Glycodermorphins: opioid peptides with potent and prolonged analgesic activity and enhanced blood-brain barrier penetration

1. In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O-βglucosylated analogs ([Ser⁷-O-βGlc]dermorphin and [Ser⁷-O-βGlc(Ac)₄]-dermorphin) and C-αgalactosylated analogs ([Ala⁷-C-αGal]dermorphin and [Ala⁷-C-αGal(Ac)₄]-dermorphin).
2. O- and C-glycosylation of dermorphin halved the peptide affinity for brain μ-opioid receptors and the biological potency in guinea-pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8–40 pmol) and subcutaneously (s.c., 0.5–3 μmol kg^?1) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5–10 times both biological activity on GPI and μ-receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two-three times lower (s.c.) than dermorphin potency.
3. Blood-Brain Barrier Permeability Index (BBB-PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O-β-linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT-1 of the endothelial barrier. However the calculated BBB-PI for the C-αgalactoside was about two times higher than that of the O-βglucoside, excluding the implication of GLUT-1 that is known to be selective for O-β-links and preferring for the exose glucose.
4. The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.

     

HUMAN β-ENDORPHIN: SYNTHESIS AND BIOLOGICAL ACTIVITY OF ANALOGS WITH SUBSTITUTIONS IN POSITIONS 2, 9, 18, 27 AND 31

Four analogs of the opioid peptide human β-endorphin (β_h-EP) have been synthesized: [d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, [d -Phe¹⁸,Phe²⁷,Gly³¹)-β_h-EP, [d -Thr²,d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, and [d -Thr²,d -Phe¹⁸,Phe²⁷,Gly³¹]-β_h-EP. All are practically indistinguishable from β_h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.     

The conformational and biological analysis of a cyclic anti‐obesity peptide from the C‐terminal domain of human growth hormone

Abstract: The three‐dimensional solution structure of anti‐obesity drug (AOD), a 15‐residue, disulfide‐bonded, cyclic peptide, cyclo(6,13)‐H₂N‐Leu‐Arg‐Ile‐Val‐Gln‐Cys‐Arg‐Ser‐Val‐Glu‐Gly‐Ser‐Cys‐Gly‐Phe‐OH, derived from the C‐terminal domain of the human growth hormone (hGH) (residues 177–191) was determined using two‐dimensional ¹H NMR spectroscopy. AOD stimulates lipolysis and inhibits lipogenesis, in vitro, in rodent, porcine and human adipose tissues. These biological effects suggest that AOD is a potential therapeutic candidate for the treatment of obesity. Conformational studies of AOD were conducted in aqueous solution and in water/dimethylsulfoxide mixtures. In general, spectral quality was superior in the water/dimethylsulfoxide mixtures. The cyclic region of AOD in water/dimethylsulfoxide adopts type I β‐turns at residues Ser⁸‐Val⁹‐Glu¹⁰‐Gly¹¹ and Ser¹²‐Cys¹³‐Gly¹⁴‐Phe¹⁵, each preceded by loop‐like structures. Comparison of the conformation of this peptide with residues 177–191 in the native hGH protein X‐ray crystal structure indicates that the synthetic peptide retains some structural similarity to the intact protein. This study provides evidence that the C‐terminal region of hGH is a specific functional domain of the multifunctional hGH protein.     

Biological and conformational study of β‐substituted prolines in MT‐II template: steric effects leading to human MC5 receptor selectivity*

Abstract: To investigate the molecular basis for the interaction of the χ‐constrained conformation of melanotropin peptide with the human melanocortin receptors, a series of β‐substituted proline analogs were synthesized and incorporated into the Ac‐Nle‐c[Asp‐His‐d ‐Phe‐Arg‐Trp‐Lys]‐NH₂ (MT‐II) template at the His⁶ and d ‐Phe⁷ positions. It was found that the binding affinities generally diminished as the steric bulk of the p‐substituents of the 3‐phenylproline residues increased. From (2S, 3R)‐3‐phenyl‐Pro⁶ to (2S, 3R)‐3‐(p‐methoxyphenyl)‐Pro⁶ analogs the binding affinity decreased 23‐fold at the human melanocortin‐3 receptor (hMC3R), 17‐fold at the hMC4R, and eight‐fold at the hMC5R, but selectivity for the hMC5R increased. In addition, the substitution of the d ‐Phe⁷ residue with a (2R, 3S)‐3‐phenyl‐Pro resulted in greatly reduced binding affinity (10³–10⁵) at these melanocortin receptors. Macromodel's Large Scale Low Mode (LLMOD) with OPLS‐AA force field simulations revealed that both MT‐II and SHU‐9119 share a similar backbone conformation and topography with the exception of the orientation of the side chains of d ‐Phe⁷/d ‐Nal (2′)⁷ in χ space. Introduction of the dihedrally constrained phenylproline analogs into the His⁶ position (analogs 2 – 6 ) caused topographical changes that might be responsible for the lower binding affinities. Our findings indicate that hMC3 and hMC4 receptors are more sensitive to steric effects and conformational constraints than the hMC5 receptor. This is the first example for melanocortin receptor selectivity where the propensity of steric interactions in χ space of β‐modified Pro⁶ analogs of MT‐II has been shown to play a critical role for binding as well as bioefficacy of melanotropins at hMC3 and hMC4 receptors, but not at the hMC5 receptor.     

