4C1 Chair Research Articles

Chemical Cross-Linked Gelation of Hydroxypropyl Guar (HPG) under Shear Flow

A chemical modified polysaccharide hydroxylpropyl giar (HPG) derived from the guar which is a galactomannan polysaccharide obtained from seed endosperm was used to investigate the chemical cross-linked gelation. The backbone of HPG consists of 1,4 linked β-D-mannopyranosyl (4C1 chair conformation) residues to form a polymer chain with irregular and incomplete pendant 1,6 linked α-D-galactopyranosyl (4C1 chair conformation) side groups. A viscosity enhancement was found a borate-cross-linked HPG gels which have experienced a shear flow at the early stage of chemical cross-linking. The enhancement of viscosity seems to relate with a conformational change from a spherical shape to an elongated rod-like or/and an ellipsoidal shape at the early stage of chemical cross-linking processes.

Synthesis and Characterization of Non‐standard Nucleosides and Nucleotides Bearing the Acceptor‐Donor‐Donor Pyrimidine Analog 6‐Amino‐3‐methylpyrazin‐2(1H)‐one

Abstract6‐Aminopyrazin‐2(1H)‐one, when incorporated as a pyrimidine‐base analog into an oligonucleotide chain, presents a H‐bond acceptor‐donor‐donor pattern to a complementary purine analog. When paired with the corresponding donor‐acceptor‐acceptor purine in oligonucleotides, the heterocycle selectively contributes to the stability of the duplex, presumably by forming a base pair of Watson‐Crick geometry joined by a non‐standard H‐bonding pattern. Aspects of the nucleoside chemistry, including syntheses of the β‐furanosyl ribonucleoside 1, the ribonucleoside triphosphate 2 and the ribonucleoside bisphosphate 3 of 6‐aminopyrazin‐2(1H)‐one are reported here. In aqueous solution, the ribonucleoside 1 was found to undergo acid‐ and base‐catalyzed rearrangement with an apparent half‐life of ca. 63 h at neutral pH and 30°. The rearrangement appears to be specific acid‐ and base‐catalyzed. The thermodynamically most stable compound formed during this rearrangement reaction was isolated by HPLC and shown to be the β‐pyranosyl form 4 of the 6‐aminopyrazin‐2(1H)‐one nucleoside in its 4C1 chair conformation. This reactivity of 1 under physiological conditions may explain why Nature does not use this particular heterocyclic system to implement an acceptor‐donor‐donor H‐bonding pattern in the genetic alphabet.

NMR solution conformation of heparin-derived tetrasaccharide.

The solution conformation of the homogeneous, heparin-derived tetrasaccharide delta UA2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S (residues A, B, C and D respectively, where IdoA is iduronic acid) has been investigated by using 1H- and 13C-NMR. Ring conformations have been defined by J-coupling constants and inter-proton nuclear Overhauser effects (NOEs), and the orientation of one ring with respect to the other has been defined by inter-ring NOEs. NOE-based conformational modelling has been done by using the iterative relaxation matrix approach (IRMA), restrained molecular dynamics simulations and energy minimization to refine structures and to distinguish between minor structural differences and equilibria between various ring forms. Both glucosamine residues B and D are in the 4C1 chair conformation. The 6-O-sulphate group is oriented in the gauche-trans configuration in the D ring, whereas in the B ring the gauche-gauche rotomer predominates. Uronate (A) and iduronate (C) residues are mostly represented by 1H2 and 2S0 twisted boat forms, respectively, with small deviations in expected coupling constants and NOEs suggesting minor contributions from other A and C ring conformations.

1-O-Benzyl-β-L-arabinopyranose, 1-O-Benzyl-3,4-O-isopropylidene-β-L-arabinopyranose and 1-O-Benzyl-2-O-benzoyl-3,4-O-isopropylidene-β-L-arabinopyranose

The sugar ring of 1-O-benzyl-β-l-arabinopyranose (C12H16O5), (I) adopts the 4C1 chair conformation. In 1-O-benzyl-3,4-O-isopropylidene-β-l-arabinopyranose (C15H20O5), (II) and 1-O-benzyl-2-O-benzoyl-3,4-O-isopropylidene-β-l-arabinopyranose (C22H24O6), (III), the pyranosyl rings have a slightly distorted chair 4C1 towards 0H1 conformation. The five-membered isopropylidene ring in (II) and (III) adopts a slightly distorted envelope conformation. The methylene carbon atoms of the benzyl group are slightly displaced out of the plane of the phenyl ring in all three crystal structures. All three hydroxy groups in (I) participate in hydrogen bonds each as donor and acceptor simultaneously, forming a two-dimensional hydrogen-bond network. In (II), molecules linked by hydrogen bonds form polymeric chains along the b axis.

Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group.

Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysproum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, has been determined by X-ray crystallography at a resolution of 2.7 A utilizing the 4-fold noncrystallographic symmetry present in the asymmetric unit. The electron density map clearly reveals the presence of three glucosyl units of the inhibitor, consistent with the known number of sugar-binding subsites, located at the active site of the enzyme in the -2, -1, and +1 subsites, i.e., actually spanning the point of enzymatic cleavage. The pyranose ring at the point of potential enzymatic cleavage is clearly distorted from the standard 4C1 chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L.O., Johnson, L.N., Machin, P. A., Phillips, D.C., & Tijan, T. (1974) J. Mol. Biol, 88, 349-371]. The distortion observed goes beyond the "sofa" conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.

Crystal structure and molecular packing analysis of 1,5-anhydropentitols and per-O-acetyl derivatives

Abstract The crystal structures of three 1,5-anhydropentitols of formulae C5H10O4 and their per-O-acetyl derivatives of formulae C11H16O7 are reported. 1,5-anhydro-D-arabinitol (ADA), 1,5-anhydroribitol (AR), 1,5-anhydroxylitol (AX), 2,3,4-tri-O-acetyl-1,5-anhydro-D-arabinitol (AcADA), 2,3,4-tri-O-acetyl-1,5-anhydroribitol (AcAR) and 2,3,4-tri-O-acetyl-1,5-anhydroxylitol (AcAX) have the following space group and unit cell parameters: ADA, orthorhombic, P212121, a = 6.521(1) Å, b = 6.826(1) Å, c = 13.557(3) Å; AR, monoclinic, P21/n, a = 6.515(1) Å, b = 12.003(1) Å, c = 8.272(1) Å, β = 105.65(1)°; AX, monoclinic, P21/n, a = 6.652(2) Å, b = 9.224(2) Å, c = 10.548(2) Å, β = 101.80(1)°; AcADA, orthorhombic, P212121, a = 8.182(2) Å, = 10.768(2) Å, c = 15.111(3) Å; AcAR, monoclinic, P21/c, a = 15.178(1) Å, b = 10.339(1) Å, c = 8.038(1) Å, β = 92.50(1)°; and AcAX, monoclinic, P21/c, a = 12.092(1) Å, b = 9.066(1) Å, c = 12.492(2) Å, β = 102.39(1)°. The structures were solved using direct methods and refined by full-matrix least-squares techniques to R indices of 0.033, 0.042, 0.045, 0.066, 0.055 and 0.048, respectively. The pyranoid ring adopts 4C1 chair confirmation in the molecules of AR, AX and AcAX, and the 1C4 one in the molecules of ADA, AcADA and AcAR. Theoretical methods were used to examine lattice energetics and molecular packing in the crystals formed.

3-O-benzoyl-4,6;4',6'-di-O-benzylidene-2,2'-dideoxy,alpha,alpha-ribo- trehalose.

The low-temperature X-ray structure of an asymmetrically substituted derivative of alpha,alpha-trehalose, 3-O-benzoyl-4,6;4',6'-di-O-benzylidene-2,2'-dideoxy-alpha,alpha-ribo-tre halose (3-O-benzoyl-4,6-O-benzylidene-2-deoxy-alpha-D-ribo-hexopyranosyl 4,6-O-benzylidene-2-deoxy-alpha-D-ribo-hexopyranoside, C33H34O10), is reported. The hexopyranosyl rings and the 1,3-dioxane rings have normal 4C1 chair conformations, so that each half of the molecule has a double-chair conformation, resembling a trans-decalin ring system. Each benzylidene acetal group takes the form of the thermodynamically more stable (R)-diastereomer with its phenyl group attached to the 1,3-dioxane ring in an equatorial orientation. The conformations about the glycosidic linkages are stabilized by the anomeric effect and by an intramolecular hydrogen bond between the lone hydroxy group and the glycosidic O atom.

