Structural Requirements for CNS Active Opioid Glycopeptides

Article pubs.acs.org/jmc

Structural Requirements for CNS Active Opioid Glycopeptides Mark Lefever,† Yingxue Li,† Bobbi Anglin,† Dhanasekaran Muthu,† Denise Giuvelis,§ John J. Lowery,§ Brian I. Knapp,‡ Jean M. Bidlack,‡ Edward J. Bilsky,§ and Robin Polt*,† †

Carl S. Marvel Laboratories, Department of Chemistry and Biochemistry, BIO5, The University of Arizona, Tucson, Arizona 85721, United States ‡ Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642-8711, United States § Department of Biomedical Sciences, COM and Center for Excellence in the Neurosciences, University of New England, 11 Hills Beach Road, Biddeford, Maine 04005, United States S Supporting Information *

ABSTRACT: Glycopeptides related to β-endorphin penetrate the blood−brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [DAla2,N-MePhe4,Gly-ol5]enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful. A series of panagonists was evaluated for antinociceptive activity (55 °C tail flick) in mice. A flexible linker was required to maintain antinociceptive activity. Circular dichroism (CD) in H2O, trifluoroethanol (TFE), and SDS micelles confirmed the importance of the amphipathic helices (11s → 11sG → 11) for antinociception. The glycosylated analogues showed only nascent helices and random coil conformations in H2O. Chemical shift indices (CSI) and nuclear Overhauser effects (NOE) with 600 MHz NMR and CD confirmed helical structures in micelles, which were rationalized by molecular dynamics calculations. Antinociceptive studies with mice confirm that these glycosylated endorphin analogues are potential drug candidates that penetrate the BBB to produce potent central effects.

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to function effectively as CNS drugs.10−13 Polt and co-workers synthesized the cyclic glycopeptide H-Tyr-c[D-Cys-Gly-Phe-DCys]-Ser(β-Glc)-Gly-NH2, which showed only modest μ/δselectivity and modest μ-binding affinity relative to morphine but displayed high and prolonged analgesic efficacy after intraperitoneal (ip) injection.14 Horvat showed that N-terminal glycosylation of a Leu-enkephalin analogue significantly enhanced proteolytic enzyme stability in human serum.15 Negri and co-workers glycosylated dermorphin and deltorphin I, producing glycopeptides with much greater analgesic potency than the parent compounds when injected subcutaneously (sc) in mice.16 Rocchi and co-workers showed that a nociceptin analogue, glycoside [Ser-O-β-Gal]-NC(1−13)-NH2, displayed agonist activity comparable to that of NC(1−13)-NH2.17 Hruby has suggested that glycosylated bifunctional peptide derivatives with improved metabolic stability show picomolar-level affinity for the hNK1 receptor and partial but effective agonist activity at the μ/δ opioid receptors. 18 Ballet et al. studied pharmacologically active glycosylated Dmt-DALDA dermorphin analogues in the context of Caco-2 monolayers and suggested that active transport is not involved in the transport process.19

INTRODUCTION Since the discovery of the endogenous pentapeptides Metenkephalin and Leu-enkephalin in 1975, more than 250 neuropeptides have been identified, and there appears to be a broad vista for the application of endogenous peptides in pharmacology, especially for the opioids that are anatomically widespread throughout the CNS and involved with ascending1 and descending pain-modulating pathways.2,3 Considerable advances have been made in the understanding of opioid receptors and their endogenous ligands in the CNS. Many potent and selective peptide agonists have been developed for the opioid receptors, but instability in vivo and poor blood− brain barrier (BBB) penetration still present problems.4,5 Opioid peptides are generally degraded before they penetrate the CNS and the ability of drugs to penetrate biological membranes has been linked to molecular size, charge, hydrogen bonding, and lipid solubility.6 Furthermore, the physicochemical determinants for predicting oral bioavailability are not necessarily applicable to peptides or glycopeptides targeting the CNS.4,7 To increase BBB penetration, several peptide drug strategies have been developed, including structural modifications for enhanced stability and various methods of formulation.8 Glycosylation of peptides9 has been shown to increase penetration of the BBB to allow the resulting glycopeptides © XXXX American Chemical Society

Received: January 5, 2015

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DOI: 10.1021/acs.jmedchem.5b00014 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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(Edmundson diagram),23 such that the water-soluble residue side chains were exposed on one face and lipophilic residue side chains were exposed on the opposite face as shown in Figure 1b. Among the hydrophobic amino acids, leucine was believed to have a superior propensity for helix formation. The presence of residues containing side chains capable of forming hydrogen bonds with the main chain carbonyl or amide NH when placed at the beginning (N-cap) or end (C-cap) of α-helices has been shown to induce and stabilize helical conformations in proteins and peptides. 24 In naturally occurring protein helices, asparagine is the usual N-cap residue, forming i, i + 2 or i, i + 3 type hydrogen bonds with backbone NH hydrogens.25 As such, asparagine was installed immediately following the linker to nucleate helix formation.26 The hydrophilic residue serine, which serves as the point of glycoside attachment, was positioned preceding the C-terminal residue. An additional series of glycopeptides (712) was synthesized10 using an analogue of the δ-selective message DTLET (D-Thr-Leu-enkephalin-Thr, YtGFLT-OH) developed by Roques et al.,27 in which the C-terminal residue (threonine) of the message was replaced with a linker of variable length and rigidity from which the helical address was extended. In this set of glycopeptides, the linkers proline, D-proline, glycine, alanine, β-alanine, γ-aminobutyric acid (GABA), and δ-aminovaleric acid (DAVA) (Figure 2) were inserted to study the length and flexibility requirements for the linker. A more flexible hydrophobic linker was expected to allow simultaneous interaction between the cell membrane and the opioid receptor. For this series of glycopeptides, the helical address remained the same as that used in the initial series of analogues (1−6a) for all but three control compounds. The three exceptions include a nonglycosylated short address (11s), a glycosylated short address (11sG), and the full-length address with the monosaccharide glucose replaced by the disaccharide lactose (11L). This triad of peptides and glycopeptides was examined in order to verify the importance of glycosylation as well as the amphipathic helical address segment on BBB penetration and antinociceptive action, to correlate the effect of the biousian character9,28,29 of the glycopeptide with BBB penetration. In addition to opioid receptor binding affinities, A50 values were obtained by employing the mouse tail-flick test for antinociceptive activity after icv (central) administration and after iv (peripheral) administration. Glycopeptide MMP2200 is included for comparison of receptor binding affinities in a glycopeptide with no linker present (Table 1).30 Finally, besides evaluating the “linker effect” of various flexible linkers on BBB penetration and pharmacologic activity, the effects of both charge density and salt bridge formation occurring along the amphipathic helix address were investigated for opioid receptor binding affinity. Using the μ-selective DAMGO message as a pharmacological probe, a series of glycopeptides (6a−6g, Table 1) having the Gly-Gly linker and helices with charges ranging from +3 to −1 were evaluated for opioid binding affinity. For comparison, ligand 7 was included. This glycopeptide has the δ/μ-agonist DTLET based message, a rigid Pro linker, and the same helical address domain as 6a.