Synthesis of glycosylated tuftsins and tuftsin-containing IgG fragment undecapeptide

Syntheses are described of two new tuftsin derivatives containing a 2-acetamido-2-deoxy-D-galac-topyranosyl unit α- or β-glycosidically linked to the threonine's hydroxy side chain function and of the glycosylated undecapeptide corresponding to the tuftsin region of the heavy chain of IgG (amino acid sequence 289–299). The glycosylated tuftsins were synthesized by the solution procedure. Fmoc-[Gal NAc(Ac)₃α]Thr-OH and Fmoc-[GalNAc(Ac)₃β]Thr-OH were allowed to react with H-Lys(Z)-Pro-Arg(NO₂)-OBzl by the mixed anhydride procedure and the resulting glycosylated tetrapeptides were fully deblocked by catalytic hydrogenation followed by treatment with potassium cyanide, purified by ion exchange chromatography and characterized by analytical HPLC, elemental and amino acid analyses, optical rotation, and proton NMR spectroscopy. Synthesis of the glycosylated undecapeptide was achieved by the continuous flow solid phase procedure on 4-hydroxymethylphenoxyacetyl-norleucyl derivatized Kieselguhr-supported resin. Fmoc-amino acid symmetrical anhydrides or pentafluorophenyl esters, in the presence of N-hydroxybenzotriazole, were used as the acylating agents. To mimic the native sequence of the tuftsin region at the Fc-domain of immunoglobulin G a 2-acetam～do-2-deoxy-β-D-glucopyranosyl unit was N-glycosidically linked to the amide side chain of Asn 297. The glycosylated asparagine residue was introduced as N²-fluorenylmethyloxycarbonyl-N⁴-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopy-ranosyl)-asparagine pentafluorophenyl ester. After cleavage from the resin the glycopeptide was deprotect-ed, purified by ion exchange chromatography, and characterized by analytical HPLC, amino acid analysis, high voltage electrophoresis, and proton NMR. The conformational features of the glyco-undecapeptide were determined by circular dichroism measurements both in water and in 98% trifluoroethanol. Results of biological assays will be published elsewhere.     

Synthesis and biological activity of [L-hydroxyproline]3-tuftsin analogue and its α- or β-O-D-glucosylated derivatives

Syntheses are described of the Hyp³-tuftsin analogue and of its derivatives α- or β-O-glycosylated at the side chain function of the hydroxyproline residue. The carbohydrate-free tetrapeptide was prepared by reacting Z-Thr-Lys(Z)-OH with H-Hyp-Arg(NO₂)-OBzl by the mixed anhydride procedure. In the synthesis of the α-glycosylated analogue the O-glycosyl amino acid was incorporated by reacting Boc-(Glcα+β)Hyp-OH with H-Arg(NO₂)-OBzl through the same procedure. The α-glucosylated dipeptide was isolated from the diastereomeric mixture, selectively deblocked, and acylated with Z-Thr-Lys(Z)-OH by the mixed anhydride procedure. In the preparation of the β-glucosylated analogue the BOP procedure was used for reacting Boc-[Glc(Ac)₄β]Hyp-OH with H-Arg(NO)₂-OBzl was well as for the final coupling to tetrapeptide. Removal of protecting groups from crude tetrapeptides was achieved by catalytic hydrogenation. Deacetylation of the sugar moiety of the β-glucosylated tetrapeptide was achieved by treatment with sodium methoxide in methanol. The synthetic compounds were isolated by ion exchange chromatography, and characterized by elemental analysis, amino acid analysis, optical rotation and proton NMR. Their capacity to evoke the release of interleukin 1 from mouse peritoneal macrophages and to modulate immunogenic activity of antigen-fed cells was evaluated, in comparison with tuftsin and rigin. All of the analogues were found to possess tuftsin-like activity.     

