Solution Structure of Heparin Pentasaccharide: NMR and DFT

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The Solution Structure of Heparin Pentasaccharide: NMR and DFT Analysis Milos Hricovini J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07046 • Publication Date (Web): 04 Sep 2015 Downloaded from http://pubs.acs.org on September 5, 2015

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The Journal of Physical Chemistry

The Solution Structure of Heparin Pentasaccharide: NMR and DFT Analysis

Miloš Hricovíni

Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, Slovakia

Running title: structure and spin-spin coupling constants in heparin pentasaccharide

Corresponding author. Tel: +421-2-59410323, Fax: +421-2-59410222 E-mail address: [email protected]

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ABSTRACT: High-resolution NMR and density functional theory (DFT) calculations have been applied into analysis of heparin pentasaccharide 3D structure in aqueous solution. The fully optimized molecular geometry of two pentasaccharide conformations (differing from each other in the form, one 1

C4 and the other 2S0, of the sulfated iduronic acid residue) were obtained using the B3LYP/6-

311+G(d,p) level of theory in the presence of solvent, the latter included as explicit water molecules. The presented approach enabled the insight into variations of the bond lengths, bond angles and torsion angles, formations of intra- and intermolecular hydrogen bonds and ionic interactions in the two pentasaccharide conformations. A rather complex hydrogen bond network is formed, including interresidue and intra-residue bonds between the NH group in the GlcN,3,6S with oxygens linked to C–2 at the IdoA2S residue, and the glycosidic O–1 and the neighboring OSO3– group linked to C–3 in the same residue. On the other hand, as the first hydration shell is strongly influenced by strong ion-ion and iondipole interactions between sodium ions, sulfates, carboxylates, and –OH groups, ionic interactions play an important role in the stabilization of the 3D structure. The DFT-computed three–bond proton–proton coupling constants also showed that best agreement with experiment was obtained with a weighted average of 15:85 (1C4:2S0) of the sulfated iduronic acid forms indicating that the ratio is even more shifted towards the

2

S0 form than previously supposed. The DFT-computed pentasaccharide

conformation differs compared to the previously published data, with the main changes at the glycosidic linkages, namely, the ψ1 torsion angles and the φ3 angle. The comparison of the glycosidic linkage torsion angle values in solution with the antithromin-pentasaccharide complex also indicates that the pentasaccharide conformation changes upon binding to antithrombin III. The data supports the assumption that the protein selects the more populated 2S0 conformer of heparin pentasaccharide and, consequently, the binding process of heparin pentasaccharide with antithrombin III is energetically more favorable than formerly expected. 2 ACS Paragon Plus Environment

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Keywords: heparin pentaccharide; structure; DFT calculations; NMR spin-spin coupling constants;



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INTRODUCTION Determination of the structure and dynamics of carbohydrates and their complexes with proteins plays crucial role in rational explanation of many processes in glycobiology. NMR spectroscopy is widely used for carbohydrate structural analysis but experiments often also require theoretical analysis, especially when complex conformational equilibria are present in solution. Theoretical calculations of medium-sized biomolecules, such as oligosaccharides, often utilize a density functional theory (DFT) approach for its moderate requirements for computer time and memory.1−8 In addition to optimization of the molecular geometry, NMR parameters, such as spin-spin coupling constants, also provide important information for structural determination as well.9 Among the most studied carbohydrate derivatives are glycosaminoglycans (GAGs) due to their important biological functions.10−13 GAG molecules are constituted by uronic acids and hexosamines. Heparin, probably the best known GAG, is composed of repeated disaccharidic sequences of the L-iduronic (to a smaller extent

D-glucuronic)

acid and

D-

glucosamine (most of these units are sulfated to a varying degree), linked through (1→4)-glycosidic bonds. Heparin is known mainly for its anticoagulant properties and its biological activity is mostly due to the unique pentasaccharide sequence (Scheme 1). 10,14 The structure of heparin oligosaccharides has been the subject of a number of experimental14−27 and theoretical28−34 studies. Although conformations at the glycosidic linkages and pseudorotation of the pyranose rings have been studied2,4,14,19,28,29,35−36

many 3D saccharide structures have not been

adequately described. In addition, there are several phenomena influencing carbohydrate structures and their interactions with proteins that are not yet fully understood, including solvent effects, influence of counterions and hydrogen bonding. The combination of NMR experiments and DFT calculations can give detailed insight into the molecular geometry and the effects of solvent, counterions as well as the formation of intra- and intermolecular hydrogen bonding. It is also of interest to analyze NMR 4 ACS Paragon Plus Environment

