way. Moreover, unlike the approaches based on 1H- and 13 C-chemical shifts that need multiple supportive 2D NMR experiments for complete assignments, 15N-based NMR analysis of GAGs usually requires just a single experiment type, the 15N-HSQC spectrum or related heteronuclear pulse sequence versions. However, a downside exists. The low natural abundance and magnetic susceptibility of 15N (Table 2), its negative NOE, and long T1 relaxation times make NMR detection based solely on this nucleus very challenging. In order to overcome this obstacle, the insensitive nuclei enhanced by polarization transfer (INEPT), which transfers the magnetization back to the proton for acquisition with a gain of sensitivity proportional to the difference between the sensitivities of the heteronucleus and proton (in the case of 15 N, an enhancement of 10-fold occurs, see Table 2), becomes a crucial technical strategy for extracting structural information of GAGs, or derivatives, when 15N is the nucleus of interest.27,28,114,151,152,154 The INEPT can be seen in pulse sequences involving heteronuclear correlations such as those of detection for the 1H/15N pairs in 15N-HSQC experiments or related pulse sequences. This explains why 15N-filtered INEPTcontaining pulse sequences, like 15N-gHSQC, are necessary for the 15N-related data acquisition of GAG samples. As a consequence, some 15N-gHSQC spectra of the most studied native CS types and CS-derived oligosaccharides have been recorded (Figures 4 and 5). From these spectra, structural information based on derived 1H- and 15N-chemical shifts can be raised, as described below. Note from the panels of Figure 4 together with the information displayed at Table 3 that 4-sulfated GalNAc units show amide resonances at the upfield 15N-chemical shifts (somewhere between δN 120.4 and 121.1 ppm) as opposed to the downfield 15N-chemical shifts of the 6-sulfated sites (somewhere between δN 121.5 and 122.0 ppm). The 4,6disulfated GalNAc units, presented in OSCS, for example, has shown a single amide resonance with a curious 15N-chemical shift midway of the two monosulfated sites (∼121.2 ppm) (Figure 4C and Table 3). These resonances with different chemical shifts allow assignments and recognition of the sulfation types in a very quick and straightforward way. The upfield 15N-chemical shift (Figure 4) but downfield 13Cchemical shift (Figure 3) of 4-sulfation, and the contrary orientations for 6-sulfation, occur likely due to the opposite signs of the gyromagnetc ratios of these isotopes (Table 2). On the basis of the spectra of Figure 4, the resonances from GalNAc units of CS and DS are also very distinguishable from each other (see Table 3). DS has downfield 1H-resonance (∼ 8.09 ppm) (Figure 4D, Table 3) since their most GalNAc units are linked to IdoA units rather than linked to GlcA units as commonly found in CS-A and CS-C (Figure 1A), which in turn show upfield 1H-chemical shift resonances at ∼7.9 ppm (panels A and C of Figure 4 and Table 3). This is another clear differentiation based on 1H/15N pairs. This enables easy identification of the GalG subfamilies. The quantitative measurements based on cross-peak integrals are also indicative of rough amounts of the major structural components. For example, CS-C is much more structurally homogeneous than 77



Review

Figure 6. Fully assigned 1D 1H NMR spectra (expansions from δH 6.1 to 1.8 ppm, panels A−D or from δH 5.8 to 3.1 ppm, panel E, which cover all signals) of a HS example, and the main types of Hp: (A) HS from the bivalve N. nodosus,24 (B) Hp from bovine source, (C) Hp from porcine source, (D) enoxaparin (LMWH), and (E) fondaparinux (Arixtra), all in their sodiated forms. Spectra were recorded at pH 7.0 at (A) 323 K, (B−E) 298 K, in (A) Bruker 400 MHz, (B−D) Bruker 800 MHz, or (E) Bruker AMX 500 MHz NMR instruments, all samples dissolved in 99.9% D2O. The composing units IdoA2S, IdoA, GlcA, GlcNS-6S, GlcNAc6S (or GlcNAc), GlcNS, and GlcNS-3S-6S are labeled as I, I′, G, A, B, C, and A*, respectively. In panel A, G1(SO3−) means a anomeric proton from a GlcNAc that can be either 2- or 3-sulfated.24 The invertebrate HS has sulfation at the GlcA, which is unusual for mammal HS samples. In addition, this invertebrate HS has an averaged MW of 36 kDa. The lower MWs of the other samples (around 15 kDa for UFH, ∼7 kDa for LMWH, and ∼1.7 kDa for fondaparinux) lead to sharper 1H-signals and thus a more resolved 1D 1H spectrum. This occurs primarily because of higher τc values in longer polymers. In addition to that, a magnetic instrument of lower strength has been used for the HS sample (A), 400 MHz rather than 800 MHz for the Hp samples (B−D) and 500 MHz for fondaparinux (E). Note the differences in the signal quality. Fondaparinux has a methylated GlcNAc unit at the reducing end, noted as AM. See Figure 1C for the structure. The signal from this methyl group (∼3.4 ppm) is triply intense due to the amounts of 1H-nuclei. The numbers that follow the letters represent the 78



Review

Figure 6. continued position of the 1H within the sugar ring. The following letters represent the covalently bound adjacent unit. O-CH3 and CH3 represents the 1Hsignals of the O-methyl protons of fondaparinux (E) and of the acetyl groups from HS/Hp (A−D). HOD means the signal from residual protonated water, although suppression of the water signal was set by a presaturation hard pulse before acquisition. All spectra were recorded, except the one of panel E, and assigned by the author, although these spectra have already been published elsewhere. Assignments were based on reference values from the literature. Peaks labeled with asterisks in panel E denote minor units in the fondaparinux. Data reprinted with permission from ref 24 (Copyright 2010 American Society for Biochemistry and Molecular Biology) and from ref 166 (Copyright 2000 American Society for Biochemistry and Molecular Biology).

Table 4. 1H- and 13C-Chemical Shifts (ppm)a of the Major Glucuronic Acid and Glucosamine Units of the Invertebrate HSb nucleus H1 H2 H3 H4 H5 H6-SO3− H6-6OH C1 C2 C3 C4 C5 C6-SO3− C6-6OH

G

G(2SO3−)

G(3SO3−)

4.61 3.37 3.73 3.86 3.82

4.76 4.14 NA 3.92 3.84

4.71 3.63 4.58 NA NA

105.1−102.0 74−8−70.2 77.3−72.9

105.0−100.9 75.3−72.0 NA 77.3−72.9 77.3−72.9

103.2−101.0 73.0−70.5 85−80.4 NA NA

GlcNAc (B)

GlcNS (C)

5.42−5.38 4.09−3.95 3.82 NA 4.04 4.48/4.22 3.88 98.2−95.3 54.8−51.5 69.8−67.0 NA 71.8−66.9 66.9−64.5 62.1−58.9

5.30 3.27 3.79 3.9 4.05 4.24/4.34 3.76/3.90 98.1−95.9 59.0−56.2 69.0−67.2 82.8−81 4.19−4.03 65.9−65.1

Chemical shifts are relative to external trimethylsilylpropionic acid to 0 ppm for 1H and to methanol for 13C at 50 °C. G stands for glucuronic acid, while NA, not assigned. O-sulfation, N-sulfation, and glycosylation sites are highlighted in bold, bold/italic, and italic, respectively. bThe 13C-HSQC spectrum of this GAG is shown in Figure 7E for verification on chemical shifts of both nuclei. Data reproduced with permission from ref 24. Copyright 2010 American Society for Biochemistry and Molecular Biology. a

rely solely on the contributions from natural abundant 15N in GAGs. For the latest item, signal averaging must be accumulated through the use of multiple numbers of the scan set during the acquisition process. This will increase the total time of NMR experiment acquisition. Moreover, for this case of unlabeled materials, a reasonable concentration of GAG samples must be reached to keep a good signal-to-noise ratio. This is easy for the case of GAGs from animal extractions rather than those of cell cultures.28 Heparin and Heparan Sulfate. Figure 6 depicts the fully assigned 1D 1H NMR spectra of a multitude of HS and Hp samples in order to show recognition of their 1H-chemical shifts in relation to their respective varying composing units and structural features. The HS utilized was extracted from the bivalve Nodipecten nodosus, which has previously been fully characterized based on multiple NMR experiments.24 This GAG isolated from the marine invertebrate has been taken here as just an illustrative example (Figure 6A). All typical units of the commoner HS molecules are included therein, and this HS sample can be assumed as a reliable example of HS, regardless of its origin. 1D 1H NMR spectra for the principal types of Hp have been recorded as well, followed by proper assignments of their major resonances (see Figure 6B−E) based on typical chemical shift values from the literature. They are unfractionated heparin (UFH) sodium salt from bovine intestinal mucosa (Figure 6B)39,112 and from porcine intestinal mucosa (Figure 6C),39,112 enoxaparin sodium salt (a LMWH sample) (Figure 6D), and the AT-high-affinity pentasaccharide fondaparinux sodium salt (trademark name Arixtra) (Figure 6E). All samples are clinically relevant and were mostly obtained from the Brazilian pharmaceutical market.

