Effects of glycosylation on peptide conformation: a synergistic experimental and computational study

Asparagine-linked glycosylation, the co-translational covalent attachment of carbohydrates to asparagine side chains, has a major effect on the folding, stability, and function of many proteins. The carbohydrate composition in mature glycoproteins is heterogeneous due to modification of the initial oligosaccharide by glycosidases and glycosyltransferases during the glycoprotein passage through the endoplasmic reticulum and Golgi apparatus. Despite the diversity of carbohydrate structures, the core beta-D-(GlcNAc)(2) remains conserved in all N-linked glycoproteins. Previously, results from our laboratory showed that the molecular composition of the core disaccharide has a critical and unique conformational effect on the peptide backbone. Herein, we employ a synergistic experimental and computational approach to study the effect of the stereochemistry of the carbohydrate--peptide linkage on glycopeptide structure. A glycopeptide derived from a hemagglutinin protein fragment was synthesized, with the carbohydrate attached to the peptide with an alpha-linked stereochemistry. Computational and biophysical analyses reveal that the conformations of the peptide and alpha- and beta-linked glycopeptides are uniquely influenced by the attached saccharide. The value of computational approaches for probing the influence of attached saccharides on polypeptide conformation is highlighted.     

Synthesis of 3,4-di-O-acylated glucose-derived furanoid sugar amino acids (Gaa): conformational analysis of a Leu-enkephalin analog containing di-O-myristoylated Gaa

3,4-Di-O-acylated derivatives 1-3 of a glucose-derived furanoid sugar amino acid (Gaa) were synthesized as novel peptide building blocks to study their effects on peptide conformation. Structural analysis of the di-O-myristoylated Gaa 3-containing Leu-enkephalin analog 4 by various NMR techniques and constrained molecular dynamics (MD) simulation studies established a well-defined β-turn structure in DMSO-d₆ with an intramolecular hydrogen bond between PheNH → TyrCO.     

Stabilization of β-turn conformations in Pro-X sequences by disulphide bridging. Synthesis and solution conformations of five cyclic cystine peptides

Five cyclic peptide disulphides of the type

have been synthesized, where X = Gly (1), L-Ala (2), D-Ala (3), Aib (4) and L-Leu (5). ¹H NMR studies at 270 MHz in CDCl₃ and (CD₃)₂SO provide evidence of a Pro-X β-turn conformation, stabilized by a transannular 4→1 hydrogen bond involving the Cys(4) NH, in all the peptides. In addition peptides 2, 4 and 5 also possess a second intramolecular hydrogen bond involving the -NHMe group. The spectroscopic data are consistent with a consecutive type III β-turn conformation for peptides 2, 4 and 5, a type I(III) β-turn structure for 1 and a type II turn for 3.     

m-Aminobenzoic acid inserted β-turn in acyclic tripeptides: a peptidomimetic design

X-ray diffraction studies show that peptides Boc-Leu-Aib-m-ABA-OMe (I) (Aib, α-aminoisobutyric acid; m-ABA, meta-aminobenzoic acid) and Boc-Phe-Aib-m-ABA-OMe (II) adopt a type-II β-turn conformation, solely stabilized by co-operative steric interactions amongst the amino acid residues. This type of β-turn without any intramolecular hydrogen bonding is generally referred to as an open turn. Although there are some examples of constrained cyclic peptides in which o-substituted benzenes have been inserted to mimic the turn region of the neurotrophin, a nerve growth factor, peptides I and II present novel two examples where m-aminobenzoic acid has been incorporated in the β-turn of acyclic tripeptides. The result also demonstrates the first crystallographic evidence of a β-turn structure containing an inserted m-aminobenzoic acid, which can be considered as a rigid γ-aminobutyric acid with an all-trans extended configuration.     

Studies of β-turn opening with model peptides containing non-coded α-amino isobutyric acid

Single crystal X-ray diffraction studies show that among the three terminally protected model tripeptides I-III, Boc-Ile-Aib-Xx-OMe (Xx in peptide I: Val; II: Leu; III: Phe) with a centrally placed non-coded amino acid Aib (Aib: α-amino isobutyric acid), peptide I displays a conformational preference for β-turn, peptide II forms a hydrated β-turn representing the solvent mediated intermediate for the interconversion between β-turn and β-strand and peptide III adopts a completely unfolded β-strand like structure. By varying the steric bulk of the third residue, Xx(3), various conformations related to the structural interconversion between the β-turn and β-strand have been isolated. The peptide conformations in the solution phase have been probed by solvent dependent NMR titration and CD spectroscopy. Morphological studies with scanning electron microscopy (SEM) reveal that among the three peptides only peptide III can form filamentous fibrils in the solid state.     