β-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human β-endorphin (β-EP) which contain cystine bridges, [Cys¹⁵-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (I) and [Cys¹⁶-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2–2.5 times the opiate receptor binding activity of β-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg^{9,19,24,28,29}Cys(Cam)^11,26, Phe²⁷,Gly³¹] and [Arg^{9,19,24,28,29},Cys-(Cam)^12,26, Phe²⁷, Gly³¹], were shown to have approximately the same opiate receptor activity as β-endorphin.     

Antifibrotic peptide N‐acetyl‐Ser‐Asp‐Lys‐Pro (Ac‐SDKP): Opportunities for angiotensin‐converting enzyme inhibitor design

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Solvolysis and aminolysis on peptidyl‐Kaiser oxime resin assisted by Ca2+and Eu3+: a mild procedure to prepare α‐methyl and ‐ethyl esters of protected peptides

Abstract: Ca²⁺ and Eu³⁺ were able to assist solvolysis on peptidyl‐Kaiser oxime resins generating α‐methyl and ‐ethyl esters of protected peptides. The methanolysis assistance was at least twice as effective as that of acetic acid, the common catalyst used in aminolysis of the ester oxime linkage. No molar excess of Ca²⁺ or Eu³⁺ was needed to enhance this reaction efficiency. Ca²⁺ also assisted aminolysis on peptidyl‐Kaiser oxime resins. Solvolysis and aminolysis rates depended on the nature of theC‐terminal residue attached to the resin and on the alcohol used. Both reactions were selective to the ester oxime linkage since no significant amount of secondary products, resulting from rearrangements or simultaneous transesterification of the β‐benzyl or cyclohexyl esters, was detected in the reaction media. The α‐methyl and ‐ethyl esters of Ac‐Ala‐Gly‐X [where, X = Gly, Ala, Phe or Lys (2‐Cl‐Z)] and of Ac‐Ile‐Ser (Bzl)‐Asp(OZ) (where, Z = Bzl or cHex) were essentially the only products formed in the solvolyses performed. Ac‐Ile‐Ser(Bzl)‐Asp(OcHex)Arg(HCl)‐OMe and Ac‐Ile‐Ser(Bzl)‐Asp(OcHex)Arg (HCl)‐OEt were the major products formed in the aminolysis reactions. In the presence of the metal ions, the resin‐cleavage yields were > 50%. In their absence, they were < 15%.     

Importance of the C‐terminal phenylalanine of gastrin for binding to the human CCK2 receptor

Abstract: The importance of the C‐terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK₂ receptor were assessed using analogs of [Leu¹⁵]G(11?17). The following peptides were synthesized, Ac[Leu¹⁵]G(11?17), Ac[Leu¹⁵]G(11?16)NH₂, [Leu¹⁵]G(11?17), [Leu¹⁵,Ala¹⁷]G(11?17), [Leu¹⁵,Abu¹⁷]G(11?17), [Leu¹⁵,Val¹⁷]G(11?17), [Leu¹⁵,Leu¹⁷]G(11?17), [Leu¹⁵,Cha¹⁷]G(11?17), [Leu¹⁵,Trp¹⁷]G(11?17), [Leu¹⁵,Tic¹⁷]G(11?17), [Leu¹⁵, d ‐Phe¹⁷]G(11?17) and [Leu¹⁵,p‐X‐Phe¹⁷]G(11?17), where X = F, Cl, Br, I, OH, CH₃, NH₂ and NO_2. Competition binding experiments with [³H]CCK‐8 were performed using human CCK₂ receptors stably expressed in CHO cells. Phe¹⁷ was shown to be important for binding. A hydrophobic side‐chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side‐chain either in the g(+) or g(–) conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d ‐Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des‐Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK₂ receptor.     

Synthesis of a cyclic retro analogue of somatostatin suitable for photoaffinity labelling

Cyclic somatostatin analogues containing the modified retro sequence of the amino acids Phe⁷ to Phe¹¹ of the natural compound have been found to exhibit high activity for cytoprotection of rat hepatocytes against cell poisons such as phallotoxins and galactosamine. Cyclo(-Phe(p-NH(1-¹⁴C)Ac)-Thr-Lys(CO(p-N₃)C₆H₄)-Trp-Phe-d -Pro-), a photoreactive and radioactive analogue of one of the most active cyclohexapeptides, was synthesized by a combination of solid phase technique and classical solution peptide synthesis. This peptide labels the same proteins in rat liver cell membrane that are modified by photolysable derivatives of bile acids, phalloidin and antamanide.     