Crystal Structure of Nystose Trihydrate

Abstract A tetrasaccharide, O-α-d-glucopyranosyl-(1→2)-O-β-d-fructofuranosyl-(1→2)-O-β-d-fructofuranosyl-(1→2)-O-β-d-fructofuranoside (nystose) trihydrate (C24H42O21·3H2O), was crystallized from its aqueous solution by using seed crystals. The crystal structure has been determined by an X-ray diffraction method. The crystal data are as follows: orthorhombic, P212121, Z = 4, a = 13.582(3), b = 23.385(6), and c = 10.198(2) Å. The α-d-glucopyranose ring has the normal type 4C1 chair conformation, and three fructofuranose rings have the 4T3, E4, and 4T3 (or E3) conformations. As in the 1-kestose crystal, all of the hydrogen bonds found made connections between adjacent molecules. Although the linkage conformation (∠C1O1C1–2C1–1, ∠C2C1O1C1–2) of the sucrose component in nystose (100.3°, −137.1°) was different from that in 1-kestose (53.3°, −152.9°), the linkage conformation (φ,ψ,ω) of the inulobiose component at the end of nystose (71.3°, −165.9°, 68.0°) was very similar to that of 1-kestose (80.5°, −169.6°, 56.5°).

Stereochemistry of sugar units in glycosylamine ligands of octahedral complexes derived from tris(trimethylenediamine)-nickel(II) and natural aldohexoses

Some natural aldohexoses reacted with [Ni(tn)3]2+(tn = trimethylenediamine) to yield octahedral nickel(II) complexes containing glycosylamine ligands. The complexes were characterized by elemental analyses, magnetic moments, electronic and CD spectra. The molecular structure of [Ni(L-Rha-tn)2]Br2·2H2O·MeOH [L-Rha-tn = 1-(3-aminopropylamino)-1,6-dideoxy-L-mannose]1 has been determined by single-crystal X-ray techniques: orthorhombic, space group P212121, a= 12.373(1), b= 21.614(2), c= 11.272(1)A, least-squares refinement led to a final R factor of 0.044 using 2657 reflections having Fo > 3σ(Fo). From X-ray crystallographic studies of glycosylamine complexes, two typical co-ordination structures of the aldose unit have been found in 1 and [Ni(D-GlcN-en)2]2+[D-GlcN-en = 2-amino-1-(2-aminoethylamino)-1,2-dideoxy-D-glucose], which can be discriminated by the two geometrical isomeric forms arising from the two substituents on C1 and C2 of the pyranoside ring, namely cis and trans. The sugar unit of 1 has cis geometry containing equatorial and axial substituents on C1 and C2 of the pyranoside ring. In contrast, the sugar unit of the D-GlcN-en complex has trans geometry at these positions, which are both equatorial. Assuming the 4C1(chair) conformers to be the most stable and based on some stereochemical considerations of both the sugars and the chelate rings, the structures of the sugar units in the glycosylamine complexes from all natural aldohexoses can be classified into either of the above two types, the only difference being the relative absolute configuration at C2 and C5. This classification is well correlated with the CD spectral patterns obtained in methanolic solutions.

Preparation of heptakis(2,6-di-O-ethyl)-beta-cyclodextrin and its nuclear magnetic resonance spectroscopic characterization.

Heptakis(2,6-di-O-ethyl)-beta-cyclodextrin (DE-beta-CyD) was prepared and its 1H and 13C nuclear magnetic resonance (NMR) signals in DMSO-d6 were unequivocally assigned by two-dimensional COSY and ROESY. The results on 1H coupling constants indicated that all ethylated glucose units are in a 4C1 chair conformation. The average spin-lattice relaxation times (T1) of ring carbons of DE-beta-CyD were only slightly shorter, and their standard deviations from the mean T1 value were larger, than those of beta-cyclodextrin (beta-CyD) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CyD), suggesting the presence of slightly irregular internal motion in the ethylated glucose units. The temperature dependence of chemical shift of DE-beta-CyD in DMSO-d6 suggested that the C3 hydroxyl protons may participate as proton donor in the intramolecular hydrogen bond to the C2 ethoxyl groups of neighboring glucose, and the intramolecular hydrogen bond of DE- and DM-beta-CyDs is much stronger than that of beta-CyD, suggesting the stable macrocyclic ring structure of DE-beta-CyD.