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parameters, such as proton-proton and proton-carbon spin-spin coupling constants that provide important knowledge about molecular structure and can be used for interpretation of experimental data. 3D heparin pentasaccharide has been studied several times14,15,19,27,28,35−37 as it has important biological properties.10,11 NMR and molecular modelling showed that the molecule is quite flexible in solution due to pseudorotation of the iduronic acid residue and internal motions at the glycosidic linkages.14,36,38 Two conformers of the iduronic acid residue are present is aqueous solution, namely 1C4, and 2S0. Molecular mechanics calculations indicated that the ratio of the two conformers is about 35:65 (1C4 : 2S0)36 and that the main conformer, 2S0, is adopted when pentasaccharide binds to protein antithrombin.39,40 In addition, several changes at the glycosidic linkages between the solution state and the complex occur during the binding process.40 The free energy changes during binding of heparin-pentasaccharide to proteins thus depends upon conformational properties of this pentasaccharide in solution. The present paper aims at deeper insight to 3D properties of heparin pentasaccharide which represents the binding site of heparin for antithrombin III (Figure 1) using NMR and DFT. In addition to the analysis of the fully optimized pentasaccharide molecular structure in the presence of solvent (the latter included as explicit water molecules), NMR spin-spin coupling constants were computed and compared with measured experimental values. Analysis of contributions to spin-spin coupling constants, Fermi-contact contributions and spin-orbit contributions, has also been performed.

Figure 1. Chemical structure of heparin pentasaccharide. 5 ACS Paragon Plus Environment


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METHODS NMR spectroscopy. Heparin pentasaccharide methyl glycoside (GlcN,6S-GlcA-GlcN,3,6S-IdoA2SGlcN,6S-OMe, Scheme 1) was purchased from Glaxo Wellcome Production, Notre Dame de Bondeville, France. High-resolution NMR spectra were recorded in a 5 mm cryoprobe at 15 °C and 25 °C in D2O on either Varian 600 VNMRS or at 950 MHz (D2O, 25 °C) Bruker Avance III HD spectrometers. One-dimensional 600 MHz 1H and 150 MHz

13

C NMR spectra, as well as two-

dimensional COSY, HSQC and HMBC were used for determination of 1H and

13

C chemical shifts. 1H

and 13C chemical shifts were in agreement with those previously published.27,38 1H spectrum collected on a 950 MHz spectrometer and spin-simulation (MestReNova software)41 have been used for determination of three-bond proton-proton coupling constants (3JH-H). Three-bond proton-carbon coupling constants (3JC-H) were measured at 14 T by gradient-selected high-resolution HMBC42 applying band-selective pulses (sinc-type pulses with duration of 4.47 ms) in the

13

C frequency region.43 The J-

scaling factor was 15, t1= 256 points, t2= 2048 points, spectral widths 10500 Hz (f1) and 3600 Hz (f2), number of transients =256 and the recycle time=1.6 s. Geometry optimization. The molecular structure of heparin pentasaccharide has been fully optimized without any constraints applying the ONIOM approach. The B3LYP44 functional and the 6311+G(d,p)45 basis set were applied for the solute (including sodium ions) and the universal force field (UFF)46 for the solvent using GAUSSIAN09.47 Explicit solvent was used as hydration of the polar groups in heparin-pentasaccharide (sulfates and carboxylates) is better described this way than with a continuum model.48 The initial positions of the 98 water molecules were based on coordinates of oxygen atoms in water molecules in the published crystal data of sulfated monosaccharides.49,50 The geometry optimization was performed for two conformations of the L-IdoA2S residue (1C4, 1, and 2S0, 2, Figure 2) whereas all other residues were in the 4C1 conformations. The starting conformations at the glycosidic