Enkephalin-based drug design: conformational analysis of O-linked glycopeptides by NMR and molecular modeling

Glycosylation provides an effective means of enhancing penetration of the blood–brain barrier by pharmacologically active peptides. Glycosylated enkephalin analogues demonstrate much greater analgesic effects than their unglycosylated counterparts when administered peripherally. The solution conformations of glycopeptide enkephalin analogues with the sequences H-Tyr-c-[d-Cys-Gly-Phe-d-Cys]-Ser(β-O-Glcp)-Gly-NH₂, 2, and H-Tyr-c-[d-Cys-Gly-Phe-d-Cys]-Ser(α-O-Glcp)-Gly-NH₂, 3, have been determined by NMR and molecular modeling, and were compared to the unglycosylated peptide H-Tyr-c-[d-Cys-Gly-Phe-d-Cys]-Ser-Gly-NH₂, 1, to determine the impact of glycosylation on peptide conformation. The only observed conformational effects were on the residue of attachment, Ser⁶, and on the adjacent Gly⁷-amide. This has important implications in peptide-based drug design in that strategically placed glycosylation can improve transport without destruction of the receptor selectivity of a pre-existing non-glycosylated peptide pharmacophore.     

Solid-phase synthesis of glycopeptide carrying a tetra-N-acetyllactosamine-containing core 2 decasaccharide

A novel synthesis of tetralactosaminyl O-glycoamino acid is described. The stereoselective assemblage of a lactosaminyl unit was performed by 2-trichloroacetamido group-assisted β-glycosylation. Initial investigation into the synthesis of decasaccharyl threonine 2 showed limited success because of the low yield in the step concerning the removal of 4-O-chloroacetyl groups. In contrast, 4-O-benzylated decasaccharyl threonine 50 was efficiently synthesized from key LacNAc derivative 35 carrying a 3-O-allyl protecting group at the Gal residue by reiterative glycosylation using the (N-phenyl)trifluoroacetimidate method. Decasaccharide 50 was used as a building block in the solid-phase synthesis of a MUC1-related glycopeptide. Synthetic glycopeptide was obtained through two acidic processes: cleavage from resin with reagent K at a lowered temperature and debenzylation with a diluted cocktail of low-acidity TfOH. Desired glycopeptide 54 was isolated as the major product, while a series of the saccharide-shortened minor products were generated due to the acid-labile property of the β-GlcNAc glycosidic linkages.     

Synthesis of sidechain adapted β-turn mimics for modifying the C-terminus of substance P

Sidechain adapted β-turn mimics of type 1 characterised by a fixed cis-conformation of the peptide chain in the aminopiperidinonecarboxylate scaffold have been synthesised from pyrazinones in order to perform a β-turn scan of the messenger region of substance P. The synthesis of a substance P peptide analogue is also described.     

Synthesis and conformational analysis of 18-membered Aib-containing cyclohexapeptides

The synthesis and conformational analysis of two Aib-containing cyclic hexapeptides, cyclo(Gly-Aib-Leu-Aib-Phe-Aib) 1 and cyclo(Leu-Aib-Phe-Gly-Aib-Aib) 2, is described. The linear precursors of 1 and 2 were prepared using solution phase techniques, and the cyclization efficiency of three different coupling reagents (HATU, PyAOP, DEPC) was examined. The success of the cyclization was found to be reagent dependent. Solid-state conformational analysis of 1 and 2 was performed by X-ray crystallography and has revealed some unusual features as all three Aib residues of 1 assume nonhelical conformations. Furthermore, the residue Aib⁴ adopts an extended conformation (?=−175.9(3)°, ψ=+178.6(2)°), which is, to the best of our knowledge, the first observation of an Aib residue adopting an extended conformation in a cyclopeptide. The structure of 1 is also a rare example in which an Aib residue occupies the (i+1) position of a type II′ β-turn, stabilized by a bifurcated hydrogen bond. The cyclic peptide 2 adopts a more regular conformation in the solid state, consisting of two fused β-turns of type I/I′, stabilized by a pair of intramolecular hydrogen bonds. In addition, the conformational study of the cyclic peptide 1 in DMSO-d₆ by NMR spectroscopy and molecular dynamics simulations revealed a structure, which is very similar to its structure in the crystalline state.     