Crystal structure and n.m.r. analysis of lactulose trihydrate

The 13C CPMAS n.m.r. spectrum of 4-O-β-d-galactopyranosyl-d-fructose (lactulose) trihydrate, C12H22O11·3 H2O, identifies the isomer in the crystals as the β-furanose. This is confirmed by a crystal structure analysis, using CuKα X-ray data at room temperature. The space group is P212121, with Z = 4 and cell dimensions a = 9.6251(3), b = 12.8096(3), c = 17.7563(4) Å. The structure was refined to R = 0.031 and Rw 0.025 for 1929 observed structure amplitudes. All the hydrogen atoms were unambigously located on difference syntheses. The conformation of the pyranose ring is the normal 4C1 chair and that of the furanose ring is 4T3. The 1 → 4 linkage torsion angles are O-5′−C-1′−O-1′−C-4 = −79.9(2)° and C-1′−O-1′−C-4−C-5 = − 170.3(2)°. All hydroxyls, ring and glycosidic oxygens, and water molecules are involved in the hydrogen bonding, which consists of infinite chains linked together by water molecules to forma three-dimensional network. There is a three-centered intramolecular, interresidue hydrogen bond from O-3-H to O-5′ and O-6′. The n.m.r. spectrum of the amorphous, dehydrated trihydrate suggests the occurrence of a solid-state reaction forming the same isomeric mixture as was observed in crystalline anhydrous lactulose, although the mutarotation of the trihydrate when dissolved in Me2SO is very low.

Structure elucidation of cyanidin‐3‐sambubioside and assignments of the 1H and 13C NMR resonances through two‐dimensional shift‐correlated NMR techniques

AbstractThe anthocyanin cyanidin‐3‐sambubioside was isolated from the acidified methanolic extract of the fruits of Sambucus nigra by successive application of an ion exchange resin, droplet countercurrent chromatography and gel filtration. By means of chemical degradation and spectroscopy (especially one‐ and two‐dimensional NMR techniques) the structure has been determined to be cyanidin‐3‐O‐β‐D‐xylopyranosyl‐(1±2)‐β‐D‐glucopyranoside with both the sugar rings assuming a 4C1 chair conformation.The combination of several NMR techniques such as double‐quantum filtered proton‐proton shift correlation (DQF‐COSY), homonuclear Overhauser enhancement (NOE) difference spectroscopy, heteronuclear coupling‐modulated spin echo, proton‐carbon shift correlation optimized for direct (HSC) and long‐range coupling (COLOC) provided assignments of the 1H and 13C signals. The coupling constants of the sugar protons were obtained by iterative spin simulation. This is the first report of a heteronuclear shift correlation experiment applied on anthocyanins.

Structure of lactitol (4-O-β-D-galactopyranosyl-D-glucitol) monohydrate: an artificial sweetener

C12H24O11.H2O, Mr = 362.4, orthorhombic, P2(1)2(1)2(1), a = 7.808 (2), b = 12.685 (2), c = 15.931 (3) A, V = 1577.9 (6) A3, Z = 4, Dx = 1.525 Mg m-3, lambda(Mo K alpha) = 0.71073 A, mu = 0.13 mm-1, F(000) = 776, T = 295 K, R = 0.031 for 1781 unique reflections with I greater than 2.5 omega (I). The galactopyranosyl ring has the 4C1 chair conformation and the conformation of the bent glucitol C-atom chain is MAA. The torsion angles characterizing the conformation of the glycosidic linkage are -86.3 (2) degrees [O(5)--C(1)--O(1)--C(14)] and 116.8 (2) degrees [C(1)--O(1)--C(14)--C(13)]. All hydroxyl groups act as donors in hydrogen bonds; three bonds are intramolecular. With the exception of O(1) of the glycosidic link which is not an acceptor and O(6) of the glucitol residue which is a double acceptor, all O atoms accept one hydrogen bond. The water molecule donates two and accepts one hydrogen bond.