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linkages Φ (H1'–C1'–O1'–C4) and Ψ (C1'–O1'–C4–H4) were based on previously published data,14 thus Φ1, Ψ1 = -32°, -27° (GlcN,6SNR-GlcA), Φ2, Ψ2 = 44°, 14° (GlcA-GlcN,3,6S), Φ3, Ψ3 = -39°, -33° (GlcN,3,6S-IdoA2S), Φ4, Ψ4 = 24°, -48° (IdoA2S-GlcN,6SR) for 1, and Φ1, Ψ1 = -34°, -27° (GlcN,6SNRGlcA), Φ2, Ψ2 = 47°, 6° (GlcA-GlcN,3,6S), Φ3, Ψ3 = -35°, -42° (GlcN,3,6S-IdoA2S), Φ4, Ψ4 = 43°, 6° (IdoA2S-GlcN,6SR) for 2. Positions of counterions were based on the x-ray data for structurally similar compounds;50 the positions of Na+ ions close to the 2–SO3- group at the IdoA2S residues were based on the starting structures of heparin trisaccharide.48 3JH-H and nJC-H , as well as the individual contributions to coupling constants (i.e. Fermi contact term, FC; spin-dipolar, SD; paramagnetic spin-orbit, PSO; diamagnetic spin-orbit, DSO contributions), were then DFT-computed (B3LYP functional) using the DGDZVP51 basis set. The choice of this set was based on previous DFT calculations48 as this approach gave satisfactory 3JH-H values in a reasonable time. 3JH-H were also computed from the previously described dependence upon torsion angles.52 Averaged coupling constants were obtained by fitting either the DFT-computed coupling constants to experimental values, considering the contributions of both conformers 1 and 2 or the coupling constants obtained from the 3JH-H dependence upon torsion angles.

RESULTS AND DISCUSSION

A. Geometry. The 3D structure of heparin pentasaccharide in aqueous solution was analyzed by NMR spectroscopy combined with DFT calculations. Selected DFT-optimized bond lengths are listed in Table 1. The interatomic distances in the pyranose rings were mostly comparable to each other in 1 and 2. Small variations in magnitudes were found in the C1-O1 bond lengths at the glycosidic linkages in GlcN,3,6S and IdoA2S residues. Bond lengths in the carboxylate groups, C6–O51 and C6–O52, were influenced by the positions of the Na+ ions in both uronate residues, resembling similar effects in



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heparin trisaccharide.48 Hydrogen bonds between carboxylate oxygens and water molecules are another factor that could also affect the carboxylate bond lengths. Such effects have already been discussed in QM/MM dynamics studies of CH3COO––water hydrogen bonds.53 However, the influence of water molecules upon the pentasaccharide structure seems less significant than in the aforementioned acetic acid–water complex, as can be inferred from the computed water–COO– distances (2.806 Å and 2.973 Å in GlcA carboxylate in 1) and is comparable to heparin trisaccharide (~2.8 Å – 3.1 Å).


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Figure 2. DFT-optimized structure of heparin pentasaccharide. The two forms, 1 and 2, have different conformations of the IdoA2S residue. The IdoA2S residue is in the 1C4 conformation in 1 and in the 2S0 conformation in 2. Other residues are in the 4C1 conformation. Violet dots represent sodium ions. The numbering of atoms is the same in all residues. The numbering of oxygen atoms in N-sulfate or Osulfate groups is also the same (O21, O22 and O23); they are differentiated by which pyranose ring unit they occur in, and by the carbon number in the pyranose ring to which they are linked. The same is valid for carboxylate oxygen atoms (O51 and O52). Solvent (water) molecules are not shown for clarity.

Much larger differences between 1 and 2 were in the formation of hydrogen bonds (H-bonds). There are various intra-residue, inter-residue and intermolecular (solute-solvent) H-bonds in 1 and 2. For example, H-bond formation by the NH group in the GlcN,3,6S residue depends upon the IdoA2S conformation: inter-residue and intra-residue bifurcated H-bonds are formed between the NH group and two oxygens – the oxygen linked to C–2 at the IdoA2S residue (2.312 Å; inter-residue) and the glycosidic O–1 (2.320 Å) in the same residue in 1 (Figure 3A). In 2, however, an H-bond (2.294 Å) is formed between the NH group and the neighboring OSO3– group linked to C–3 (Figure 3B). Formation 9 ACS Paragon Plus Environment