Helicity can play an important role in opioid binding for the larger endorphins and deltorphins. According to our previous studies, a series of glycopeptides formed nascent helix-random coil structures in H2O and a membrane-induced highly amphipathic helix in the presence of anionic and zwitterionic membrane mimics, which could modify agonism at the opioid receptor and likely influence the kinetics of opioid binding.10 Amphipathic binding to the membrane also seems to influence BBB transport rates. Kaiser suggested that the hydrophilic linker region appears to have little propensity for the formation of a secondary structure according to conformation predictive parameters. Subsequent design of two model compounds with a similar β-endorphin amphipathic address segment but with different linkers, a nonhomologous (to endorphin) hydrophilic linking region, S-γ-amino-γ-hydroxymethylbutyric acid (HOMe-GABA) and an alternating glycine/serine sequence from residues 612, examined how hydrophilic linkers influenced bioactivity of β-endorphin analogues.20 These peptides showed δ-receptor binding and guinea pig ileum (GPI) activities that were similar to natural β-endorphin, and a significant decrease in μ-receptor binding, as well as greater resistance to proteolysis in rat brain homogenates. Considering this, using linkers of various lengths that possess different degrees of flexibility may lead to variations in opioid receptor binding affinity and selectivity by virtue of positioning the message sequence at varying orientations and depths in the membrane surrounding the receptor. In the present study, Olinked glycopeptides were designed to investigate opioid receptor binding affinities and selectivity generated by linker variants. Initially, glycopeptides (16a, Table 1) using the μ-selective [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) message21 (YaG(N-Me)F) were synthesized to explore the effect of the different linkers on the promotion and stabilization of the helical address domain and the effect of the linker on opioid receptor binding affinity. The linkers (Figure 2) ranged from single residues like the flexible glycine (1) or the rigid proline (2) to the dipeptide combinations Gly-Pro (3), β-Ala-Pro (4), Pro-Pro (5), and Gly-Gly (6a). To stabilize the helical C-terminal amphipathic address, a number of previously effective strategies were employed including the incorporation of an α-methylated amino acid (α-aminoisobutyric acid, Aib = B), the use of helix stabilizing alanine residues, and addition of salt bridges between residues separated by one α-helical turn (Figure 1a).22 The sequence of hydrophilic and hydrophobic residues was determined by plotting the C-terminal helical segment in a helical wheel plot

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RESULTS AND DISCUSSION Receptor Binding Studies, Ligands 1−6a. From receptor binding studies (Table 1), even though the DAMGO analogues showed 5−10 times lower receptor affinity than DAMGO, they all retained μ-opioid receptor selectivity.

Figure 1. (a) The model sequence of the glycopeptide analog amphipathic helix for 16a and 713. (b) The hydrophilic residues are shown in red and the hydrophobic residues in blue. (B9 = αaminoisobutyric acid or Aib). B



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Figure 2. Structures of amino acids linkers with various lengths and flexibilities.

Table 1. Peptide Sequence and Opioid Receptor Binding Activitiesa

a

Glycopeptides 7 and 1112 include analgesic A50 doses after icv and iv administration. Binding studies of the cyclic tetrapeptide 13 were performed by McFayden, et al.50. * = glucoside; **= lactoside; † = %-saturation of the receptor at 10 μM; nb‡ = no binding, 0%-saturation of the receptor at 10 μM; ¶ = single measurement.

DAMGO analogue 6a, bearing the more flexible Gly-Gly linker, had greater μ-opioid receptor selectivity than the other analogues. The C-terminal group on DAMGO, equivalent to the linker and address for our synthetic peptides, is the polar glycinol (∼NHCH2CH2OH) group. This small water-soluble moiety in combination with the YaG(N-Me)F message interacts most strongly with the μ-opioid receptor (MOR), with 500 times higher affinity than that to κ-opioid receptor (KOR) and almost 2000-times higher than that to the δ-opioid receptor (DOR). The DAMGO-based glycopeptide series demonstrated the expected μ selectivity. The effect of the linker on receptor binding affinity revealed the Gly-Gly (6a) linker had the highest MOR affinity with a Ki value of 2.8 ± 0.23 nM, but that was at only 20% of DAMGO. The Gly-Pro (3) agonist showed similar activity at 3.2 nM (17.5% of DAMGO). The single β-Ala (1) residue as the linker showed 4.8 ± 0.42 nM binding to MOR, (11.7% of DAMGO), and βAla-Pro (4) was at 5.6 nM (10% of DAMGO). From these data, it appears flexibility favors MOR binding and that “breaking” the helix permits β-turn formation at the Nterminus. The rigidity of a single Pro (2) or the Pro-Pro (5)