The 1D 1H spectrum of the N. nodosus HS (Figure 6A) shows besides a reasonable level of sample homogeneity (not as many peaks as in the Hp spectra are seen, even though taking into account the lower spectral resolution), equivalent amounts of anomeric protons of α-glucosamines (noted as A and B) and of β-glucuronic acid (noted as G) residues. This is the major evidence indicating unequivocally that the invertebrate GAG sample is a HS rather than a Hp-like molecule. Hp is preponderantly composed of α-anomeric protons in its backbone (Figure 1B) rather containing β-anomers. It is worthy saying at this point that α-1H1 resonances of GAGs have δH typically somewhere between 4.9 and 5.5 ppm, while the β-anomeric 1H resonances are typically somewhere between 4.4 and 5.0 ppm (Table 4). This difference is likely due to anisotropic chemical shift behaviors. The most important at this point in our analytical perspective is that this differential spectral range allows distinction of anomericity based on 1Hchemical shifts. 15N-related experiments are also able to allow such distinction (Table 3). In regards to the structural characterization of the invertebrate HS based on 1H-chemical shift assignments from its 1D 1H NMR spectrum (Figure 6A), we can observe considerable amounts of 1H-anomerics (δH 5.56−5.44 ppm, Table 4) attributable to α-D-glucosaminyl units whose N-positions are substituted by acetyl groups (resonances labeled as B in the spectra of Figure 6A). This substitution is present in ∼60% of all α-residues of the mollusk GAG. This demonstrates that the invertebrate HS contains a significant amount of N-acetylated glucosamine units. This data additionally supports the fact that the invertebrate GAG is a HS,24 because Hp usually possesses on average up to 20% of GlcNAc units (see Figure 1B and Heparin and Heparan Sulfate). 79



Review

Table 5. 1H- and 13C-Chemical Shifts (ppm) for α-IdoA and α-GlcNX Residues of Bovine and Porcine Hp112,a IdoA2S(I) → GlcNS,6S(A)

IdoA2S(I) → GlcNAc6S(B)

signalb

current spectra

literature datac

signalb

current spectra

I1-A I2-A I3-A I4-A I5-A A1 A2 A3 A4 A5 A6R A6S

5.19 4.34e 4.20 4.10 4.81 5.39 3.28 3.68 3.76 4.04 4.38 4.27

5.21 4.34 4.20 4.12 4.79 5.39 3.29 3.67 3.77 4.03 4.39 4.27

I1-B I2-B I3-B I4-B I5-B B1 B2 B3 B4 B5 B6R B6S

5.23 NDf ND ND ND 5.38 3.86 ND ND ND ND ND

I1-A I2-A I3-A I4-A I5-A A1 A2 A3 A4 A5 A6R A6S

101.3 80.0 71.3 78.0 71.4 98.6 60.0 ND 77.9 ND 68.4 68.4

101.9 78.7 71.8 78.5 72.2 96.9 60.3 72.0 78.5 71.6 68.7 68.7

I1-B I2-B I3-B I4-B I5-B B1 B2 B3 B4 B5 B6R B6S

101.3 ND ND ND 70.4 ND 55.5 ND ND ND ND ND

IdoA2S(I) → GlcNS(C)

literature datad

IdoA(I′) → GlcNS-6S

signalb

current spectra

literature datad

literature datac

I1-C I2-C I3-C I4-C I5-C C1 C2 C3 C4 C5 C6R C6S

5.23 4.34 4.20 4.04 4.84 5.30 3.27 3.69 4.04 4.23 3.89 3.76

5.26 4.35 4.25 4.06 4.84 5.31 3.27 3.71 3.70 3.89 3.86 3.88

5.0 3.8 4.1 4.1 4.8 5.3 3.3 3.7 3.8 4.0 4.2 4.4

I1-C I2-C I3-C I4-C I5-C C1 C2 C3 C4 C5 C6R C6S

101.3 ND ND 77.8 70.4 99.1 60.0 ND ND ND 62.1 62.7

102.0 77.6 70.7 78.7 71.4 100.0 60.8 72.4 80.5 73.8 62.6 ND

104.5 72.0 72.0 77.5 72.5 98.5 60.0 71.9 79.6 71.3 68.6 ND

1

H-Chemical Shifts 5.20 4.37 4.31 4.08 4.91 5.36g 3.91 3.77 3.80 4.05 4.24 4.34 13 C-Chemical Shiftsh 102.2 76.8 67.3 74.2 70.8 96.6i 56.2 73.0 79.3 72.3 69.6 69.6

Data from Figure 6B,C and from literature references. bSee legend of Figure 6 for nomenclature of the NMR signals. Units of β-GlcA were not included in the table. cData from ref 166. dData from ref 167. eThe O-sulfonated, N-sulfonated, N-acetylated, and glycosylated sites are highlighted in bold, bold/underscore, bold/italic, and italic, respectively, for rapid notation. fND, not determined. gData from ref 168. hData from ref 112. iData from ref 165. a

4-linked N,6-disulfated α-glucosamine units (labeled as A). On the other hand, the Hp sample of bovine origin revealed a complex mixture of disaccharide types in addition to the commonest trisulfated disaccharide, as discussed next. The 1Hsignals noted as A1 and I1 are the anomeric protons of the residues N-sulfated 6-sulfated α-glucosamines and 2-sulfated αIdoA, whose δH are at 5.39 and 5.19 ppm, respectively (Figure 6B,C and Table 5). Besides this major component of trisulfated disaccharide unit in both UFH, the bovine Hp has two extra signals that belong to different α-glucosaminyl units, as noted by the upfield anomeric 1H-resonances with δH at 5.38 and 5.30 ppm for B1 and C1, respectively (Figure 6B,C and Table 5). The residue assigned as B denotes N-acetylated α-glucosamine units, regardless the 6-substitution, due to the downfield shift of the H2 around 0.5 ppm (from 3.28 ppm of GlcNS to 3.86 ppm of GlcNAc, Table 3), which is typical of acetylation-related shift. The 6-substitution (sulfate ester or hydroxyl group) has a long-range chemical influence on the ring; therefore, the assignment is dubious based solely on the 1H-chemical shifts obtained from 1D NMR experiments (see B6 assignments at Table 5 as not determined). Conversely, the 6-position of the C unit, noted as C6, is relatively easy for assignment (Figure 6B,C). A 0.5 ppm upfield shift is typical of nonsulfation (Table 5). This undoubtedly indicates an N-sulfated and 6-unsulfated α-glucosaminyl residues for the unit denoted as C. The 6-unsulfation and N-acetylation features of α-glucosamine units can affect the 1H-chemical shifts of adjacent 2sulfated α-IdoA residues in different ways. The anomeric 1H-

Surprisingly, the 1D 1H spectrum reveals considerable amounts of 1H-anomeric of GlcA residues with sulfate esters located at the less common 2- and 3-positions (G2S at δH 4.73−4.8 ppm and G3S at δH 4.61−4.72 ppm; Table 4). The amounts of sulfated GlcA units are higher than the nonsulfated units since the nonsulfated GlcAs with upfield anomeric 1H-chemical shift at around 4.6 ppm (see 1H-signals labeled as G1 in the spectra of Figure 6A) have smaller intensity when compared to the sulfated GlcAs labeled as G1(SO3−) in the spectrum of panel A, Figure 6, even though water-resonance disturbance may be seen. These assignments are in full accordance with previously published data.165 The spectral width shown in Figure 6B and 6C (expansions from 6.1 to 1.8 ppm for HS/Hp samples) can cover all 1Hsignals of the two types of therapeutic UFH samples. These signals are quite useful to assess the chemical features of Hp structures and can be used to establish comparison between therapeutic samples coming from different sources of origin. In the current case, we are showing the 1D 1H NMR spectral profile of Hp samples from bovine and porcine intestinal mucosa (parts B vs C of Figure 6). This tissue is the main source explored for large industrial scale extraction and production of Hp material destined to the global pharmaceutical market. The 1D 1H NMR spectrum of the clinical porcine Hp preparation is consistent with a chain mostly composed of trisulfated disaccharide repeating units of alternating 4-linked 2sulfated α-IdoA (notated in the 1D 1H NMR spectra as I) and 80