Synthesis of a β-GlcN-(1→4)-MurNAc building block en route to N-deacetylated peptidoglycan fragments

Some bacteria present a variation in their peptidoglycan structure that is the absence of the N-acetyl substituent in the glucosamine residue. Very recently, this structural modification was demonstrated to be critical for host innate immune evasion in Listeria monocytogenes. To shed light on the molecular details of the evasion mechanism, the synthesis of some N-deacetylated peptidoglycan fragments is needed. En route to this goal a high-yielding synthesis of a GlcN-MurNAc disaccharide building block has been accomplished. A careful study of the optimal protecting groups and reaction conditions was done to have a complete β-stereoselectivity in glycosylation as well as to ensure a high versatility to the disaccharide building block.     

One-pot stereoselective synthesis of β-N-aryl-glycosides by N-glycosylation of aromatic amines: application to the synthesis of tumor-associated carbohydrate antigen building blocks

We studied the stereoselective synthesis of several β-N-aryl-glycosides by glycosylation of aromatic primary amines using unprotected carbohydrates in aqueous solution. This was the first report showing an efficient method for the synthesis with one step of β-N-glycosyl-para-amino-phenyl alanine building blocks for the tumor-associated carbohydrate antigen (TACA) glycopeptides synthesis. Analysis of products by ¹H and ¹³C NMR indicated that the Amadori rearrangement had not occurred after formation of the stereoselective β-N-glycoside bond (natural N-glycoprotein linkage). The study of the chemical and enzymatic stability in aqueous media of β-N-aryl-glycosides synthesized was also investigated. For the first time we have shown that the N-glycosidic bond was relatively stable at pH near to 7 and more stable than the O-glycosidic bond to enzymatic hydrolysis. This higher enzymatic and chemical stability of the N-glycosidic bond is essential to envisage further development of stable TACA building blocks.     

A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes

Aberrantly truncated immature O-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immature O-glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognises specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its solution NMR structure, an extended conformation induced by site-specific glycosylation. We demonstrate that this method accelerates dramatically the development of a new class of designated antibodies targeting a variety of “dynamic neoepitopes” elaborated by disease-specific O-glycosylation in the immunodominant mucin domains and mucin-like sequences found in intrinsically disordered regions of many proteins.

We developed new class of designated antibodies targeting of “dynamic neoepitopes” elaborated by disease-specific O-glycosylation at the immunodominant mucin domains.     

Regioselective lipase acylation as a useful tool for separation and selective protection of β-d-Gal(1→4)-d-GlcNAc and β-d-Gal(1→3)-d-GlcNAc disaccharides

Supported lipase from Candida antarctica (Chirazyme®) was employed for a regioselective protection of the 2-azido derivatives 1 and 2, synthetic equivalents of β-d-Gal(1→3)-d-GlcNAc and β-d-Gal(1→4)-d-GlcNAc (N-acetyl lactosamine), respectively. The selectivity of the enzyme towards 1 and 2 was also exploited for an easy separation of the mixture of the two compounds obtained from a straightforward synthetic approach.     

Amyloid-like fibril-forming supramolecular β-sheets from a β-turn forming tripeptide containing non-coded amino acids: the crystallographic signature

The crystal structure of a terminally protected tripeptide Boc-Leu-Aib-β-Ala-OMe 1 containing non-coded amino acids reveals that it adopts a β-turn structure, which self-assembles to form a supramolecular β-sheet via non-covalent interactions. The SEM image of peptide 1 exhibits amyloid-like fibrillar morphology in the solid state.     

Design and synthesis of a new series of amphiphilic peptide-β-cyclodextrins as phase transfer carriers for glucosamine

A new series of amphiphilic β-cyclodextrins were designed and synthesized by grafting peptide chains on to all primary hydroxyl groups via ester bond formation. The desired amphiphilic structures have been produced from ester connection between the C-6 of β-cyclodextrin and the carboxyl group of N-acetylated resides: H₂N-Leu-COOH, H₂N-Leu-Gly-COOH, H₂N-Leu-Gly-Leu-COOH, and H₂N-Leu-Gly-Leu-Gly-COOH (3a-d). The synthetic pathway involves selective bromination of all primary hydroxyls of β-cyclodextrins and then substitution with the carboxylate moiety of the mentioned N-acetyl residues in the presence of DBU (1,8-diazabicyclo[5,4,0]undec-7-ene). The ability of the synthetic compounds for extraction and phase transfer of glucosamine, as a hydrophilic organic compound, was then studied. The results showed a considerable ability of these amphiphilic compounds for extraction and a selective tendency of 3c for phase transfer of glucosamine.     