Effect of sulfate position on rnyotropic activity of the gastrin/CCK-like insect leucosulfakinins

Leucosulfakinin and leucosulfakinin-II are sulfated insect neuropeptides with homology to human gastrin II and cholecystokinin. Biological evaluation of the analog [Ser(SO₃H)², Tyr⁵]leucosulfakinin-II, two cholecystokinin analogs containing key leucosulfakinin amino acid replacements, and a Tyr(SO₃H) position-analog series has demonstrated that while the presence of a sulfate group is critical to leucosulfakinin myotropic activity, its position is not. Most strikingly, the analog [Tyr (SO₃H)⁵,Phe⁶,Nle⁹]leucosulfakinin, in which the sulfate moiety is shifted one position towards the N-terminus relative to the parent peptide, retains a very significant 38% of the parent's threshold activity. A shift in the location of the sulfate group by two to five positions toward the N-terminus led to reduction of potency to a significant but low plateau of activity, whereas a shift by more than one position towards the C-terminus led to a complete loss of activity. The introduction of a single residue (Arg) in the 7 position of CCK-8 transforms this inactive mammalian hormone into a myotropically-active leucosulfakinin analog on the isolated cockroach hindgut.     

Synthetic protein design: construction of a four‐stranded β‐sheet structure and evaluation of its integrity in methanol–water systems

Abstract: The characterization of a four‐stranded β‐sheet structure in a designed 26‐residue peptide Beta‐4 is described. The sequence of Beta‐4 (Arg‐Gly‐Thr‐Ile‐Lys‐^Dpro‐Gly‐Ile‐Thr‐Phe‐Ala‐^DPro‐Ala‐Thr‐Val‐Leu‐Phe‐Ala‐Val‐^DPro‐Gly‐Lys‐Thr‐Leu‐Tyr‐Arg) was chosen such that three strategically positioned ^DPro‐Xxx segments nucleate type II′β‐turns, which facilitate hairpin extension. A four‐stranded β‐sheet structure is determined in methanol from 500 MHz ¹H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal J_CαH‐NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four‐stranded sheet and a nonpolar cluster involving Ile⁸, Phe¹⁰, Val¹⁵ and Phe¹⁷. Circular dichroism studies in water–methanol mixtures provide evidence for melting of the β‐sheet structure at high water concentrations. NMR analysis establishes that the four‐stranded sheet in Beta‐4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three β‐turn nuclei appear to be maintained.     

Synthesis and properties of dermorphin and an analog of β-endorphin containing the dermorphin sequence

Dermorphin (I) and [D-Ala², Phe³, Gly⁴, Tyr⁵, Pro⁶]-β_c-EP (II) have been synthesized by the solid-phase method (β-EP, camel β-endorphin). Positions I through 7 of II correspond to the sequence of I. Relative potencies of synthetic peptides in the mouse tail-flick test for analgesia by the intracerebroventricular route were: human β-endorphin, 100; camel β-endorphin, 164; I, 450; II, 440. The dermorphin was about 670 times more potent than morphine in the assay. Peptide II represents a rare instance where the enkephalin moiety of β-endorphin has been altered to produce a more potent analgesic.     

β-Endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays

Three synthetic analogs of human β-endorphin (β_h-EP) (I, [Gln⁸, Gly³¹]-β_h-EP-Gly-Gly-NH₂; II, [Arg^{9,12,24,28,29}]-β_h-EP and III, [Cys^11,26, Phe²⁷, Gly³¹]-β_h-EP), which have been shown to possess potent inhibiting activity to β_h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were β_h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: β_h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.     

Multiple anchoring of myelopeptides on sequential oligopeptide carriers (SOCn): synthesis,conformation and studies in human leukemia cells

Abstract: Myelopeptides, MP‐6 (Val‐Asp‐Pro‐Pro) and MP‐4 (Phe‐Arg‐Pro‐Arg‐Ile‐Met‐Thr‐Pro), induce metabolic changes in human leukemia cells, HL‐60, characteristic of the differentiation process, which should be regarded as a promising therapeutic approach in cancer and related diseases. With the aim to optimize the differentiation effect of MPs, they were coupled to the Lys‐N^εH₂ groups of a sequential oligopeptide carrier Ac‐(Lys‐Aib‐Gly)₄, SOC₄, and the constructs obtained were studied. The rigid 3₁₀ secondary structure of the carrier is preserved even after linkage of the MPs, which also maintain their initial conformations without interacting either with each other or with the carrier, as demonstrated by ¹H nuclear magnetic resonance (NMR) spectroscopy. It is concluded that the carrier accommodates the presentation of MPs, thus improving their differentiation effect on human leukemia cells.     