Review

and H5-chemical shifts from GlcNS-linked α-IdoA2S units (noted as I1-C and I5-C, respectively) are slightly downfield shifted due to the lack of 6-sulfation (Table 5) likely because of β-effects. In contrast, anomeric 1H (I1-B), but not H5 (I5-B), is downfield shifted due to the presence of N-acetylation (Table 5). Consistently, the peak areas from the C1-signal (δH at 5.30 ppm) are equivalent to those from I5-C (δH at 4.84 ppm), those of B1 + C1 (δH at 5.23 ppm) are similar to I1-B + I1-C (δH at 5.23 ppm), and those from A1 + B1 (δH at ∼5.39 ppm) are similar to I5-A + I5-B (δH at ∼4.09 ppm) (see Figure 6B and Table 5), as expected based on nuclear content. Hp may also contain small amounts of β-GlcA units113 (Figure 1B). The occurrence of these residues in both bovine and porcine UFH can be seen by the 1H anomeric signal that resonates at 4.65 ppm (G1 in Figure 6B,C), which is more upfield than the I1 resonances. If integrals are the parameter of interest in the 1H NMR-based analysis, an underestimation of the G1 signal can occur as opposed to I1 signals because of the closer resonant frequency of G1 to the residual water signal (4.7 ppm at 298 K). The consequential side effect of the HOD suppression by presaturation utilized in the 1D 1H NMR experiments is the attenuation of G1 signal intensity. One way to overcome this resonance overlap is to play with different temperatures. This was accomplished previously, and the correct integral values were obtained.112 Therefore, on the basis of 1H-chemical shift assignments and the respective integral values of related signals, the following conclusion about the structures of Hp samples from the two sources can be reached: porcine intestinal Hp is composed mostly by the trisulfated disaccharide units of [→ 4)α-IdoA2S-(1 → 4)-α-GlcNS-6S-α-(1 →], while the α-glucosamine units of bovine Hp vary significantly: ∼50% are 6- and N-disulfated, as in porcine Hp, while ∼36% are 6-unsulfated, and ∼14% are N-acetylated, instead of carrying the N-sulfation. On the basis of this data, the statement that Hp compositions may vary accordingly to organisms of extraction regardless of the tissue type is entirely true. In addition to most of the above-described resonances of the 1D 1H NMR spectra of UFH samples (Figure 6B,C), the spectrum of enoxaparin has the signals labeled as ΔU1 (δH at 5.46 ppm), ΔU4 (δH at 5.95 ppm), AnM1 (δH at 5.53 ppm), and AnA1 (δH at 5.58 ppm), as shown in Figure 6D. These resonances come from residues that are generated during the depolymerization process of UFH. Enoxaparins are produced by chemical β-elimination reactions, which generates 4,5unsaturated uronic acids (ΔU) located at the nonreducing terminus.169 This residue is 2-sulfated since it derives from the IdoA2S unit. However, nonsulfated unsaturated residues can be also observed, but mostly through the 13C-HSQC spectrum.111 N-Sulfated, 6-sulfated glucosamine units prevailed at the reducing end as either glucosamine (A)- or mannosamine (M)-anhydro (An) derivatives (AnA and AnM). Hence, the nomenclature AnA and AnM correspond, respectively, to Nsulfated 1,6-anhydro β-glucosamine or β-mannosamine. These cyclic structures are originated from alkaline hydrolysis of the benzoyl ester of UFH.170 Minor signals associated with undersulfated or unusual Hp sequences can also be observed in the NMR spectra of enoxaparin samples, and a more detailed discussion and NMR analysis are found in ref 111. On the basis of the 1D 1H NMR profile of fondaparinux, it becomes clear that better spectral resolution with sharper peaks and well-resolved splitting (Figure 6E) can be reached as compared to the polymeric Hp materials (Figures 6A−D). These two characteristics are likely derived from the lower-MW

of the oligomeric structure of fondaparinux, which results in a faster tumbling of this molecule during the NMR experiment and thus lower rotational correlation time (τc) values. This faster motion allows a rapid relaxation process that will end up in producing narrower 1D 1H-signals (Figure 6E). This is additionally enhanced by the simpler structure of fondaparinux. Better spectral resolution makes more suitable NMR chemical shifts assignments, since resonance overlap is significantly decreased in this case. Nonetheless, we can still observe some superimpositions but not as dramatic as in the spectra of polymers (Figure 6A−D). The 1H-chemical shifts of fondaparinux were tabulated accordingly to the resonance positions within the spectrum (Figure 6E), using the correspondent nomenclature of protons per units following the residue labels in Figure 1C (Table 6). The 13 C-chemical shifts of fondaparinux that are shown in Table 6 were extracted from ref 170 with permission. Copyright 1995 Elsevier. Table 6. 1H- and 13C-Chemical Shifts (ppm)a of Fondaparinux, the AT-High-Affinity Hp Pentasaccharide unitb

signal

δ (ppm)c

signal

δ (ppm)d

A

H1 H2 H3 H4 H5 H6/H6′ H1 H2 H3 H4 H5 H1 H2 H3 H4 H5 H6/H6′ H1 H2 H3 H4 H5 H1 H2 H3 H4 H5 H6

5.63 3.26e 3.61 3.59 3.90 4.17/4.38 4.63 3.43 3.84 3.85 3.78 5.52 3.46 4.35 4.00 4.17 4.50/4.27 5.18 4.31 4.16 4.16 4.75 5.02 3.29 3.66 3.79 3.97 4.41/4.16

C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C1 C2 C3 C4 C5 C6

99.65 60.06 73.21 71.01 71.79 68.60 103.30 74.84 78.17 78.91 79.14 98.13 58.74 78.26 74.90 71.61 68.31 101.68 79.40 72.50 78.12 72.43 100.29 59.79 71.85 78.37 70.60 69.05

G

A*

I

AM

a

Chemical shifts are relative to external trimethylsilylpropionic acid to 0 ppm for 1H at 25 °C. bSee Figure 1C for labeling of units. cChemical shifts obtained from spectrum of Figure 6E. dChemical shifts obtained from ref 170. eSites of O-, N-sulfation, and glycosylation are highlighted in bold, bold/italic, and italic, respectively, for rapid notation.

In summary, a lot of useful information regarding structure and integrity of Hp materials destined to the pharmaceutical market can be reached by 1D 1H NMR spectra. This type of NMR experiment is structurally information-rich and very precise in terms of measuring the peak intensities and integrals. This is because no considerable magnetization losses happen in 81



Review

Figure 7. Assigned 15N- (A−C) and 13C- (D and E) HSQC spectra of HS (A, C, and E), and UFH (B and D). Spectral expansions from δH 8.5 to 8.1 ppm and δN 118.0 to 124.6 ppm (A−C) and from δH 6.1 to 3.1 ppm and δC 45.0 to 116.0 ppm (D and E). The HS in panel A was from cultures of chinese hamster ovarian cells,28 the UFH in panels B and D was from a pharmaceutical supplier, and the HS in panels C and E was extracted from the bivalve N. nodosus.24 This type of invertebrate HS is shown here just for illustrative purposes to prove the applicability of the 15N-HSQC method in structural determination of the IdoA/GlcA ratio in Hp/HS samples. The HS has considerably low amounts of IdoA, as opposed to Hp. Note that UFH and HS have, respectively, low and high amounts of GlcA as well as, respectively, high and low amounts of IdoA, as demonstrated by the tiny or lacking color-coded peaks in their respective 13C-HSQC spectra (panels D and E). In these panels, the letters used are A for glucosamine (GlcNX), I for iduronic acid (IdoA), G for glucuronic acid (GlcA), NAc for N-acetyl, NS for N-sulfation, 6X for 6-(un)substituted, 2S for 2-sulfation, XS for 2- and/or 3-sulfation, and 3S for 3-sulfation. For panels D and E, the numbers after the capital letters designate the ring position of the substitutions. For example, I52s means the cross-peak from the 1H5/13C5 pair of a 2-sulfated IdoA ring. The colors indicate blue for GlcNAc, red for GlcNS, green for IdoA, and yellow for GlcA. The expected adjacent residues are indicated between parentheses. The 15N-gHSQC spectra (A−C) were recorded at Varian 800 MHz, with samples dissolved at 50 mM sodium acetate, pH 4.5, at 298 K, whereas the 13C-HSQC spectra were recorded from samples dissolved at 99.9% D2O, at 298 K, either with the (D) Varian 800 MHz or (E) Bruker 400 MHz (note the difference in resolution quality due to different laboratory magnetic fields and also different MWs). The plot D is just partially assigned. All data adapted with permission from ref 24 (Copyright 2010 American Society for Biochemistry and Molecular Biology) and data adapted from ref 28 (Copyright 2010 American Chemical Society).

1D 1H NMR as opposed to those that occur during the polarization transfer in multidimensional experiments. In addition, when the signals are placed far away from the residual water resonance, trustworthy information can be raised. 1D 1H NMR is also the fastest NMR experiment to be acquired among all types of the NMR experiment. As mentioned before, the detection of the 15N-related properties of GAGs is dependent on the INEPT step in HSQC experiments. This allows proper polarization transfer between 1 H and the heteronucleus, which will result therefore in the 1 H/15N connectivities observed in the resultant spectra. As above-stated, the 15N NMR method has high applicability in analysis of GAGs due to the small number of peaks, associated with the wealth of structural information that can be raised from this NMR method. However, at pH 4.5 and 25 °C, as

used to construct plots A−C of Figure 7, the sulfamate (NHSO3−) groups of HS and Hp (Figure 1B) have their proton exchange occurring rapidly on the NMR time scale, resulting, therefore, in loss of magnetization to the bulk water and signal attenuation. The 1H/15N pairs of sulfamate-attached sites occur just too fast and thus become undetectable under this experimental condition. This might be taken as an analytical limitation to this type of GAGs under this condition and methodology employed. Because of the fast-exchangingrate regime of the sulfamate protons with the solvent protons, no resonances other than those exclusively related to the GlcNAc can be seen in spectra of panels A−C of Figure 7, under the above-mentioned experimental conditions. Other works regarding 15N NMR research on Hp and HS using different experimental conditions to overcome this lack of NH 82