1H and 13C NMR analysis of D‐amygdalin: Oligosaccharide assignment and sequencing

AbstractThe 1H and 13C NMR spectra of D‐amygdalin, a cyanogenetic disaccharide accumulated in food plants, were assigned using a combination of NMR techniques. Methods applied include standard 1H–1H COSY, proton zero quantum coherence (ZQCOSY), 1D NOE, broad‐band decoupled 1H–13C chemical shift correlation and long‐range 1H–13C chemical shift correlation. ZQCOSY was especially useful in assigning the closely spaced sugar resonances, and long‐range 1H‐13C chemical shift correlation allowed sequencing of the monosaccharide rings. Coupling constants useful in conformational analysis are derived from highly digitized 1H 1D spectra and 2D J‐resolved spectroscopy. The 1H and 13C chemical shifts and coupling constants indicate that the β‐D‐glucopyranose rings of D‐amygdalin adopt a 4C1 chair conformation.

Conformational effects in 2‐deoxyglucopyranos‐1‐yl radicals

2-Unsubstituted and 2-substituted (fluoro, tosylamino, n-propyl) 3,4,6-tri-O-acetyl-2-deoxypyranosyl radicals are obtained by reaction of the corresponding pyranosyl bromides or phenylselenides with photolytically generated trimethyltin radicals in benzene solution. Analysis of the ESR hyperfine splittings reveals that the 2-deoxy-2-fluoroglucopyranosyl radical exists in a boat-like conformation at room temperature, whereas the others retain the 4C1 chair conformation of the starting material. The observed conformational effects are explained by a “quasi-homo-anomeric” frontier orbital interaction of the nx lone pair of the ring oxygen atom with the σ* MO of the β-C-O bond, in which the singly occupied p orbital at C-1 acts as a mediator for this 1,3-anomeric interaction.

Conformational analysis of the tumor-associated carbohydrate antigen 19-9 and its Lea blood group antigen component as related to the specificity of monoclonal antibody CO19-9.

A structural comparison between the synthetic, tumor-associated 19-9 tetrasaccharide, NeuAc alpha 2----3Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 and its Lea blood group antigen component, Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 was carried out by two-dimensional 1H NMR spectroscopy and hard-sphere energy calculations. Significant chemical shift differences between the two molecules were detected only for protons at or near the linkage site of NeuAc to the Lea trisaccharide core. Coupling constants for the ring protons of both molecules did not suggest major deviation from the 4C1 chair conformation for Gal and GlcNAc, the 1C4 conformation for Fuc, or the 2C5 conformation for NeuAc. Two-dimensional nuclear Overhauser enhancement experiments revealed through-space, inter-proton interactions that corresponded to some extent with those predicted by diffraction data and hard-sphere energy minimization programs for both saccharides. However, a significant number of interactions did not obey the distance dependence predicted from a rigid structure model. These data suggest that, while the average conformation of the 19-9 antigen's Lea core may be invariant to NeuAc alpha 2----3Gal linkage, the dynamics of the Lea trisaccharide are altered upon sialylation. Data also indicate that the terminal NeuAc linkage is more flexible than the inter-residue bonds of the core trisacharide. This analysis, in combination with the fact that the monoclonal anti-19-9 antibody CO 19-9 does not cross-react with the Lea antigen, provides evidence in favor of NeuAc as an epitope-creating unit involved directly at the antibody binding site. However, given the possible role of variable dynamics in epitope formation, these results do not preclude crucial roles in antibody recognition for regions on the 19-9 antigen that are distanced from NeuAc.

Three‐Dimensionnal structures of complexes of Lathyrus ochrus isolectin I with glucose and mannose: Fine specificity of the monosaccharide‐binding site

The structure of the methyl-alpha-D-mannopyranoside-LOL I complex has been solved by the molecular replacement method using the refined saccharide-free LOL I coordinates as starting model. The methyl-alpha-D-mannopyranoside-LOL I complex was refined by simulated annealing using the program X-PLOR. The final R-factor value is 0.182 [Fo greater than 1 sigma(Fo)]. The isostructural methyl-alpha-D-glucopyranoside-LOL I complex was refined by X-Ray coupled energy minimization using the methyl-alpha-D-mannopyranoside-LOL I structure as a starting model to an R factor of 0.179 (all data). In both crystal forms, each dimer binds two molecules of sugar in pockets found near the calcium ions. The two saccharide moieties, which are in the C1 chair conformation, establish the same hydrogen bond pattern with the lectin. However, the van der Waals contacts are different between the O2, C2, C6, and O6 atoms of the two molecules and the backbone atoms of residues 208-211. Mannose, due to its axial C2 conformation, encloses the backbone atoms of the protein in a clamplike way. Van der Waals energy calculations suggest that this better complementarity of the mannoside molecule with the lectin could explain its higher affinity for isolectin I.