Integrin α5β1: a new purification strategy based on immobilized peptides

Abstract: Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C‐terminally modified peptide Ac‐Gly‐Ala‐c‐(Cys^SS‐Arg‐Arg‐Glu‐Thr‐Ala‐Trp‐Ala‐Cys^SS)‐Gly‐Ala‐O(CH₂CH₂O)₂CH₂CH₂‐NH₂ allows for the affinity purification of the integrin α₅β₁. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α₅β₁. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well‐characterized affinity chromatography material with a site‐specifically immobilized peptide allows to obtain integrin α₅β₁ in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein‐ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.     

Synthesis of O-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety

Synthesis is described of four tuftsin derivatives containing a D-ghcopyranosyl or a D-galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an α or βO-glycosidic linkage. Fmoc-threonine derivatives containing the suitable unprotected sugar were used for incorporating the O-glycosylated amino acid residue. Z-Thr[α-Glc(OBzl)₄]-OBzl and Z-Thr[α-Gal(OBzl)₄]-OBzl were prepared from the tetra-O-benzylated sugar and Z-Thr-OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate. The α glycosylated threonine derivatives were converted into Fmoc-Thr(α-G1c)-OH and Fmoc-Thr(α-Gal)-OH by catalytic hydrogenation followed by acylation with Fmoc-OSu. β-Glucosylation and β-galactosylation of threonine were carried out by reacting the proper per-O-acetylated sugar with Z-Thr-OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the β-O-glycosylated threonine derivatives followed by acylation with Fmoc-OSu and deacetylation with methanolic hydrazine yielded Fmoc-Thr(β-Glc)-OH and Fmoc-Thr(β-Gal)-OH, respectively. The O-glycosylated threonine derivatives were then reacted with H-Lys(Z)-Pro-Arg(NO₂)-OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and ¹H NMR. The β-galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.     

Chemical synthesis and receptor binding of catfish somatostatin: a disulfide‐bridged β‐d‐Galp‐(1→3)‐α‐d‐GalpNAc O‐glycopeptide*

Abstract: The glycopeptide hormone catfish somatostatin (somatostatin‐22) has the amino acid sequence H‐Asp‐Asn‐Thr‐Val‐Thr‐Ser‐Lys‐Pro‐Leu‐Asn‐Cys‐Met‐Asn‐Tyr‐Phe‐Trp‐Lys‐Ser‐Arg‐Thr‐Ala‐Cys‐OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains d ‐GalNAc and d ‐Gal O‐glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc‐Cys(Trt)‐PAC‐PEG‐PS support, using stepwise Fmoc solid‐phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin‐22 were accessed by incorporating as building blocks, respectively, N^α‐Fmoc‐Thr(Ac₃‐α‐D‐GalNAc)‐OH and N^α‐Fmoc‐Thr(Ac₄‐β‐D‐Gal‐(1→3)‐Ac₂‐α‐D‐GalNAc)‐OH. Acidolytic deprotection/cleavage of these peptidyl‐resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl‐protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N^α‐dithiasuccinoyl (Dts)‐glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2‐fold tighter binding.     

Probing the conformational and dynamical effects of O‐glycosylation within the immunodominant region of a MUC1 peptide tumor antigen

Abstract: MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSA PDTRP APGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under‐glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl α2–6 GalNAc) and TF (Gal β1–3 GalNAc) carbohydrates. Here, we report the results of ¹H NMR structural studies, natural abundance ¹³C NMR relaxation measurements and distance‐restrained MD simulations designed to probe the structural and dynamical effects of Tn‐glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine‐residue MUC1 peptide of the sequence, Thr1‐Ser2‐Ala3‐Pro4‐Asp5‐Thr6‐Arg7‐Pro8‐Ala9, and a Tn‐glycosylated version of this peptide, Thr1‐Ser2‐Ala3‐Pro4‐Asp5‐Thr6(αGalNAc)‐Arg7‐Pro8‐Ala9. The results of these studies show that a type I β‐turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar β‐turn within the PDTRP core peptide epitope of the under‐glycosylated cancer‐associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I β‐turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.