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of the latter H-bond was discussed in a recent NMR study.15 Present data indicate that the NH group in the central GlcN,3,6S residue is involved in a rather complex H-bond network, both intra-residue and bifurcated inter-residue, where the major conformer (IdoA2S residue is in the 2S0 form, i.e. 2; see later discussion) has the intra-residue NH…O(3)SO3– H-bond. Theoretical data also showed that the NH group at the non-reducing end the GlcN,6SNR residue does not form any H-bond, which is in agreement with NMR experiments as well.15,37 On the other hand, the NH group at the GlcN,6SR residue could be involved in transient H-bonds (in both 1 and 2) with OMe. Differences are also found in O–H…O H-bonds in 1 and 2. The H-bond spanning the GlcA and IdoA2S residues was formed between the OH group at C–2 in the GlcA residue and the carboxylate group in the IdoA2S residue in 1 (1.664 Å) (Figure 4A). In 2, the same OH group formed a bifurcated H-bond: intermolecularly with a water molecule (2.743 Å), and intramolecularly with the glycosidic oxygen (2.509 Å) in the same residue (Figure 4B). Another H-bond was formed from the OH group at C–3 to the neighboring O–2 in the same residue in 2. This is in also in agreement with NMR data, but is different to the H-bond observed in MD simulations.15 Fairly different H-bonds were also seen in the IdoA2S residue: the inter-residue H-bond (1.834 Å) was formed between the OH group at C–3 and the neighboring O–6 (linked to C–6) in the reducing end GlcN,6SR residue in 1. In 2, however, the OH group at C–3 showed a bifurcated weak inter-residue H-bond with the ring O–5 oxygen in the central GlcN,3,6S residue (2.886 Å) and with a water molecule (2.973 Å). Other H-bonds were between the OH group at C–4 and the neighboring OSO3- group (linked to C–6) in the GlcN,6SNR residue in 1 (1.833 Å), whereas the same OH group formed a weak intermolecular H-bond with water molecule (3.066 Å) in 2. Several O–H…O H-bonds differed from those described in a recent MD study.15 The differences are mainly due to the differences in some torsions angles at the glycosidic linkages and likely the positions of counterions. For example, the sodium ion is 6-fold coordinated with oxygens from the NSO3- group in


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the GlcN,6SNR residue, the OSO3- group (linked to C–6) in the GlcN,3,6S residue, two oxygens (O–2 and O–3) in the GlcA residue and two water molecules in the DFT-computed structure 2. Such arrangement favors the formation of intraresidue (GlcA) H-bond between the OH3 and the neighboring O–2 residue rather than the H-bond between the OH group at C–3 (GlcA) and the NSO3- group (GlcN,6SNR), as mentioned above. The theoretical data thus clearly show that different H-bonds are competing with each another in the two different conformations of heparin pentasaccharide and play an important role in the internal dynamics of pentasaccharide in solution. It should also be taken into account that H-bonds are not static54 as they are breaking and reforming in new configurations whenever the conformation changes between 1 and 2 (other H-bonds between solute and solvent as described here are also possible). In addition, strong ion-ion and ion-dipole interactions among sodium ions, sulfates, carboxylates and the – OH groups in the solute and the solvent are also present. As small ions (e.g. Na+) have high charge densities,55 they have a strong influence on the first hydration shell of heparin pentasaccharide. Ionic interactions are in general considered stronger than H-bonds and, consequently, they play significant role in the stabilization of the 3D structure. In agreement with this, DFT calculations showed that the more stable conformer in solution (structure 2, see later discussion) has less intra-molecular H-bonds than 1 and that conformer 2 seems mainly stabilized by ionic interactions. Similarly to previous observations,30 Na+ ions showed a tendency toward 6-fold coordination with oxygens from sulfates, carboxylates and water molecules in both 1 and 2. The interatomic distances between sulfate or carboxylate oxygens and sodium ions were typically ~2.2 Å and ~2.6 Å, respectively; water oxygen…Na+ ion separations were ~2.7–3.6 Å.



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A

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B

Figure 3. Hydrogen N–H…O-type H-bonds in pentasaccharide. Inter-residue and intra-residue bifurcated H-bonds in 1 from the NH group in the GlcN,3,6S residue to two oxygens: the oxygen linked to C–2 at IdoA2S (2.312 Å) and the glycosidic O–1 (2.320 Å) in the same residue (A). A H-bond (2.294 Å) is formed between the NH group in the GlcN,3,6S residue and the neighboring OSO3– group linked to C–3 in 2 (B). Solvent (water) molecules are omitted for clarity.


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A

B

Figure 4. Hydrogen O–H…O-type H-bonds in pentasaccharide. The H-bond between the OH group at C–2 in the GlcA residue and the carboxylate group in the IdoA2S residue in 1 (1.664 Å) (A). The same OH group forming a bifurcated intermolecular H-bond with water molecule (2.743 Å) and intramolecular H-bond with the glycosidic oxygen (2.509 Å) in the same residue in 2 (B).