Review

Figure 8. (A) Fully assigned 1D 1H NMR spectrum (expansion from δH 5.5 to 1.0 ppm, which covers all signals) of sodium salt HA from Lifecore Biomedical CO. (Chaska, MN). The letters N and U indicate GlcNAc and GlcA units, respectively. The numbers after these letters indicate positions of hydrogen atoms within the respective monosaccharide ring. The HA sample was dissolved in deuterated dimethyl sulfoxide, and the spectrum was recorded at 310 K in a Bruker 500 MHz. Data modified with permission from ref 91. Copyright 2009 Elsevier. (B and C) Assigned 1H/15N-HSQC spectra of HA-derived oligosaccharides, from tetrasaccharide (HA4) to decasaccharide (HA10). Expansions from (B) δH 8.31 to 8.0 ppm and δN 121.8 to 123.0 ppm and (C) from δH 8.08 to 8.01 ppm and δN 121.92 to 122.16 ppm. The Greek letters indicate amide signals from GlcNAc units differentially positioned within the HA oligosaccharide chains (see Table 7 for chemical shifts and position recognition). Green, magenta, selective yellow, and royal blue are the colors utilized to distinguish resonances from HA4, HA6, HA8, and HA10, respectively. Samples were dissolved in 10% D2O, 0.02% NaN3, pH 6.0, and spectra were recorded at 298 K in a 750 MHz instrument. Data modified with permission from ref 157. Copyright 2004 Oxford University Press.

signal have been conducted.151−153 Nevertheless, both uronic acid types in HS and Hp can be easily identified based on differential 1H-/15N- chemical shifts, even using the limited experimental conditions in which N-sulfated 1H/15N pairs are undetectable. The 1H/15N cross-peak of GlcNAc-linked GlcA resonates at δH/δN of 8.37/123.7 ppm, while the 1H/15N crosspeak of GlcNAc-linked IdoA resonates at δH/δN of 8.28/123.8 ppm. These resonances are also fairly quantized based on their integrals (Figure 7A−C). The distinct IdoA/GlcA ratio is one of the major structural differences between HS and Hp, and this can be proved based on differential intensities seen in the spectra of these GAG types. There is an intense GlcA-related signal in both 15N- and 13C-HSQC spectra of HS (Figure 7, panels A, C, and E) as opposed to a very low or absent GlcArelated signals and intense IdoA-related signals in both 13C- and 15 N-HSQC spectra of Hp (Figure 7, panels B and D). The major problem in the use of the 15N-related analytical methodology on analysis of GAGs is that this approach works out only for those acidic units necessarily linked to the GlcNAc units, since the adjacent units linked to GlcNS units are unobserved at the used conditions, as opposed to the 13Crelated HSQC method in which all signals are detected regardless the N-substitution and experimental conditions. It is

worth saying that the IdoA/GlcA ratio is highly used to distinguish HS from Hp and vice versa, if not, the main characteristic employed to separate them analytically. Even though structural information is coming only from the residual GlcNAc units, the integrals of the 1H/15N cross-peaks are still very diagnostic and fair enough to allow proper identification of the GAG subfamilies Hp versus HS (Figure 1B) when they are subjected to the 15N-HSQC method (Figure 7A−C). Hyaluronic Acid. The 1D 1H NMR spectrum of HA is shown in Figure 8A. From the 1H-resonance profile of this spectrum, a high degree of chemical shift degeneracy can be noted. Only protons from the anomerics (N1 for GlcNAc H1 with δH at 4.54 ppm and U1 for GlcA H1 with δH at 4.42 ppm), carbon-2 (N2 and U2 with δH at 3.90 and 3.31 ppm), and methyl groups (noted as CH3 δH at ∼2.0 ppm) are resolved and readily available for assignments. Conversely, the remaining eight peaks are still quite squeezed between 3.87 and 3.42 ppm and their chemical shift assignments are impaired by resonance overlap and dubious notation. For these cases, spin connectivities by 2D homonuclear experiments are essential to make definitive chemical shift assignments, as detailed further. 83



Review

HA seems the GAG type of 1D 1H NMR spectrum with the lowest spectral resolution, since low levels of 1H-resonance dispersion is observed therein. This is likely a consequence of (i) broader resonances together with (ii) low peak dispersion. The first feature is originated from the longer τc values of this GAG type, since HA molecules show very a high degree of polymerization together with very low hydro-solubility. The low water-solubility is caused by the absence of sulfation. Highmass HA forms gel easily in solution.140 This significantly restricts the molecular tumbling at the same time it increases the τc values of HA. The lower degree in structural complexity of this GAG type, besides facilitating assignment, is conversely a great contributor to resonance overlap. This is the reason for the second feature mentioned above. Anyway, all these two features collaborate together to make the 1H NMR spectral resolution of HA the poorest among all GAG types. In order to resolve high levels of resonance degeneracy in NMR spectra of HA, Colebrooke and co-workers have prepared 13C-enriched HA-tetra and hexasaccharides for 13Crelated NMR experiments to allow assignments of carboxylaterelated chemical shifts.171 Two modified pulse sequences have been used to extract structural information from chemical shifts of the carboxylate moieties of HA-derivatives. This strategy can filter out the signals from N-acetylated sugars and produce simple spectra containing resonances solely from the acidic sugars in HA samples. One pulse sequence allows one-bond couplings to be built between the carboxylate carbon with the adjacent carbon and it’s directly attached proton, while another pulse sequence exploits a long-range coupling to correlate the carboxylate carbon with anomeric proton and carbon of the same residue. In addition, inclusion of isotropic mixing block into these sequences allows resonances from the otherwise degenerate ring protons to be resolved. A series of spectra of 13 C-labeled HA-tetra and hexasaccharides have shown that all composing GlcA units in these samples can be resolved and assigned from one another, allowing nuclear chemical shifts to be assigned in a sequence-specific manner.171 Besides 2D 13C-related NMR spectra of 13C-enriched HAderivatives171 or 13C-direct detection,140 another way to overcome this problem of 1H-NMR chemical shift degeneracy in HA that makes difficult both spin and structural assignments is to employ the novel 15N NMR approach. It will select resonances only from the amide groups of the GlcNAc units in HA molecules. This fact can be even ameliorated by using lowMW HA-derivatives that might have higher hydro-solubility properties in addition to faster tumbling and motions in solution which would help to shorten the rotational correlation time. As mentioned earlier, even though few in numbers, the 1 H-/15N-chemical shifts from 15N-HSQC experiments of GAGs are still very useful and diagnostic for structural features, particularly for oligosaccharides (see again Figure 5 for the example of CS oligosaccharides). This successful application can be seen in Figure 8B,C of the 15N-HSQC spectra of HA oligosaccharides (4- to 10-mers).157 Note that on the basis of differential 1H- and 15N-chemical shifts (Figure 8B,C and Table 7), the amide groups can be easily assigned and recognized in terms of the specific positions of their GlcNAc units within the oligomeric chains (Figure 8C and Table 7). The anomeric configurations of the reducing end GlcNAc units can be easily assigned and properly recognized as well (Figure 8B and Table 7). Keratan Sulfate. KS is the least GAG type studied by NMR spectroscopy and perhaps by any other analytical technique.

Table 7. 1H- and 15N-Chemical Shifts (ppm) of HA Oligosaccharides with Varying Lengthsa HA oligosaccharide ring position α β γ δ ψ ω

nucleusb 1

H 15 N 1 H 15 N 1 H 15 N 1 H 15 N 1 H 15 N 1 H 15 N

HA4

HA6

HA8

HA10

8.188 122.943 8.271 122.089

8.189 122.947 8.271 122.098 8.050 121.990

8.191 122.953 8.271 122.101 8.052 121.996

8.044 122.099

8.042 121.954 8.042 122.097

8.191 122.951 8.271 122.098 8.053 121.993 8.040 121.942 8.043 121.950 8.042 122.093

8.055 122.134

a

Reprinted with permission from ref 157. Copyright 2004 Oxford University Press. b1H ±0.005 ppm, 15N ±0.005 ppm.

This is likely a consequence of its limited abundance together with the considerably lower number of biological actions of KS when compared to the other GAG families, especially HS and CS. As stated before, an additional peculiarity of KS is it’s absence of an acidic unit, which is replaced by the neutral sugar Gal (Figure 1E). This structural peculiarity gives rise to a differential 1D 1H NMR spectrum (Figure 9A). 1H NMR spectrum of KS showed a distinct peak distribution of 1Hsignals as compared with those from the other GAGs (Figures 2, 6, and 8A). The 1H/1H correlation spectroscopy (COSY) spectrum54 and especially the 1H/13C-HSQC spectrum (Figure 9B) of KS enable proper assignments of several signals attributable to this particular GAG type. Through the edited 1 H/13C-HSQC spectrum, in which the phased CH signals (orange signals in Figure 9B) can be easily distinguished from antiphased CH2 signals (in green), a series of structural information can be generated as discussed next. First, two characteristic β-anomeric 1H/13C-signals with δH/ δC at 4.68/102.85 and 4.49/102.93 ppm can be identified and, respectively, attributed to residues of GlcNAc (noted as N1) and β-Gal (denoted G1) (Figure 9B and Table 8). These signals are in approximately equimolar proportions, as conceived by the pattern of disaccharide repeating unit of GAG-like molecules, which for the case of KS is composed of alternating 4-linked GlcNAc and 3-linked Gal residues, both at their β-anomeric configurations (Figure 1E). Second, two clear signals involved in glycosydic bonds with δH/δC of 3.70/78.91 and 3.69/82.28 ppm (denoted N4 and G3, respectively, in Figure 9B and Table 8) can be assigned. Aside from the anomerics that contain the most downfield 13C-chemical shifts, the glycosydic linkage-related resonances have also typical downfield 13C-chemical shifts when compared to the other remaining signals (Table 8). This is an intrinsic aspect of glycosidic bonds noted by 13C-chemical shifts, since the 13C of the acetal group seen by the glycosidic bond is placed nearby the electronegative oxygen of the linkage. The two cross-peaks (N4 and G3) seen from spectrum of Figure 9B belong necessarily to the 1H/13C-signals of 4-linked GlcNAc and 3linked Gal residues. The assignments of both these signals are consistent with the glycosidic linkage types of KS molecules (Figure 1E). Finally, the H2 assigned at ∼3.55 ppm through the 1 H−1H COSY spectrum of KS (see ref 54) unequivocally 84



Review

Figure 9. (A) Partially assigned 1D 1H NMR spectrum (expansion from δH 5.45 to 1.50 ppm, which covers all signals) of bovine cartilage KS.54 The letters N and G indicate GlcNAc and Gal units, respectively. The numbers after these letters indicate positions of hydrogen atoms within the sugar ring. (B) Partially assigned 1H/13C multiplicity edited HSQC spectrum of KS (expansions from δH 5.0 to 3.0 ppm and δC 50.0 to 104.0 ppm). This spectral window shows all resonances except that related to the methyl group. The numbers following letters indicated position of 1H/13C crosspeaks. SO3− and non-SO3− stand for sulfated and nonsulfated sites. Green and orange peaks denote antiphased (negative) CH2 signals and phased (positive) CH peaks, respectively. Data adapted with permission from ref 54. Copyright 2012 Elsevier.