Synthesis of multivalent N-acetyl lactosamine modified quantum dots for the study of carbohydrate and galectin-3 interactions

Ternary core/shell CdSeS/ZnS-QDs coated with N-acetyl lactosamine was prepared as a fluorescent probe to study the interactions of N-acetyl lactosamine and galectin-3. The synthesis of N-acetyl lactosamine was achieved through the ‘azidoiodoglycosylation’ method. The amount of ligand coated on QDs was determined by ¹H NMR and ICP-OES. The interactions between carbohydrates and galectin-3 were measured using SPR. The results revealed that the affinity of galectin-3 with di- and multivalent N-acetyl lactosamine increased 20 and 184-fold, respectively. The prepared glyco-QDs could be used as an efficient fluorescent probe to study carbohydrates and galectin-3 interactions.     

The Role of Ligand Density in the Enzymatic Glycosylation of Carbohydrates Presented on Self-Assembled Monolayers of Alkanethiolates on Gold

The biological activity of immobilized carbohydrates can show a dramatic dependence on the density of carbohydrate. This is the result of investigations with self-assembled monolayers that present N-acetylglucosamine groups as a model substrate for glycosylation by bovine β-1,4-galactosyltransferase (GalTase; see picture). Surface plasmon resonance spectroscopy and carbohydrate-binding lectins were used to characterize the reaction at the interface. UDP-Gal=uridine diphosphogalactose     

Stereoselective synthesis of novel N-(α-l-arabinofuranos-1-yl)-l-amino acids

In lead detoxification, the α-anomer of N-glycocyl-l-amino acid is more potent than its β-anomer. Here a six-step-reaction route for stereoselectively preparing N-(α-l-arabinose-1-yl)-l-amino acids is reported. Treating l-arabinose with acetic anhydride and sodium acetate provided 1,2,3,5-tetra-O-acetyl-l-arabinofuranose in 90% yield. After removing the 1-acetyl group, the thus formed 2,3,5-tri-O-acetyl-l-arabinofuranose and N-(2-nitrophenylsulfonyl)-l-amino acid t-butylesters were treated with triphenylphosphine to perform Mitsunobu dehydration and form 2,3,5-tri-O-acetyl-l-arabinofuranosyl-l-[N-(2-nitrophenylsulfonyl)]amino acid t-butylesters 2a–f, and the ratios of their α- to β-anomer ranged from 8/1 to 9/1. Chromatographic separation provided epimerically pure 2a–f-α and 2a–f-β. In the presence of CF₃CO₂H, 2a–f-α and 2a–f-β were converted to α- and β-anomers of N-(2,3,5-tri-O-acetyl-l-arabinofuranosyl)-N-(2-nitrobenzenesulfonyl)-l-amino acids, 3a–f-α and 3a–f-β, in 87–92% yields. While in the presence of NaOCH₃, 3a–f-α and 3a–f-β were converted to α- and β-anomers of N-(l-arabinofuranosyl)-N-(2-nitrobenzenesulfonyl)-l-amino acids, 4a–f-α and 4a–f-β, in 90–96% yields. Treating 4a–f-α and 4a–f-β with N-ethyldiisopropylamine (DIPEA) and thiophenol, their 2-nitrophenylsulfonyl groups were removed, and the α- and β-anomers of N-(l-arabinose-1-yl)-l-amino acids were formed in 70–79% yields. The bioassay confirmed that the lead detoxification activity of the α-anomer was significantly higher than that of the β-anomer.     

Synthesis and conformational properties of model dipeptides containing novel axially chiral α,β-didehydroamino acids at the (i+1) position of a β-turn conformation

A series of model dipeptides containing some novel axially chiral α,β-didehydroamino acids at the (i+1) position has been synthesised by reaction of the corresponding 4-(4-alkylcyclohexylidene)-2-phenyl-1,3-oxazol-5(4H)-one with (S)-phenylalanine cyclohexylamide. The conformations of two dipeptides in the crystal state have been studied by X-ray diffraction crystallographic analysis. The backbone torsion angles suggest that both peptides adopt similar type-II′ β-turn conformations. NMR spectroscopy has revealed that relatively rigid β-turn structures also persist in solution and that the absolute configurations of the axially chiral α,β-didehydroamino acids do not significantly influence the conformation of the peptide chain. Both heterochiral and homochiral dipeptides are found to accommodate the same βII′-turn conformation. Axially chiral α,β-didehydroamino acids (R_a)- and (S_a)-4-methyl-, 4-phenyl- and (4-tert-butylcyclohexylidene)glycine can be considered as elongated structural analogues of alanine, phenylglycine and tert-leucine of R and S configuration since, in these chiral α,β-didehydroamino acids, the methyl, phenyl and tert-butyl groups are located about 4.3 Å away from the peptide backbone in which they are incorporated.     

Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.