dipeptide linker further diminished MOR binding affinity, their respective Ki values being 8.8 ± 0.23 nM and 15.0 ± 0.10 nM. Notably, with the exception of the Pro (2) linker, all other linkers improved binding to the KOR compared with that of DAMGO, the Pro-Pro (5, 19.0 nM), Gly-Pro (3, 45 nM), and Gly-Gly (6a, 59 nM) demonstrating the largest increases in affinity. DOR affinity remained essentially unchanged compared with that of DAMGO. Receptor Binding and Antinociception Studies, Ligands 712. β-Endorphin and Leu-enkephalin inspired analogues 7, 8, and 9 with proline, glycine, and alanine linkers, respectively; 8 and 9 had Ki values of greater than 20 nM for δand μ-opioid receptors and between 9 and 22 nM for the κopioid receptor. Compared with the L-proline linker on glycopeptide 7, the D-proline linker in 10 clearly showed reduced binding. Presumably, this is because the D-proline directed the message segment of glycopeptide 10 away from the opioid receptor pocket and resulted in significant decrease (10−30-fold) in affinity to all three opioid receptors. In general, glycopeptides 11 and 12 with GABA and DAVA linkers and the full-length address retained low single digit nanomolar C



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Figure 3. Intracerebroventricular (left) and intravenous (right) antinociceptive dose− and time−response curves for peptides in the mouse using the 55 °C warm water tail-flick test. Bars represent the standard error of the mean for each dose at the various time points after injection.

affinity Ki values for binding to the μ- and δ-opioid receptors. There was a modest increase in icv antinociceptive potency between peptide 7 and 12 and a decreased icv potency from peptide 11 to 12 as shown in Figure 3. Peptide 11 had a prolonged duration of action, producing significant analgesia for over 90 min after icv administration at a lower dose compared with peptide 7. Compared with peptide 11, there is almost an order of magnitude increase in A50 concentration after icv administration for peptide 12 (Table 2), the only difference being a one carbon extension of the linker. Intracerebroventricular administration of all three peptides produced maximum

Table 2. Comparison of Antinociceptive Effect of DTLETBased Glycopeptides with Different Length Linkers

D

name

linker type

icv A50 (nmol)

iv A50 (μmol/kg)

7 11s 11sG 11 11L 12

Pro GABA GABA GABA GABA DAVA

0.74 0.06 0.06 0.046 0.165 0.371

0.36 >11.3 2.54 0.31 1.13 0.36



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Figure 4. Intravenous antinociceptive dose− and time−response curves for peptides in the mouse 55 °C warm water tail-flick test. Bars represent the standard error of the mean for each dose and the various time points after injection.

the receptor or cell membrane (off rate) because of the increased hydrophilic nature of lactose compared with glucose. Glycopeptide 11 had a more sustained antinociceptive action using a smaller dose than the other peptides as measured using the tail-flick assay. Ligands 6a−6g. Binding data for this series show strong MOR selectivity and a wide range of receptor affinities correlating with the overall helix charge. In this series, only 6a (2.8 nM), 6c (0.24 nM), and 6g (5.2 nM) (Table 1) show potent μ-opioid receptor selective binding. Peptide 6c even has a higher MOR affinity than DAMGO (0.56 nM). The only differences among the structures of this series are the respective amino residue positions 10 and 11, with Glu-Lys in peptide 6a, Lys-Asn in peptide 6c, and Asn-Asn in 6g. Changing the acidic glutamate to the polar but neutral asparagine produced a net increase in positive charge for peptide 6c and elimination of the salt bridge between Glu10 and Lys14. This change is thought to be responsible for improved binding through increased membrane association. It is expected that the intramolecular salt bridge formed by Glu10 and Lys14 of peptide 6a contributes to the stabilization of the helix. Even though there is still hydrogen bonding between the side chains in the peptide 6c, the loss of the salt bridge and relaxation of the helix into a coil enables the message segment of the peptide to

antinociception within 10−20 min. After iv administration, the maximum analgesic effect was seen within 30 min, and the duration of effect was extended compared with icv (Figure 3), with peptide 11 (GABA linker) having the longest duration of action. These antinociceptive potency changes are thought to be due to structural and conformation differences among these glycopeptides influencing receptor binding affinity and activation. Short peptide 11s and its glucoside 11sG, both lacking the amphipathic helical address, showed somewhat lower binding affinity to opioid receptors than the helical glycopeptide 11. The lactoside 11L had the same δ- and κ-receptor affinity as glucoside 11, affinities that are comparable to peptide 12 (DAVA linker), and showed enhanced μ-opioid receptor binding affinity. The antinociceptive potencies showed doseand time-related antinociceptive effects with full efficacy in the 55 °C tail-flick assay. Peptide 11s and 11sG show lower binding affinities to opioid receptors than glycopeptide 11, and unglycosylated peptide 11s did not effectively penetrate the BBB after iv administration, even at the highest dose of 10 mg/ kg (Figure 4). Glycopeptide 11L had similar antinociceptive duration to 11 of about 180 min following iv administration, but peak antinociceptive effect decreased rapidly after the maximum, perhaps secondary to a more rapid dissociation from E