Table 8. 1H and 13C-Chemical Shifts (ppm) from Signals Identified in the 2D NMR Spectra of Fraction KS Compared with Literature Dataa literature datab

values from Figure 9 signal H1 H2 H3 H4 sulfated H6 nonsulfated H6 C1 C2 C3 C4 sulfated C6 nonsulfated C6

4-GlcNAc-β-1 (N)

3-Gal-β-1 (G)

4.68 3.79

4.49 3.53 3.70

3.71 4.33/4.27 3.69 102.83 55.13

4.15 3.58/3.50 102.86 69.97 82.22

78.81 66.65 56.90

67.72 62.80

4-GlcNAc-β-1 4.76 3.83

3-Gal-β-1 5.54 3.79

∼3.80 4.32/4.39 105 57

4.22 105 85

81 69

70

a Reprinted with permission from ref 54. Copyright 2012 Elsevier. bData from ref 172. The sulfation and glycosylation sites are highlighted in bold and italic, respectively.

and to the downfield H2 of GlcNAc with δH at ∼3.8 ppm (Table 8). Information about 6-sulfation of GlcNAc and Gal residues can be easily derived from analysis of the antiphased peaks showed in orange in Figure 9B. We can identify two signals with δH/δC at 4.37−4.23/66.58 and 4.20−4.08/67.67 ppm,

confirms the presence of a Gal unit (Table 8), and KS is the only GAG type that bears this neutral sugar instead of an uronic acid unit, as previously mentioned in the section Keratan Sulfate. Gal units have a 1H-chemical shift of their H2 with δH at ∼3.55 ppm, as opposed to the upfield H2 resonances of GlcA with δH at ∼3.4 ppm (signal U2 of spectrum in Figure 3A,C) 85



Review

noted as 6-sulfated GlcNAc (N6 SO3−) and Gal (G6 SO3−) units, respectively (see also Table 8). These signals are ∼0.7 ppm shifted downfield in the 1H-scale and ∼8 ppm in the 13Cscale from a more heterogeneous group of signals assigned to nonsulfated residues. Overall, these results have clearly proved that KS have 6-sulfation at both composing residues, although 6-sulfation occurs more at GlcNAc units. See peak-intensities of 6-sulfated Gal (noted as G6 SO3−) versus 6-sulfated GlcNAc (labeled as N6SO3−) in Figure 9B. All the above-mentioned data about the structural characterization of KS by 1D 1H and 13 C-HSQC experiments is in full agreement with the structural features depicted in Figure 1E. Because of the lower amounts of NMR studies on KS and likely to the limited abundance and commercial availability of this type of GAG, published 15N NMR data of KS are virtually nonexistent. This might represent a promising and demanding avenue for future NMR studies in glycosaminoglycanomics. Pros and Cons of Using 1H-, 13C-, and 15N-Isotopes in Solution NMR Structural Analysis of GAGs. Since this material is treating the role of NMR chemical shifts on the analysis of GAGs by their three magnetically active atomic nuclei (1H, 13C, and 15N), a specific discussion concerning the benefits versus disadvantages of each particular nucleus should be reasonable at this point. 1H-based NMR methods are fast and sensitive since this nucleus has the highest natural abundance together with the highest relative NMR receptivity (Table 2). When 1D 1H-signals are resolved, their integration values are very reliable in terms of the nuclear amount. This represents an important feature in NMR analysis of GAGs since this amount means the proportion of the structural variations and substitutions in GAGs and they can be measurably quantized and expressed in terms of percentage per analyzed sample. As observed by the 1D 1H NMR spectra of GAGs (Figures 2, 6, 8A, and 9A), 1H-chemical shifts are readily diagnostic of many valuable features, however, still very complex for assignments and highly suitable for resonance overlap, especially in the case of HA (Figure 8A). For these cases of low dispersion of signals, the acquisition of the 2D NMR spectrum followed by proper spin assignments are highly necessary in order to permit decisive identification of the ambiguous peaks in the 1D spectrum. Moreover, 1H NMR linewidths of GAG samples are usually broader since relaxation times of this nucleus are longer than carbon as explained further. These broader 1H-linewidths are another contributing aggravating factor in chemical shift assignments, since they can increase the chances of peak superimposition. Nonetheless, NMR signals from 1H are still the worldwide preferential choice in NMR spectroscopy for analysis of GAGs, mainly because of the amount of structural information available in the literature and the easiness for routine analysis together with the fact that proton nucleus is the one of highest sensitivity and abundance (Table 2). It allows the fastest acquisition for NMR data collection, and no magnets of ultrahigh-fields are needed if reasonable sample concentration is not a matter of concern. Sample availability is not a serious problem in the case of GAGs coming from sources of large industrial scale production. The NMR spectrum containing carbon directly observed is rarer in structural analysis of GAGs. Besides the very low sensitivity, it only provides 13C-chemical shifts since no internuclear correlations are involved. Assignments solely based on this technique are difficult. Although 1D 13C-signals are considerably narrower than those of 1D 1H, 2D 13C-based NMR heteronuclear spectra like HSQC, heteronuclear multiple

quantum coherence (HMQC), and heteronuclear multiple bound coherence (HMBC) are under more utility in glycosaminoglycanomics because they provide a more robust approach in terms of chemical shift assignments. Correlation between 13C- and 1H-atoms and respective chemical shifts can be generated from these experiments. 13C-signals are usually observed narrower than 1H-signals because, in general, 13Catoms in sugar rings are mostly secondary carbons attached to paramagnetic oxygens. The presence of the paramagnetic oxygens speeds up the spin-relaxation of neighboring atoms, including carbon-13. Therefore, the longitudinal relaxation of 13 C-atoms in regular hexose rings is usually faster than those of 1 H. This gives rise to lower tendencies of resonance overlap in 13 C-directly observed spectra of GAGs.140,173 See ref 174 for a good example of 1D 1H- versus 13C NMR spectral resolution of HA-derived oligosaccharides. Nonetheless, it is from the 1 H/13C NMR experiments that information regarding anomericity, residue type, linkage patterns, and sulfation sites of GAG molecules is straightforwardly extracted, since the specific structure-related resonances occur in just certain regions of the 2D spectra (see Figures 3, 7D,E, and 9B). Besides the 1Hchemical shift correlation seen in these heteronuclear 13C-based NMR experiments, the magnetization transfer back to 1H during the INEPT step in those pulse sequences is a key procedure to enhance sensitivity, as mentioned before in Chondroitin Sulfate and Dermatan Sulfate. The sensitivity enhancement by INEPT in 13C-based experiments is in the factor of 4 while for 15N-related experiments are in the factor of 10. In addition, 13C-based heteronuclear spectra are much more populated and suitable for resonance overlap than 15N-based ones. Nevertheless, 13C-chemical shifts can be also extremely useful in terms of identification of structural features in GAG molecules (see information from Tables 1, 4−6, and 8). This was proven with the case of GAG oligosaccharides of welldefined chemical structures. In this review, we have chosen CS hexasaccharides as examples of an explanation for this type of NMR approach. Their 1H/13C-chemical shifts (Figure 3 and Table 1) are usefully diagnostic for the proper recognition of both sulfation types and patterns through a very straightforward way of analysis (discussion in Chondroitin Sulfate and Dermatan Sulfate). Although NMR analysis of GAGs via 15N-direct observation is virtually nonexistent, maybe some attempts using dynamic nuclear polarization might be under investigation nowadays.175,176 However, 15N NMR heteronuclear experiments have already proven its usefulness and viability for GAG analysis.28,114,151,152,154 This approach has two great advantages. First, it is based on rather simple spectra with just a few easily identifiable cross-peaks (see Figures 4, 5, 7A−C, and 8B,C). Second, although few in number, these signals and their respective chemical shifts still allow rapid assignments and identification of several structural features of GAGs such as anomericity and ratio (α versus β, Figure 8B), hexosamine type (GlcNAc versus GalNAc, see differential 1H-chemical shifts of these sugars in Figures 4 and 7), sulfation sites (Figures 4 and 5 and Table 3), and uronic acid contents (Figure 7A−C).28 This is really useful for analytical purposes in glycosaminoglycanomics. However, the 15N-based NMR approach still has some downsides. Besides the sensitivity issue discussed above, a loss in the ability to quantitatively relate 1H/15N cross-peaks to molar amounts of different constituents of GAG polymers was reported.28 As previously stated, HSQC experiments depend on 86



Review

Figure 10. Partially assigned 1D 1H NMR spectra of (A) a porcine Hp sample (400 MHz, at 315 K, in 99.9% D2O) containing considerable amounts of CS-A/CS-C, DS, and OSCS29 with 1H-acetyls assigned accordingly to each GAG type and (B) a representative pharmaceutical grade CS from shark cartilage (600 MHz, at 298 K, in 99.9% D2O) containing 16% of KS.54 In this latter case, 1H-signals at the anomeric region are diagnostic of contamination. Data modified with permission from ref 29 (Copyright 2010 Springer) and ref 54 (Copyright 2012 Elsevier).