The structure of the saccharide-binding site of concanavalin A.

A complex of concanavalin A with methyl alpha-D-mannopyranoside has been crystallized in space group P212121 with a = 123.9 A, b = 129.1 A and c = 67.5 A. X-ray diffraction intensities to 2.9 A resolution have been collected on a Xentronics/Nicolet area detector. The structure has been solved by molecular replacement where the starting model was based on refined coordinates of an I222 crystal of saccharide-free concanavalin A. The structure of the saccharide complex was refined by restrained least-squares methods to an R-factor value of 0.19. In this crystal form, the asymmetric unit contains four protein subunits, to each of which a molecule of mannoside is bound in a shallow crevice near the surface of the protein. The methyl alpha-D-mannopyranoside molecule is bound in the C1 chair conformation 8.7 A from the calcium-binding site and 12.8 A from the transition metal-binding site. A network of seven hydrogen bonds connects oxygen atoms O-3, O-4, O-5 and O-6 of the mannoside to residues Asn14, Leu99, Tyr100, Asp208 and Arg228. O-2 and O-1 of the mannoside extend into the solvent. O-2 is hydrogen-bonded through a water molecule to an adjacent asymmetric unit. O-1 is not involved in any hydrogen bond and there is no fixed position for its methyl substituent.

Dimerization of carbohydrate radicals

AbstractGlycosyl bramides and selenides 1 react with hexamethylditin to give coupling products 2–4 in 32% yield. The reactions occur by dimerization of glycosyl radicals, the radical 8 in the 4C1 chair conformation being slightly more stereoselective than the glucosyl radical 7 in the B2,5 boat conformation.

Conformation of the glycotripeptide repeating unit of antifreeze glycoprotein of polar fish as determined from the fully assigned proton n.m.r. spectrum

With its simple glycotripeptide repeating structure the antifreeze glycoprotein of polar fish may be an especially simple conformational mode for mucin glycoproteins with similar but more complex structures. The fully assigned proton n.m.r. spectrum confirms the anomeric configurations of the hexapyranosidic sugars of the side chains and the coupling constants of the alpha GalNAc and the beta Gal residues show both to be in the expected 4C1 chair conformation. The assignment of a single resonance for each proton of the (Ala-Thr-Ala)n repeat unit coupled with the observation of long range nuclear Overhauser effects (n.O.e.) implies a three-fold repeating conformation. The resonances of the two alanines are distinct and can be assigned to their correct positions in the peptide sequence by n.O.e. observed at the amide proton resonances on saturation of the alpha proton signals. The amide proton coupling constants of all three peptide residues are similar and imply a limited range of peptide backbone torsion angles, phi CN. The large n.O.e. which has been observed between the amide proton and the alpha proton of the residue preceding it in the sequence implies large positive values for the peptide dihedral angle, psi CC. Limits are placed on possible values of side chain dihedral angles by the observation of the coupling constant between the alpha and beta protons of the threonyl residue. The observation of n.O.e. between the anomeric proton of GalNAc and the threonyl side chain protons gives information on the conformation of the alpha glycosidic linkage between the disaccharide and the peptide. n.O.e. observed between the protons of the beta glycosidic linkage indicates the conformation of the disaccharide and the large amide proton coupling constant of the GalNAc residue shows a trans proton relationship. The spectroscopically derived data have been combined with conformational energy calculations to give a conformational model for antifreeze glycoprotein in which the hydrophobic surfaces of the disaccharide side chains are wrapped closely against a three-fold left handed helical peptide backbone. The hydrophilic sides of the disaccharides are aligned so that they may bind to the ice crystal face, which is perpendicular to the fast growth axis inhibiting normal crystal growth.