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rigid cyclic structure, the D-proline linker decreased the %-helix of the glycopeptide significantly more than the other linkers that were used. The flexible glycine and alanine linkers promoted greater helicity of the glycopeptide. The Pro-Pro linker showed much greater %-helix, presumably due to the formation of a “polyproline helix.” The non-natural flexible amino acid linkers β-alanine, GABA, and DAVA reduced the helical content of the conformational ensemble. Furthermore, compared with the glucoside 11, the lactoside 11L showed decreased helical content, as one would expect from the biousian hypothesis.10,28,29 In aqueous buffer, the shorter peptide 11s and glycopeptide 11sG also displayed characteristics of random coil conformation. However, in SDS micelles, the peptides 11s and 11sG yielded CD spectra (Figure 5) characteristic of a type I/III βturn conformation.10,34 NMR Analysis. CD spectra provide overall peptide conformation but no residue-specific information. On the other hand, NMR spectroscopy is well suited to study the peptide structure at the residue specific level.35 For the glycopeptides studied here, the different spin systems were assigned using TOCSY spectra, and the sequential proton assignments were made by the combined use of TOCSY and NOESY/ROESY spectra. Although a few overlapping peaks were observed, unambiguous 1H chemical shift assignments for all glycopeptides were successfully made based on sequential NOEs such as dNN(i,i+1), dαN(i,i+1), and dβN(i,i+1).36 Standard ROESY experiments yielded good quality spectra in aqueous samples but failed in SDS micelles possibly because the association of the glycopeptides with micelles generated high molecular weight assemblies, increasing the correlation times and producing poor quality spectra. For this reason, standard NOESY experiments were used for SDS samples. The signals in the spectra are well resolved with little crowding in the presence of SDS micelles, and TOCSY/NOESY spectra enabled complete sequential assignment of the glycopeptide protons. It is well established that protein secondary structures may be identified based on CαH chemical shift information. This

interact more effectively with the opioid receptor binding pocket. Circular Dichroism Studies. Circular dichroism (CD) is a powerful, simple, and fast tool for determining the secondary structure in both peptides and proteins.31 According to CD results, the helical glycopeptides displayed similar spectra that are characteristic of random coil conformation in aqueous buffer and clear helical conformations with double minima at 208 and 222 nm32 observed in the presence of TFE and SDS micelles. The calculated per-residue helicity (%-helix) based on the negative maximum at 222 nm (n → π* transition band) for each peptide in SDS micelles is given in Table 3.33 Due to the Table 3. CD Data for Glycopeptides with Variable Linkers in SDS Micellesa glycopeptide 1 2 3 4 5 6a 7 8 9 10 11 11L 12

linker used

[θ]n→π*,b,c ∼222 nm

[θ]π→π*,b,d ∼208 nm

α-helicity by CD,e % (sd)f

β-Ala Pro Gly-Pro (β-Ala)Pro Pro-Pro Gly-Gly Pro Gly Ala D-Pro GABA GABA DAVA

−13572.0 −11886.0 −11473.0 −9823.7

−17883.0 −12188.0 −12971.0 −11944.0

41.3(±2.0) 36.2(±1.4) 34.4(±3.6) 29.5(±1.9)

−15693.0 −11318.0 −12081.0 −20287.0 −18613.0 −7804.9 −11562.0 −10895.0 −11943.0

−19638.0 −13836.0 −9550.1 −21028.0 −19061.0 −9370.0 −17055.0 −17224.0 −16537.0

47.1(±1.1) 34.0(±4.8) 36.2(±0.7) 60.9(±3.4) 55.8(±5.6) 23.4(±3.3) 34.7(±1.8) 32.7(±2.8) 35.8(±4.1)

a The helical addresses are identical. Glycopeptides 1−6a use the DAMGO message and 7−13 use the DTLET message. bThe units for [θ] are deg·cm2·dmol−1. cThe negative maxima for the [θ]n→π* was observed between 222 and 225 nm. dThe negative maxima for the [θ]π→π* was observed between 205 and 209 nm. eThe %-helicity was calculated according to ref 33. fThe standard deviation based on three experiments.

Figure 5. CD spectra of analogues 11s and 11sG in 30% TFE and SDS micelles. The presence of SDS micelles has a profound effect on the conformational ensemble of both the peptide 11s and glycopeptide 11sG and suggests similar backbone conformations. Trifluoroethanol (TFE), which enhances intramolecular H-bonding at the expense of H-bonding to H2O, seems to have different conformational effects on the peptide and glycopeptide homologues. F



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Figure 6. Fingerprint area (CαH−NH) of NOESY spectra for glycopeptides 7, 10, 11, and 12 measured at pH 5.5 and 25 °C using a mixing time of 300 ms.

Figure 7. NOE summary for glycopeptides 11 and 12 in SDS micelles measured at pH 5.5 and 25 °C using a mixing time of 300 ms. The line thickness is approximately proportional to the intensity of the observed NOE cross peaks. The dashed line indicates the signal overlap of the crosspeaks.

conformational shift values in SDS micelles are summarized in the Supporting Information. Because there is no experimental random coil CαH value for glycosylated serine, the CSI value for this component is uncertain.38 The CαH−NH fingerprint area of NOESY experiments for glycopeptides 7, 10, 11, and 12 is shown in Figure 6. The linkage residue significantly influenced the chemical shift of linkage neighbor amino acid residues. Compared with the Pro linker, it is the flexible linkers GABA and DAVA that led to chemical shifts of the CαH and NH of Leu5, Aib9, and Glu10 from a lower field to a higher field; conversely Asn7 and Leu8

method, involving the chemical shift index (CSI), is more quantitative and accurate for identifying secondary structure of peptides and is qualitatively comparable to CD.37 Generally, the magnitude of CαH chemical shift difference between the observed CαH chemical shift and random coil values can significantly affect interpretation of chemical shift changes in structural terms, for example, an “ideal” α-helix appears to have an amide proton upfield shift of 0.2 to 0.3 ppm relative to a random coil ensemble. Besides, the observation of consecutive negative deviations (upfield-shift CαH resonances) from random coil indicates a helical conformation. The observed G



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Figure 8. CD spectrum of analogues (a) in aqueous solution, (b) in 30% TFE, and (c) in SDS micelles. Glycopeptide legend: 2 = 6a, 3 = 6b, 4 = 6c, 5 = 6d, 6 = 6e, 7 = 6f, 8 = 6g.