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Several differences between 1 and 2 have been observed in the bond angles (Table 2). The most pronounced differences were seen for glycosidic linkages (e.g. for GlcA–GlcN,3,6S 8.5°). Smaller, but not negligible effects, were found also within the pyranose ring units. Torsion angles (Table 3a) in the pyranose rings showed significant differences between 1 and 2. Changes were seen not only in the IdoA2S residue (which actually changes conformation) but in the “rigid” glucosamine pyranose rings as well. The torsion angles of heavy atoms, e.g. C1–C2–C3–C4 (a difference of nearly 17° between 1 and 2 in GlcN,6SR) demonstrate that pseudorotation of the IdoA2S has a significant effect upon the ring geometries of the neighboring units. The torsion angles between hydrogen atoms (Table 4) differed even more (up to about 30°, H4–C4–C5–H5, GlcN,3,6S). The φ and ψ torsion angles (H1–C1–O1–C4, H4–C4–O1–C1) at the glycosidic linkages (Table 3b) are, apart from the pseudorotation of the IdoA2S residue, the most interesting from the point of view of solution conformation and the process of binding with proteins. The computed DFT data are different to the data previously obtained from molecular mechanics14 and molecular dynamics.15 The main difference is observed in the ψ1 angles for both 1 (-61°) and 2 (-72°), compared to -27° obtained by molecular mechanics for both 1 and 2.14 It should be noted, however, these DFT-derived values are comparable with ψ1 for the AT-pentasaccharide complex in solution.40 The φ3 angle (-76°) for the major conformer 2 was also different compared with the previous data (φ3 = -35° in molecular mechanics). Interestingly, this angle is similar to that seen in the crystal state of the AT-pentasaccharide complex (-71°)39 and the solution complex with the fibroblast growth factor receptor.24 The remaining φ, ψ values for the main conformer (2) were in general close to the torsion angles obtained by MM2 method.14

B. NMR spin-spin coupling constants. Three–bond proton–proton coupling constants (3JH–C–C–H) in heparin pentasaccharide were computed using the B3LYP/6-311+G(d,p)/UFF fully optimized 14 ACS Paragon Plus Environment

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geometry (hydrated with 98 water molecules) and the DGDZVP basis set. Computed coupling constants, together with torsion angles between corresponding proton pairs and the individual contributions to coupling constants, are listed in Table 4. Major differences in 3JH–C–C–H magnitudes between 1 (Table 4a) and 2 (Table 4b) were computed for the IdoA2S residue due to differences in its conformation. As mentioned, differences in 3D structures were also seen in other pyranose rings. One example is previously mentioned H4–C4–C5–H5 array of atoms in the three sulfated glucosamine residues. In the GlcN,3,6S residue (30° difference in the H4–H5 torsion angle between 1 and 2), the geometry variations have not caused any significant differences in 3JH4–C4–C5–H5 magnitudes (9.96 Hz vs. 9.93 Hz). Similarly, comparable coupling constant magnitudes (9.14 Hz vs. 9.59 Hz) were also computed in the reducing end GlcN,6SR residue (torsions angles differed each another only marginally, by 0.6°). On the other hand, the same coupling constants in the non-reducing end GlcN,6SNR residue differ from each another considerably (11.21 Hz vs. 9.46 Hz). It should also be noted that all these coupling constants agree well with the experimental NMR data. Such variations of the 3JH–C–C–H values as a function of torsion angles have already been discussed in heparin-trisaccharide48 and explained by stereoelectronic effects. The differences in the magnitude of the Fermi-contact contributions to

3

JH4–C4–C5–H5 in these three

glucosamine residues are due to differences in the electronic structure in the neighborhood of the coupled nuclei. The presence of oxygen atoms with lone pairs causes different electron densities in the vicinity of the coupled protons and differ for different glucosamine residues. Both the central GlcN,3,6S and the reducing-end GlcN,6SR residues have glycosidic linkages at both ends of the pyranose ring (linked to C–4 and C–1) whereas GlcN,6SNR lacks the linkage at C–4. Recent analysis of Fermi-contact terms clearly showed that electrons at atoms along the coupling path, O–5 ring oxygen and the O–H group at C–3 are important for interpretation of 3JH4–C4–C5–H5 coupling constants. Apart from these atoms, however, contributions from the lone-pairs from oxygen atoms at the glycosidic linkages were