Function. GAGs destined for pharmaceutical and clinical market are usually extracted at the large industrial scale from mammalian tissues such as bovine and porcine intestinal mucosa for the case of Hp39,112 (1D 1H NMR spectra at parts B and C of Figure 6, respectively), from cartilage tissues such as those from bovine tracheal and shark for the cases of CS-A and CS-C, respectively,90 (1D 1H NMR spectra in parts A and C of Figure 3, respectively), from bovine cornea for the case of HA136 (1D 1H NMR spectrum at Figure 8A), and from bovine cornea stroma in the case of KS143,145,177 (1D 1H NMR spectrum at Figure 9A). All these GAGs are readily available commercial products, usually found in their sodium salt forms. The counterion type is influential to the chemical shifts, especially of those atoms where the cations are located nearby such as sulfated sites or the more sulfated polymers.29 NMR spectroscopy has been adopted as the main analytical technique to certify the structural integrity of the main GAG ingredient of those medical formulations as well as to assess, identify, and quantify the possible presence of contaminants, inclusively those of GAG nature, as discussed next.

the transfer and refocusing of magnetization over time intervals during the pulse sequence. Magnetization is lost during these intervals in an inverse exponential relationship to proton linewidths, something that is significant when linewidths approach the values of scalar couplings. Within a given GAG type and GAG preparation, linewidths for different amide protons, even if large, may be very similar and lead to small errors in quantitation. Even though with the existing issue in quantitation of 15N-related cross-peaks, the 15N NMR approach that provides information about the 1H-/15N-chemical shifts is undoubtedly useful in producing structural information of GAGs. The 15N-based NMR approach seems the most promising in glycosaminoglycanomics nowadays.

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USE OF CHEMICAL SHIFTS IN DETECTION OF CONTAMINANTS IN GAG-BASED THERAPEUTIC PREPARATIONS One of the major interests of GAGs is the exploration of their relevant medicinal properties, as discussed previously for CS, Hp, HA, and KS in Glycosaminoglycans: Structure and 87



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contaminant.35 These are two procedures to be carefully considered during the quality control assays of Hp samples by the N-acetyl 1D 1H-NMR fingerprint. The other beneficial factor of using 1D 1H NMR in this contamination assessment is the high sensitivity of this particular NMR method, as discussed earlier. Spectra of Hp samples can be recorded in instruments of low magnetic fields (300 MHz) from samples of milimolar concentration at natural abundance. So no additional procedures for isotopic labeling must be performed in this analysis. Moreover, peak quantitation based on integral values of resonances from 1D 1H NMR spectra seems the most reliable and trustful one among all types of NMR methods. This is relevant for the estimation of the contaminant percentages within the Hp samples.31,36 However, accurate quantitation of 1H-signals require a high-quality spectrum, which can typically be achieved using high magnetic fields (≥500 MHz), optimum solution conditions such as best temperature, pH, and counterion type and concentration, and appropriate NMR parameters such as well-calibrated 90° pulses. Some investigators have shown that even using 300 or 400 MHz NMR instruments, the levels of detection (LOD) of OSCS can be as low as 0.1% with the proper 13C-decoupling procedure.36 These authors have analyzed over 100 Hp samples using the standard addition method and monitoring the Nacetyl region. They developed a routine 1H NMR-based screening for Hp, quantified both OSCS and DS, and proved the lack of correlation between these signals. In addition to quantifying OSCS and DS, Beyer et al. also reported other impurities presented in varying amounts in pharmaceutical Hp preparations, including methanol, ethanol, and acetate.36 In a subsequent study, the investigators scrutinized the German Hp market and analyzed 145 representative samples from the year of 2008.37 The samples tested by 1H NMR were found to contain DS (51%) and OSCS (19%) as well as process-related impurities such as ethanol, methanol, acetone, formic acid, and acetate in considerable amounts. As these process impurities remain undetectable by other methods such as capillary electrophoresis and liquid chromatography, it is anticipated that NMR may be more widely exploited in future pharmacopoeias. Keire et al. have also reported a 0.1% LOD for OSCS on a 500 MHz instrument using 25 mg/700 μL Hp sample solutions.40 This group has also identified additional native and oversulfated GAGs as possible economically motivated adulterants based on their characteristic chemical shifts in the N-acetyl region and the remaining 3.0−6.0 ppm region.40 CS-A, DS, OSCS, and oversulfated DS showed unique signal patterns when spiking the Hp sample, while HS, oversulfated HS, and oversulfated Hp were found to be difficult to identify.40 Overall, OSCS-contaminated Hp samples have been reported in countries from all over the world, especially Australia, Canada, China, Denmark, France, Germany, Italy, Japan, The Netherlands, New Zealand, and the United States. For more details concerning this topic of OSCS-contaminated Hp samples see refs 30, 33, 37, 41, 47, 49, and 178 and visit the Web sites of the U.S. Food and Drug Administration (FDA) as well as of some news such as The New York Times. Besides 1D 1H NMR spectroscopy, the 15N-related NMR approach seems also of potential utility to detect and assess OSCS contamination in clinical Hp samples. The 1H- and 15Nchemical shifts of GalNAc (δH = 7.94 ppm and δN = 121.21 ppm) and GlcNAc (δH = 8.36 ppm and δN = 123.62) (Figure 4C versus Figure 7B and Table 3) are quite different. In this case, the speed of the method could be considerably improved

A severe OSCS-contamination case of Hp lots was reported in 2007−2008.30,32,36 This contamination has led to approximately 200 deaths attributed to hypotension cases triggered by anaphylactic shock.30,32 This contamination was reported as very harmful to the health of the patients since the OSCScontaminant was established to induce the release of allergyrelated kallikrein,178 a blood pressure regulator that works via the activation of bradykinin, a potent vasodilator. Many reports have appeared in the literature concerning this accident,29−53 besides meetings and conferences that have been promoted worldwide in order to allow discussion concerning this topic among internationally renowned physicians and scientists of the field. The scientific community was concerned about two major issues. First, find ways to revert possible side clinical effects provoked by OSCS. Second, establish a safety analytical procedure or method for detection of possible contamination in Hp lots distributed across the international pharmaceutical market. Since we do not want to overlap information here, we will just point out the major conclusion and agreement from these gatherings and reports that may present a common dialogue between the fields of analytical chemistry, the OSCScontaminated Hp sample cases and NMR spectroscopy utility based on chemical shifts. On the basis of a series of studies, the 1 H NMR spectra was set as the major analytical method to detect possible GAG contaminants in Hp formulations. Many papers have been published in this regard.30,35−38,40,42,49,53,179 The major conclusion was that the acetyl 1H region of the spectrum (δ H around 2.0 ppm) could be used as a fingerprint.179 This region is of a straightforward diagnostic potential of the most GAG types as represented in the expanded spectrum of panel A of Figure 10. The signals in this specific spectral region can be relatively easily assigned in a relatively easy way. From the upfield to the downfield region, the 1H from the acetyl groups of CS-A and -C appears with δH at 2.03 ppm, and the 1H from the Hp acetyl group resonates with δH = 2.05 ppm, the 1H from DS acetyl group resonate with δH = 2.08 ppm, and the 1H from OSCS acetyl groups can be detected at δH = 2.15 ppm (Figure 10A). As we can note, in a mixture of components, the signals are slightly different since the GAG species show acetyl 1H-resoances of different chemical shifts, which make them fairly reliable to be used for quality control/assurance purposes in Hp-based biomedical preparations. As stated above, 1H-chemical shifts are counterionsensitive.29 The 1H-chemical shifts of OSCS N-acetyl methyl signals vary linearly from 2.13 ppm to 2.18 ppm with increasing amounts of Ca2+ until reaching a saturation point of four Ca2+ ions per tetrasulfonated disaccharide units.46 Besides counterion type and concentration, temperature and pH are also influential on the chemical shift values and line shapes, as mentioned above. This explains why caution must be taken when using unbuffered Hp solution samples for quality control.35 Acquiring spectra of mixed GAG samples or Hp samples subjected to analysis due to contamination risks at elevated temperature can significantly improve 1H resonances line shapes for a better diagnostic.36 This is another careful aspect to be considered when low-levels of OSCS contaminations are present. The other aspect is that the OSCS N-acetyl methyl 1H resonances are very close to the 13C satellites of the 1 H-signal of the N-acetyl methyl of Hp molecules. Therefore, they have a high chance to overlap when analyzed by the acetyl fingerprint. One way to overcome this problem is to use 13Cdecoupling to discriminate the satellite peaks from the 88