characteristic of a highly α-helical conformation. Furthermore, the CD spectra of SDS micelle solution of these analogues show stronger α-helical conformation, with a double minimum at 208 and 222 nm. In Table 4, glycopeptides 6a through 6g showed helicities as measured by CD from 30% to 45% in the SDS micelle. Glycopeptide 6f with a negatively charged address segment showed the strongest CD helical conformation of 45.4% helicity in the SDS micelle membrane mimic although by NMR helicity is reported as 31.3%. Glycopeptide 6c was found to have a % helicity of close to 35% by both CD and NMR. Two-dimensional NMR experiments were used to provide detailed information about secondary structure of respective glycopeptides. Random coil structures of all analogues were observed in aqueous solution. The helical conformations of the address regions20 were confirmed by NMR experiments in the presence of SDS micelles.5,10 Spin systems were identified by total correlation spectra (TOCSY). Rotating-frame Overhauser enhancement (ROESY) in aqueous solution or nuclear Overhauser enhancement (NOESY) in SDS micelles was used to obtain interresidue connectivities and to distinguish equivalent spin systems. Although some clustering of cross-

induced a shift in the opposite direction. However, the Dproline linkage influenced chemical shift of CαH and NH of Aib9 significantly from high field to low field (helix disruption), thus decreasing affinity for binding to all three opioid receptors. All the glycopeptides exhibited consecutive strong dαN(i,i+1) NOEs, along with relatively weaker dNN(i,i+1) NOEs throughout the sequence. However, there were no additional NOEs diagnostic of any particular fold observed. Simultaneous observation of dαN(i,i+1) and dNN(i,i+1) without any other medium- or long-range NOEs is indicative of the only nascent helical natures of the glycopeptides in aqueous buffer at best. Unlike NOEs measured in H2O, SDS induces a continuous stretch of strong sequential dNN(i,i+1) NOE peaks for almost the entire length of all of the glycopeptides that were studied. In addition, other helix-specific NOEs, including dαN(i,i+3) and dαN(i,i+4), appear in the C-terminal address domain in the presence of micelles (Figure 7). All general secondary conformations of glycopeptides 6a through 6g were obtained by circular dichroism (Figure 8). The CD spectra of all these DAMGO analogues in aqueous solution reflect predominantly random coil conformation. In TFE/water 3:7 (v/v), the CD spectra of glycopeptide analogues are H



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Table 4. Circular Dichroism Data for Glucosylated Peptides with Charged Helices ranging from 3+ to 1− charge/salt bridge

solvent

[θ]n→π*,a,b ∼222 nm

[θ]π→π*a,c ∼208 nm

α-helicity by CD,d % (sd)f

6a

+1/yes

6b

+3/no

6c

+2/no

6d

+1/no

6e

0/yes

6f

−1/yes

6g

0/no

7

+1/yes

H2O TFE SDS H2O TFE SDS H2O TFE SDS H2O TFE SDS H2O TFE SDS H2O TFE SDS H2O TFE SDS H2O TFE SDS

−1768.4 −8360.0 −11318.0 −2669.9 −9484.5 −10811.0 −3240.1 −7072.6 −11898.0 −4084.5 −6120.6 −10105.0 −4320.2 −7513.4 −12416.0 −6094.4 −10604.0 −15151.0 −4285.6 −4108.3 −10712.0 −2331.0 −10801.0 −12081.0

−4783.7 −11773.0 −13836.0 −7194.8 −13135.0 −15997.0 −7601.0 −10666.0 −13223.0 −8768.8 −11363.0 −14438.0 −8885.0 −11154.0 −15652.0 −12461.0 −15728.0 −19611.0 −7943.6 −8473.2 −13053.0 −6545.4 −13720.0 −9550.1

0.0(±4.3) 25.1(±2.8) 34.0(±4.8) 0.0(±4.2) 28.5(±5.8) 32.4(±5.3) 0.0(±3.3) 21.2(±1.4) 35.7(±2.6) 0.0(±2.7) 18.4(±4.1) 30.3(±1.1) 0.0(±0.4) 29.6(±4.0) 37.2(±0.8) 0.0(±3.3) 31.8(±2.6) 45.4(±1.3) 0.0(±1.2) 12.3(±2.8) 32.1(±2.6) 7.0(±0.9)f 32.4(±1.6) 36.2(±0.7)

% α-helicity by NMRe

30.93

35.39

35.22

24.93

28.72

31.30

30.50 7.23 32.57

The units for [θ] are deg·cm2·dmol−1. bThe negative maxima for the [θ]n→π* was observed between 222 and 225 nm. cThe negative maxima for the [θ]π→π* was observed between 205 and 209 nm. dThe %-helicity was calculated according to ref 33. eSee text for the calculation method. fThe standard deviation based on three experiments. a

message domain away from opioid receptor region. Glycopeptide 6b with three positive charges showed very low μ-receptor binding activity, possibly because the net charge restricts peptide movement from the membrane bound form to the receptor pocket. Molecular Modeling Studies. Mosberg’s highly μselective agonist 1340,50 (Table 1) was used to further define requirements for the μ-receptor pocket in silico. Using the μreceptor homology model derived from the β2-adrenergic receptor (PDB code 2rh1) developed in the Mosberg laboratory,40 we aligned the message segments of glycopeptide 6a and glycopeptide 6c by initially superposing and fixing the α, β, and γ carbons atoms of the glycopeptide N-terminal tyrosine with the analogous atoms of the receptor bound Mosberg agonist and reminimized the structures using the Amber99 force field in MOE. The remaining portion of the glycopeptides were minimized, first placing a “water box” around the sugar and C-terminal three amino acids prior to minimization. Receptor residues within 4.5 Å of the intact glycopeptides were minimized along with the docked ligand. This was repeated for minimization of residues within approximately 9 Å of the docked glycopeptides, then again for the entire peptide−receptor complex. As shown in Figure 10, the message segments of all three glycopeptides could be overlapped in the μ-receptor pocket. After minimization the address domain of glycopeptide 6a (magenta) maintained the designed helical conformation while the highly μ-selective glycopeptide 6c (orange) showed a nonhelical conformation of the address domain that allowed for additional interactions with the opioid receptor. Figure 11 compares glycopeptide 6a with glycopeptide 11 (GABA linker). The address segments of both 6a and 11