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also found to be important and without them the coupling constants could not be described. Consequently, the Fermi-contact interactions contributing to the 3JH4–C4–C5–H5 in the GlcN,3,6S and the GlcN,6SR residues are different than those of the terminal GlcN,6SNR. The magnitudes of the spin-orbit contributions are comparable to Fermi-contact terms in several coupling constants, especially those in the IdoA2S residue. The diamagnetic spin-orbit (DSO) term for 3

JH4–H5 (3.30 Hz) in 1 is bigger than the Fermi term (3.15 Hz). Similar trends were seen in this residue

also for other couplings as well, 3JH1–H2 (2.18 Hz) in 1 and 3JH4–H5 (2.64 Hz) in 2. Large DSO values were computed for 3JH1–H2 in all three glucosamine residues in both 1 and 2. As previously discussed in the case of structurally similar trisaccharide,48 contributions to DSO terms are important not only from the molecular orbitals in the close vicinity of the coupled protons but also from those more distant. The position of the proton pair in the molecule is therefore an important factor that influences the magnitude of the DSO contribution to proton-proton coupling constants. The best fit of the computed averaged proton-proton coupling constants to the experimental values is shown in Table 5. The results indicate that the best agreement between theory and experiment is obtained using the weighted average of 15:85 of the IdoA2S forms (1:2, i.e. 1C4:2S0). The averaged values were computed by two methods: as a weighted average of DFT-computed coupling constants (last columns in Tables 4a and 4b) or by averaging of coupling constants determined from the dependence of coupling constants upon torsion angles52 by using the angles presented in column 3 in Tables 4a and 4b. Overall, there is not much difference between the values produced by these two approaches. In the glucosamine residues, some coupling constants are underestimated by the DGDZVP basis set. Namely, in the GlcN,6SNR residue is 8.3 Hz as the consequence of lower magnitude of the Fermi term (8.62 Hz) and the paramagnetic contribution (0.74 Hz) in 2. The 6311+G(d,p) basis set gave about 1 Hz higher value for the same coupling in the same residue in heparin


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trisaccharide. The lower theoretical value was obtained also for 3JH3–C3–C4–H4 in the IdoA2S residue in 2, with a 1.1 Hz difference between theory and experiment indicating that some discrepancies could be caused by the used basis set. On the other hand, the accordance between theory and experiment was very good in both of these cases when values were computed from the relationship between coupling constants and torsion angles. Experimental and computed proton-carbon coupling constants are listed in Table 6. Experimental 3

JH-C-C-C and 3JH-C-O-C values were determined from 2D by gradient-selected high-resolution HMBC

using both non-selective (Figure 5A) pulses and band-selective (Figure 5B, C) carbon pulses. DFTcomputed data agreed well with experimental values regarding the 15:85 (1C4, 2S0) ratio of 1 and 2 for most of the coupling constants within the pyranose rings. A noticeable difference was obtained for in the GlcN,6SNR residue (0.6 Hz vs. 2.4 Hz), likely as a consequence of distortion of the ring geometry. The unusually high value of the in the GlcN,3,6S residue (9.3 Hz vs. 6.8 Hz) could be partially caused by the effect of oxygen lone pairs in the coupling path. This effect could not be fully accounted for by the DGDZVP basis set. For the same reasons, the observed differences between theory and experiment (about 1 Hz in most cases) were obtained for the interglycosidic coupling constants. For example, the computed between the IdoA2S and the GlcN,6S residues is 1.9 Hz, i.e. 1 Hz smaller than the experimental value (2.9 Hz). One would expect higher values of the computed coupling constant based on the torsion angles (-68.6° in 1 and 56.5° in 2) from which theoretical values were computed (0.02 Hz in 1 and 2.20 Hz in 2). The reason of the lower magnitude can be explained by the same reasons as discussed earlier in heparin trisaccharide for coupling constants in the presence of a large number of oxygen lone pairs.48 The delocalization of the electron density can result in attenuation of the transmission of the Fermi term and, consequently, the overall magnitude of the coupling constant is smaller than expected. This clearly seen when comparing



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this value (i.e. 3JC1–O1–C4–H4 = 2.20 Hz in 2; the torsion angle is 56.5°) with the magnitude of the 3JC3–C4– C5–H5 H>

(4.27 Hz) in the GlcN,3,6S residue in 2 (the torsion angle is 55.5°). On the other hand, other

Solution Structure of Heparin Pentasaccharide: NMR and DFT

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