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by replacing the 2D 1H/15N-HSQC acquisitions with a 1D 15Nfiltered 1H-observed acquisition that resolves primarily based on proton chemical shifts. Interpretation of molecular characteristics of different species mixed in same GAG samples may be very straightforward by this mean. This also adds to the potentiality and usefulness of the recent 15N NMR approach. In this regard about therapeutic formulations of GAG types contaminated with GAG species other than those used as the main ingredient, we have analyzed by 1D 1H NMR spectroscopy and other analytical methods, 17 batches of pharmaceutical grade CS (14 from shark and 3 from bovine cartilages) that were obtained from Brazilian pharmaceutical companies. For our surprise, a considerable KS amount (∼16% of the total GAG amount) was detected in the CS batches, however, exclusively in those batches originated from shark cartilage sources.54 Note from plot B of Figure 10, together with Figures 2C and 9A that the 1H-anomerics signals from GalNAc (δH = 4.56 ppm) and GlcA (δH = 4.49 ppm) of shark CS-C are quite distinct from the GlcNAc 1H-anomeric signals of KS that have their δH at 4.67 ppm. This distinction is quite clear and may be used to assess possible KS-contamination in CS samples by 1D 1 H NMR spectroscopy, like for OSCS-contaminated Hp samples. Although, KS being an inert material naturally found in our body and not even closely harmful as OSCS, the presence of ∼16% of KS in CS formulations decreases significantly the amount of the real GAG ingredient specified in the label claims indicated by the suppliers.54 This contamination report urgently forewarns the following (i) manufacturers for improved isolation procedures and safety fractionation steps and (ii) the supervision agencies for better audits, in order to guarantee quality control/assurance of GAG preparations destined to the market. Since GAG materials are extracted from animal sources for a large industrial scale production, the possibility of remaining substances originated by rough isolation procedures is high. Hence, careful analysis must be done in order to avoid this possibility of carrying extra substances other than the main GAG ingredient, even though the extra substance being another GAG type and inert to human health. This was seen in the report about KS-contaminated CS formulations.54 Unfortunately, this was not the case of the more aggravating OSCScontaminated Hp-sample case.33 In this latter case in which deaths of patients have been reported, the contamination was concluded to be purposeful, since the suppliers or producers have intentionally added OSCS to the raw Hp product, resulting thus in an illegal action of drug adulteration.35

This section is dedicated to offer, through a single example, a reasonable explanation about how the changes in NMR chemical shift values can be helpful to the generation of function-related data in GAG analysis. For this, we will take as an example, a classic case in the literature involving NMR spectroscopy and GAG−protein interaction. This illustrative case describes the changes of 1H-chemical shifts of fondaparinux (Figure 1C and Table 6) upon binding with the recombinant functional extracellular domain Ig2 of the fibroblast growth factor receptor (FGFR2).180 Analyses of 1 H-chemical shifts were done for the free ligand in comparison with 10:1 ratio of the pentasaccharide/FGFR2 complex at 298 K in a buffer solution of D2O (sodium phosphate buffer (NaPi) 20 mM, NaCl 100 mM, pH 6.0). The largest chemical shift changes were found at the proton resonances of G5, which were 0.03 ppm downfield shifted, and for A*2 and A*3, which were upfield shifted, both by −0.03 ppm. These results have pointed to the specificity of residues and their respective protons in fondaparinux involved in binding with the receptor. They are the H5 of GlcA and H2 + H3 of the 3-O-sulfonated GlcNS (Figure 1C). Similar to the AT-binding event, the 3sulfated site of the GlcNS revealed itself again relevant to this GAG−protein interaction. Further analyses by NOE, Jcoupling, and molecular dynamics have confirmed the involvement of these specific protons in the binding with FGFR2 and, moreover, have proved that upon formation of the complex FGFR-Ig2/AGA*IAM (fondaparinux), the protein receptor induces a conformational selection process that favors the 2S0 geometry of the flexible IdoA (I*) unit in fondaparinux in a similar way to that previously observed for AT.181 From this single example of the literature, the biological relevance of the rare 3-O-sulfonation in GlcNS units in protein-binding events has been validated by two distinct results: (i) the interaction contribution of A*3 during contact within the protein FGFR2, as seen by its 1H-chemical shift change, and (ii) the conformational selection similar to that induced by AT, in which the 3-O-sulfonated site is a pivotal structural requirement to this event. Above these conclusions, the just mentioned example of the literature have shown the utility of NMR chemical shifts in interpreting a biological phenomenon driven by molecular complex formation. This is another application of NMR spectroscopy, via chemical shift analysis, in the studies of structural biology of GAGs.

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CHEMICAL SHIFT PREDICTION OF GAGs Chemical shift prediction is a requirement to the progress of biomolecular NMR and it has successfully been implemented to protein analysis,181−183 even at their denatured states.184 Chemical shift prediction is an advanced tool in biomolecular NMR that helps the identification of secondary structures of the molecules and the generation of torsion angle constraints that might be supportive for structure determination. Chemical shift prediction has been implemented primarily for proteins since the volume of NMR-based information related to these molecular types are fair enough to establish the relationship between structural variations and chemical shift values and to predict the tendencies of changes. However, as mentioned before, the application of NMR spectroscopy is relatively recent to carbohydrates.5 Nowadays, however, the amount of deposited NMR-based information of sugars has considerably grown, in a way to allows chemical shift prediction even for carbohydrates.185−187 A computerized approach named CASPER, an acronym for Computer Assisted SPectrum Evaluation

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NMR CHEMICAL SHIFTS IN FUNCTIONAL STUDIES OF GAGS Chemical shifts are not only useful to decipher structural properties of biomolecules in multiple experimental conditions but also to unveil some of their biological functions, especially those in which dynamics and intermolecular interactions are the contributing features to trigger the action. Migrations of chemical shift values are informative of both conformational fluctuations and dynamic behaviors. Hence, this kind of data is likely useful to generate understandings about the underlying mechanisms involved during a biological phenomenon. This may include the cases of GAG molecules with respect to their molecular interactions with proteins during cell signaling events.60,64,65,67,68,88,90 89



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deposited in the scientific bibliographic databases. In addition, a critical discussion about (i) the pros and cons of using each of the three magnetically active nucleus of GAG molecules (1H, 13 C, and 15N), (ii) the utility of NMR chemical shifts in the assessment of GAG contamination in GAG-based biomedical formulations, and (iii) the possibility of NMR chemical shift prediction of GAGs. Although thoroughly supported, this comprehensive review about NMR chemical shifts of GAGs is far from survey all tabulated 1H-, 13C-, and 15N-chemical shift values of the major GAG types and derivatives in solution. Chemical shifts of GAGs and derivatives are expected to vary accordingly to extractions, compositions, sources, and physical conditions (temperature, pH, counterion type and content, etc). Samples and conditions other than those used in this manuscript are likely to produce, although slightly, chemical shift of different values than those reported herein. Just as a simple matter of clarification, the counterion used for the majority of the GAG samples analyzed in this manuscript is sodium. Publications containing other salt forms than the sodiated form are likely to provide NMR chemical shifts with slightly different values (see ref 29 as an example). This is a matter to also be considered for calculation in the future about chemical shift prediction of GAGs. Moreover, other NMR topics such as chemical shift anisotropy have not been widely discussed here. If so, the size of this material would be rather inconvenient for a manual destined to help chemical shift-based studies of GAGs by routine solution NMR experiments. In this way, we expect that this document can clearly be useful as a guiding material to future spin and structural assignments of GAG molecules, and derivatives, in studies that may involve structural and functional properties of GAGs based on chemical shifts from solution NMR GAG samples. The solid information included here and reported elsewhere heavily support the feasibility of chemical shift prediction of GAGs in a very near future. This perspective represents a big step not only for glycosaminoglycanomics but also in the fields of biomolecular NMR, glycobiology, and glycomics in general.