peaks occurred in the amide hydrogen region, unambiguous assignments were obtained in all cases with the help of αH(i)/ NH(i+1) and other NOE connectivities characteristic of helical conformation in SDS micelles solution, as shown in Figure 9. Notwithstanding the variably charged residues of the address segments, no significant chemical shift changes of the unaltered side chains were observed among homologous residues in the different peptides. The dotted lines indicate overlap of cross peak correlation, and the bar thickness of the short-range NOE connectivites correspond to relative NOE volumes. Because of the presence of NH−NH(i,i+1) and CβH−NH(i,i+1) NOE connectivities, all glycopeptide analogues contain a significant population of conformers with dihedral angles in the α-region of Φ/Ψ space. A significant population of helical conformers was distinguished by the presence of medium-range NOE connectivities, such as CαH−NH(i,i+3) and CαH−NH(i,i+4) throughout the length of the peptide. In the 1980s, Schwyzer suggested that the lipid cellular membrane acts as a matrix for both the opioid receptors and opioid ligands.39 Furthermore, it was hypothesized that opioid receptor subtype, δ, μ, and κ, interacts with different opioid peptides based on each peptide amphiphilic moment and net charge along with hydrophobic association. These characteristics of the opioid neuropeptides were proposed to influence their conformation, orientation, and accumulation within the membrane. Positively charged peptides presumably associate more strongly with the negatively charged head groups of the lipid. We observed that negatively charged domains (glycopeptide 6f) or domains with no net charge (glycopeptides 6e and 6g) showed lower μ-receptor binding activity. It is possible that repulsion between the negative charge of the address segment and the anionic membrane repels the DAMGO analogue I



Article

Figure 9. Summary of NOE data obtained at 30 °C for all glycopeptide 6 analogues with residue substitutions in the helical domain (Tables 2 and 4): (a) 6a; (b) 6b; (c) 6c; (d) 6d; (e) 6e; (f) 6f; (g) 6g.

tide 7) directs the message segment of the glycopeptides into the opioid binding site, whereas D-Pro (glycopeptide 10) imposes a severe restriction on orientation of the message segment that decreases interaction with the receptors. Because peptide 11s could not penetrate the BBB and displayed lower opioid receptor binding activity, we also suggest that there is no specific membrane transporter for GABA to promote the penetration of glycopeptide 11s. Furthermore, the ability of glycopeptide 11sG to penetrate the BBB confirmed the important role that the glycoside plays in peptide drug delivery. Also, the lactoside 11L has similar binding affinities to all opioid receptors but has a shorter duration of action after iv administration. A possible reason is that the increased hydrophilicity of the disaccharide shifts the residency of glycopeptide 11L into the aqueous compartment, resulting in faster elimination.9,29 In summary, we suggest that a flexible linker such as GABA and a helical address segment enhances delivery of the opioid message to neurons within the CNS. Opioid selectivity is determined mainly by the identity of the message after icv

maintain the expected helical conformation. Also observed, the long flexible GABA linker on glycopeptide 11 allows the Leuenkephalin inspired message segment to interact with the opioid receptor site while maintaining the address segment interaction with the cell membrane in ideal helical conformation.

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CONCLUSIONS We conclude that the flexible linkers such as GABA and GlyGly can balance two interactions, one between the message segment and opioid receptor pocket and one between the address segment and the cell membrane, in order to achieve higher binding affinity to all three opioid receptors and result in a significant increase in centrally mediated analgesia after iv administration. In addition, GABA displays the minimal hydrophobic linker length among our glycopeptide analogues. Short linkages, such as glycine, alanine, and β-alanine, restrict glycopeptide message segment insertion into the opioid receptor pocket and result in lower opioid receptor binding activity. It seems clear that the helix-breaker L-Pro (glycopepJ



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Article

EXPERIMENTAL DETAILS

Materials. The Fmoc-protected amino acids and the Rink amide MBHA resin (4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamido MBHA, grain size 100−200 mesh, substitution 0.83 mmol/g) were obtained from CHEM-IMPEX International. Sodium dodecyl sulfate-d25 used in NMR experiments was purchased from CDN Isotopes Inc., Canada. All other highest-grade reagents and solvents were purchased from Aldrich and were used directly without any further purification. Peptide Synthesis and Purification. The glycosylated Fmoc protected amino acid serine was prepared under microwave conditions according to a modified procedure reported by Seibel and coworkers.41 The glycopeptides were synthesized manually based on established solid phase Fmoc-chemistry methodology using Rink amide MBHA resin (substitution 0.83 mequiv/G, 1% DVB). The side chain protected amino acids used in the synthesis were FmocLys(Boc)−OH, Fmoc-Glu(OtBu)−OH, Fmoc-Asn(Trt)−OH, FmocD-Thr(But)−OH, and Fmoc-Tyr(But)−OH. Coupling of all the Fmoc-protected amino acids was performed in an Emerson 900 W microwave oven (household, model number MW8992SB) at power level 1 for 10 consecutive minutes. The Fmoc-protected amino acids (2.0 equiv compared with resin) were coupled by 1-hydroxybenzotriazole (HOBt, 2.0 equiv) and N,N′-diisopropylcarbodiimide (DIC, 2.0 equiv) in a 1:1 mixture of dimethylformamide (DMF) and Nmethylpyrrolidone (NMP). The coupling reactions were monitored using the Kaiser ninhydrin test. The fluorenylmethoxycarbonyl (Fmoc) group was removed using a mixture of 3% piperidine and 2% diaza(1,3)bicyclo[5.4.0]-undecane (DBU) in DMF for 10 min under argon bubbling. The acetyl protecting groups of the sugar moiety were removed using 20% H2NNH2·H2O in CH3OH with agitation using argon bubbling three times for 1 h. The peptides and glycopeptides were cleaved from the resin with a cocktail containing F3CCOOH/Et3SiH/H2O/PhOMe/CH2Cl2 (8:0.5:0.5:0.05:1), which also removed the side chain protecting groups. The cleaved glycopeptides were precipitated in cold ether, redissolved in H2O, and then lyophilized. The crude glycopeptides were purified by RPHPLC with preparative RP (C-18) Phenomenex (250 × 21.9 mm2) column using acetonitrile−water gradient system containing 0.1% trifluoroacetic acid (TFA). Homogeneity of the pure glycopeptides (>95%) was confirmed by analytical RP-HPLC and mass spectrometry.