of Regular polysaccharides, that uses liquid state NMR data to elucidate carbohydrate structure based on agreement of 1Hand 13C-chemical shifts has been built.186,187 This program has been implemented and validated primarily based on works using N- and O-linked glycans of glycoproteins. Calculated chemical shifts built comparatively with experimental chemical shifts of glycoprotein models have shown the maximum averaged error of 0.13 ppm/shift for a pentasaccharide epitope of the O-antigen polysaccharide of Escherichia coli.187 The development of this system has led to a rapid automatic structural determination of complex carbohydrates and polysaccharides based on NMR chemical shift predictions. The additional input data needed besides unassigned and peakpicked 2D NMR spectra are few, which facilitate immensely the rapid analysis and ease of use.187 This review has proved the quality and utility of NMR chemical shifts in structural determination of GAGs despite the great structural variations and heterogeneity of these glycans. As observed, for example, through 15N-chemical shifts, the values of this NMR parameter are strictly characteristic of the structural properties of GAGs,27 and they have a tendency to resonate at just certain regions of the NMR spectrum under a small range of variation. This tendency supports the development of the chemical shift prediction of GAGs. Although not experimentally proved yet, CASPER, or other softwares, is more than likely able to make a prediction of chemical shifts of GAGs. In fact, some GAG monosaccharide building-blocks such as β-D-GalNAc as seen in CS (Figure 1A) and 3-linked βD-Gal as seen in KS (Figure 1E) have already been used to validate the CASPER software. 187 The validation was accomplished even using these sugars covalently bound to other monosaccharides in disaccharide motifs.187 The use of tabulated chemical shift values, regardless of the isotope type, as presented here or elsewhere, can help the approach of establishing NMR chemical shift prediction of GAGs. The knowledge about the relationship between NMR chemical shifts and the respective structural features of GAGs makes the idea of chemical shift prediction of GAGs very feasible although yet to be experimentally proved. This is probably the main future perspective in the field of NMR-related structural biology of GAGs. What supports this perspective is that CASPER has successfully been used for chemical shift prediction of a series of GAG-closely related monosaccharide types.185,186 The successful application of CASPER to the progress of NMRrelated bioinformatics in glycobiology through automatic chemical shift assignments has been proven in many recent works.188−192 Now, it is just a matter of time for its applicability to glycosaminoglycanomics.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +55-21-2562-2939. Fax: +55-21-2562-2090. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biography Vitor H. Pomin (M.S., Ph.D.) is a professor of Biological Chemistry, Biochemistry, Glycobiology, and NMR Spectroscopy at Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Brazil, since May 2011. He pursued his undergraduate studies in Biological Sciences and graduate studies in Biological Chemistry at the same university. He received his Diploma of Licentiate, M.S., and Ph.D. in 2003, 2005, and 2008, respectively. His M.S. and Ph.D. were supervised by the Full-Professor Paulo A. S. Mourão. After this period, he pursued a postdoctorate experience at the Complex Carbohydrate Research Center, University of Georgia, United States, until April 2011, under the supervision of the Eminent Scholar of NMR spectroscopy Prof. James H. Prestegard. Dr. Pomin has over 20 peer-reviewed published articles in high-impact journals, 11 book chapters, besides being editor of 4 academic/scientific books. He serves as an editorial member and frequent peer-reviewer of many internationally recognized journals like Biopolymers, Carbohydrate

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CONCLUSIONS On the basis of what has been stated here, a huge amount of valuable information regarding both structure and function of GAGs can be extracted from NMR chemical shifts. The main available information regardless of the GAG family and subfamily is condensed here especially in what concerns the tabulated chemical shifts versus structural features of GAGs. Furthermore, a unique document containing at a single time NMR spectra and underlying discussion of the majority of the common GAG types (CS/DS, HS/Hp, HA, and KS) seems virtually nonexistent until this review. This strongly justifies the relevance of this unprecedented type of publication, although several works about NMR-based studies of GAGs, usually those focused on one or just a few GAG types, or derivatives, do exist 90



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(23) Kono, H.; Yunoki, S.; Shikano, T.; Fujiwara, M.; Erata, T.; Takai, M. J. Am. Chem. Soc. 2002, 124, 7506−7511. (24) Gomes, A. M.; Kozlowski, E. O.; Pomin, V. H.; De Barros, C. M.; Zaganeli, J. L.; Pavão, M. S. G. J. Biol. Chem. 2010, 285, 7312− 7323. (25) Vieira, R. P.; Mourão, P. A. J. Biol. Chem. 1988, 263, 18176− 18183. (26) Vieira, T. C. R. G.; Costa-Filho, A.; Salgado, N. C.; Allodi, S.; Valente, A. P.; Nasciutti, L. E.; Silva, L. C. F. Eur. J. Biochem. 2004, 271, 845−854. (27) Pomin, V. H. Anal. Bioanal. Chem. 2013, 1−14. (28) Pomin, V. H.; Sharp, J. S.; Li, X.; Wang, L.; Prestegard, J. H. Anal. Chem. 2010, 82, 4078−4088. (29) Beyer, T.; Diehl, B.; Holzgrabe, U. Bioanal. Rev. 2010, 2, 1−22. (30) Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.; Capila, I.; Lansing, J. C.; Guglieri, S.; Fraser, B.; AlHakim, A.; Gunay, N. S.; Zhang, Z.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, J.; Langer, R.; Venkataraman, G.; Linhardt, R. J.; Casu, B.; Torri, G.; Sasisekharan, R. Nat. Biotechnol. 2008, 26, 669−675. (31) Keire, D.; Ye, H.; Trehy, M.; Ye, W.; Kolinski, R.; Westenberger, B.; Buhse, L.; Nasr, M.; Al-Hakim, A. Anal. Bioanal. Chem. 2011, 399, 581−591. (32) Blossom, D. B.; Kallen, A. J.; Patel, P. R.; Elward, A.; Robinson, L.; Gao, G.; Langer, R.; Perkins, K. M.; Jaeger, J. L.; Kurkjian, K. M.; Jones, M.; Schillie, S. F.; Shehab, N.; Ketterer, D.; Venkataraman, G.; Kishimoto, T. K.; Shriver, Z.; McMahon, A. W.; Austen, K. F.; Kozlowski, S.; Srinivasan, A.; Turabelidze, G.; Gould, C. V.; Arduino, M. J.; Sasisekharan, R. New Engl. J. Med. 2008, 359, 2674−2684. (33) Adam, A.; Montpas, N.; Keire, D.; Désormeaux, A.; Brown, N. J.; Marceau, F.; Westenberger, B. Biomaterials 2010, 31, 5741−5748. (34) Alban, S.; Lühn, S.; Schiemann, S.; Beyer, T.; Norwig, J.; Schilling, C.; Rädler, O.; Wolf, B.; Matz, M.; Baumann, K.; Holzgrabe, U. Anal. Bioanal. Chem. 2011, 399, 605−620. (35) Beni, S.; Limtiaco, J. F. K.; Larive, C. K. Anal. Bioanal. Chem. 2011, 399, 527−539. (36) Beyer, T.; Diehl, B.; Randel, G.; Humpfer, E.; Schäfer, H.; Spraul, M.; Schollmayer, C.; Holzgrabe, U. J. Pharmaceut. Biomed. Anal. 2008, 48, 13−19. (37) Beyer, T.; Matz, M.; Brinz, D.; Rädler, O.; Wolf, B.; Norwig, J.; Baumann, K.; Alban, S.; Holzgrabe, U. Eur. J. Pharm. Sci. 2010, 40, 297−304. (38) Bigler, P.; Brenneisen, R. J. Pharmaceut. Biomed. 2009, 49, 1060−1064. (39) Jones, C. J.; Beni, S.; Limtiaco, J. F. K.; Langeslay, D. J.; Larive, C. K. Annu. Rev. Anal. Chem. 2011, 4, 439−465. (40) Keire, D. A.; Trehy, M. L.; Reepmeyer, J. C.; Kolinski, R. E.; Ye, W.; Dunn, J.; Westenberger, B. J.; Buhse, L. F. J. Pharmaceut. Biomed. 2010, 51, 921−926. (41) Kishimoto, T. K.; Viswanathan, K.; Ganguly, T.; Elankumaran, S.; Smith, S.; Pelzer, K.; Lansing, J. C.; Sriranganathan, N.; Zhao, G.; Galcheva-Gargova, Z.; Al-Hakim, A.; Bailey, G. S.; Fraser, B.; Roy, S.; Rogers-Cotrone, T.; Buhse, L.; Whary, M.; Fox, J.; Nasr, M.; Dal Pan, G. J.; Shriver, Z.; Langer, R. S.; Venkataraman, G.; Austen, K. F.; Woodcock, J.; Sasisekharan, R. New Engl. J. Med 2008, 358, 2457− 2467. (42) Larive, C. K. Anal. Bioanal. Chem. 2011, 399, 525−526. (43) Laurencin, C. T.; Nair, L. Nat. Biotechnol. 2008, 26, 621−623. (44) Limtiaco, J. F. K.; Jones, C. J.; Larive, C. K. Anal. Chem. 2009, 81, 10116−10123. (45) Liu, H.; Zhang, Z.; Linhardt, R. J. Nat. Prod. Rep. 2009, 26, 313−321. (46) McEwen, I.; Mulloy, B.; Hellwig, E.; Kozerski, L.; Beyer, T.; Holzgrabe, U.; Wanko, R.; Spieser, J. M.; Rodomonte, A. Pharmeuropa bio/the Biological Standardisation Programme; EDQM: Strasbourg, France, 2008; pp 31−39. (47) Pan, J.; Qian, Y.; Zhou, X.; Pazandak, A.; Frazier, S. B.; Weiser, P.; Lu, H.; Zhang, L. Nat. Biotechnol. 2010, 28, 203−207.

Polymers, Biochimica et Biophysica Acta, General Subjects, Marine Drugs, Phytochemistry, the Journal of Biological Chemistry, and others. He conducts research on glycobiology (especially sulfated polysaccharides), structural biology, and NMR spectroscopy.

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ACKNOWLEDGMENTS This review is dedicated to my colleague Prof. Joab Trajano Silva for all of his academic and teaching contributions at the Institute of Chemistry and Institute of Biology of the Federal University of Rio de Janeiro. I am immensely grateful to Prof. Paulo A. S. Mourão and Prof. Ana Paula Valente (both from the Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Brazil) and Prof. James H. Prestegard (from the Complex Carbohydrate Research Center, University of Georgia, United States) for their great academic contributions in which they have enabled me to discover my own steps in the science of NMR spectroscopy and structural biology of sulfated polysaccharides, including GAGs. I am also thankful to FAPERJ and CNPq for their respective financial supports, and Gustavo Santos (M.S.), Roberto Fonseca (Ph.D.), and Angélica Gomes (M.S.) (all from Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Brazil) for having provided me with the various Hp, OSCS, and invertebrate HS samples.

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