Figure 10. Peptide 13 (green), peptide 6a (magenta), and peptide 6c (orange). Tyrosine and phenylalanine of 6a and 6c aligned in the μopioid receptor pocket with Mosberg’s peptide40,50 13. The helix is maintained in glycopeptide 6a where salt bridge formation stabilizes the helix while 6c relaxes into a random coil.

administration, but the address segment and linker become more important for delivery after peripheral iv administration.

Figure 11. Ribbon diagram of the μ-opioid receptor showing (a) glycopeptide 6a (green) and (b) Glycopeptide 11 (green). In each image, the peptide message segment is in the receptor pocket defined by Mosberg’s μ-agonist40 and the helical address portion of the peptide extends outside the pocket. K



Article

Receptor Binding Studies. To determine the affinity and selectivity of the peptides for the μ-, δ-, and κ-opioid receptors, Chinese hamster ovary (CHO) cells that stably expressed one type of human opioid receptor were used as previously described.42 Cell membranes were incubated at 25 °C with the radiolabeled ligands in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5. Incubation times of 60 min were used for the μ-selective peptide [3H]DAMGO and the κselective ligand [3H]U69,593, and a 3 h incubation was used with the δ-selective antagonist [3H]naltrindole. The final concentrations of [3H]DAMGO, [3H]naltrindole, and [3H]U69,593 were 0.25, 0.2, and 1 nM, respectively. Nonspecific binding was measured by inclusion of 10 μM naloxone for κ-opioid receptor binding and 100 μM for δopioid receptor binding. The binding was terminated by filtering the samples through Schleicher & Scheull No. 32 glass fiber filters with a Brandel 48-well cell harvester. The filters were washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL of ScintiSafe 30% scintillation fluid (Fisher Scientific, Fair Lawn, NJ). For [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 30 min before use. IC50 values will be calculated by least-squares fit to a logarithm-probit analysis. Ki values of unlabeled compounds were calculated from the equation Ki = (IC50)/(1 + S) where S = (concentration of radioligand)(Kd of radioligand).43 Circular Dichroism. All CD spectra were obtained on OLIS DSM20 automatic recording spectrophotometer equipped with temperature controller. The glycopeptide stock solutions were prepared by weighing the lyophilized powder, using Cahn/Ventron Instruments model 21 automatic analytical electrobalance. The samples were prepared by diluting the stock solution to 30 μM. All CD spectra were the average of three scans recorded with baseline correction between 190 and 250 nm by using integration time of 3 s and a scan step of 0.5 nm in a cell with a path length of 0.1 cm at 20 °C. All spectra were smoothed by using KaleidaGraph software (Synergy Software, USA). The molar ellipticities were calculated using the equation [θ] = [θ] obs (MRW)/(10lC), where [θ]obs is observed ellipticity in millidegrees, MRW is the mean residue weight, l is the cell path length in centimeters, and C is the glycopeptide concentration in mg/ mL. The percent α-helicity was determined by using the equation % helix = {[θ]n→p*/[−40000(1 − 2.5/n)]} × 100, where n represents the number of amide bonds (including the C-terminal amide) in the glycopeptides and [θ]n→p* is molar ellipticity of the n → p* transition band at 222 nm.33 NMR Spectroscopy. All NMR spectra were obtained from a Bruker DRX600 600 MHz spectrometer. The concentration of glycopeptide samples for the NMR experiments varied from 2.5 to 3 mM. The micelle samples were prepared by dissolving the peptide and 50 equiv of perdeuterated SDS in 0.5 mL of phosphate buffer (10 mM)/D2O (9:1 ratio by volume). The pH of the each sample was adjusted to 5.5 by using NaOH as necessary. 3-(Trimethylsilyl)-d4propionic acid (TSP) was added as internal standard reference. Rotating-frame Overhauser enhancement (ROESY),44 nuclear Overhauser enhancement (NOESY), and total correlation spectra (TOCSY)45 were acquired using standard pulse sequences and processed using XWINNMR (Bruker Inc.) and FELIX2000 (Accelrys Inc., San Diego, CA). Mixing times for TOCSY spectra were 100 ms; those for ROESY and NOESY spectra were 300 ms. All of NMR experiments were 750 increments in t1, 24/32/32 scans each, and 1.5 s relaxation delay. The WATERGATE pulse sequence was employed to suppress the water signal.46 Conformational Analysis. Molecular distance constraints for the structure calculation were obtained from integral volumes of the ROESY or NOESY peaks with using software FELIX2000 and the NOE integral volumes were classified into strong, medium, and weak with 1.0, 2.5, and 3.5 Å as upper bound distance. Molecular dynamics simulation was performed on MOE (Molecular Operating Environment, Chemical Computing Group, Canada) using standard protocol available within the system.48 Distance constraints are placed between protons identified through NMR-determined NOE corresponding upper boundary distances of 3 Å (strong), 4 Å (medium), and 5 Å (weak). A 25 kcal/mol energy penalty is used for the constraints. The

structure is minimized initially using steepest descent, followed by the conjugate gradient algorithm. Molecular Modeling. With MOE, the message segment of the glycopeptide, for example, Tyr-D-Ala-Gly-(N-methyl)Phe-Gly, was placed in the receptor pocket by initially superposing and fixing three atoms of the glycopeptide N-terminal tyrosine, the α, β, and γ carbons, with the analogous atoms of the receptor bound agonist (Tyrc(S-CH2CH2-S)[D-Cys-Phe-D-Pen]NH2)40,47 then aligning the remaining glycopeptide message residues to achieve the best fit. Placement of this segment was checked for clashes with receptor residues, then energy-minimized in the fixed pocket using the Amber99 force field. The remaining portion of the glycopeptide was reattached to the message sequence. This section was minimized, first placing a water box around the sugar and C-terminal three amino acids prior to minimization. Receptor residues within 4.5 Å of the glycopeptide were selected and minimized along with the docked ligand. This was repeated for residues within approximately 9 Å of the docked glycopeptide, then again for the entire peptide−receptor complex. The potential energies of the ligand and the ligand plus receptor were then determined, and ligand interactions with the receptor binding pocket residues were noted. Animals. Male CD-1 mice (27−30 g; Charles River Laboratory, Wilmington, MA) were used for all studies with the exception of the respiration studies. A total of 680 mice (8−10 mice per group) were used for the experiments. All mice were housed in groups of five in individually ventilated Innovive cage racks with food and water available ad libitum. All animals were maintained on a 12-h light/dark cycle (lights on at 7:00 AM) in a temperature- and humidity controlled animal colony. All animal experiments were performed under an approved protocol in accordance with institutional guidelines and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Injections. All drugs were dissolved in distilled water for intracerebroventricular (icv) injections and in physiological saline (0.9% NaCl) for intravenous (iv) injections. The icv injections were performed as described previously.48 In brief, mice were lightly anesthetized with ether, and a 5 mm incision was made along the midline of the scalp. An injection was made using a 25 μL Hamilton syringe (Hamilton Co., Reno, NV) at a point 2 mm caudal and 2 mm lateral from bregma. All iv injections were given in a volume based on the weight of the animal (0.1 mL/10 g bodyweight). Briefly, mice were restrained in a Plexiglas holder, and the distal portion of the tail was dipped into 40 °C warm water for approximately 10 s to dilate the tail vein. The injection was made into the tail vein using a 30 gauge needle and a 1 mL syringe. Tests for Antinociception. Antinociception was assessed using the 55 °C warm-water tail-flick test. The latency to the first sign of a rapid tail flick was taken as the behavioral end point.49 Each mouse was first tested for baseline latency by immersing its tail in the water and recording the time to response. Mice not responding within 5 s were excluded from further testing. Mice were then administered the test compound and tested for antinociception at various time points afterward. Antinociception was calculated using the following formula: percentage of antinociception = 100(test latency − control latency)/ (10 − control latency). To avoid tissue damage, a maximum score was assigned (100%) to animals that failed to respond within 10 s.

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ASSOCIATED CONTENT

S Supporting Information *

Receptor coordinates (pdb format), as well as a description of the in silico calculations with MOE, details regarding the synthesis of the glycopeptides, MS spectra, Ki values for the glycopeptides, CD spectra, and NMR analysis. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00014. L



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Article

(14) Bilsky, E. J.; Egleton, R. D.; Mitchell, S. A.; Palian, M. M.; Davis, P.; Huber, J. D.; Jones, H.; Yamamura, H. I.; Janders, J.; Davis, T. P.; Porreca, F.; Hruby, V. J.; Polt, R. Enkephalin glycopeptide analogues produce analgesia with reduced dependence liability. J. Med. Chem. 2000, 43, 2586−2590. (15) Jakas, A.; Horvat, S. The effect of glycation on the chemical and enzymatic stability of the endogenous opioid peptide, leucineenkephalin, and related fragments. Bioorg. Chem. 2004, 32, 516−526. (16) Negri, L.; Lattanzi, R.; Tabacco, F.; Orru, L.; Severini, C.; Scolaro, B.; Rocchi, R. Dermorphin and deltorphin glycosylated analogues: synthesis and antinociceptive activity after systemic administration. J. Med. Chem. 1999, 42, 400−404. (17) Biondi, B.; Goldin, D.; Giannini, E.; Lattanzi, R.; Negri, L.; Melchiorri, P.; Ciocca, L.; Rocchi, R. Novel nociceptin analogues: Synthesis and biological activity. Int. J. Pept. Res. Ther. 2006, 12, 139− 144. (18) Yamamoto, T.; Nair, P.; Jacobsen, N. E.; Vagner, J.; Kulkarni, V.; Davis, P.; Ma, S. W.; Navratilova, E.; Yamamura, H. I.; Vanderah, T. W.; Porreca, F.; Lai, J.; Hruby, V. J. Improving metabolic stability by glycosylation: bifunctional peptide derivatives that are opioid receptor agonists and neurokinin 1 receptor antagonists. J. Med. Chem. 2009, 52, 5164−5175. (19) Ballet, S.; Betti, C.; Novoa, A.; Tomboly, C.; Nielsen, C. U.; Helms, H. C.; Lasniak, A.; Kleczkowska, P.; Chung, N. N.; Lipkowski, A. W.; Brodin, B.; Tourwe, D.; Schiller, P. W. In vitro membrane permeation studies and in vivo antinociception of glycosylated Dmt(1)-DALDA analogues. ACS Med. Chem. Lett. 2014, 5, 352−357. (20) Taylor, J. W.; Kaiser, E. T. The structural characterization of beta-endorphin and related peptide hormones and neurotransmitters. Pharmacol. Rev. 1986, 38, 291−319. (21) Handa, B. K.; Lane, A. C.; Lord, J. A.; Morgan, B. A.; Rance, M. J.; Smith, C. F. C. Analogues of b-LPH61−64 possessing selective agonist activity at m-opiate receptors. Eur. J. Pharmacol. 1981, 70, 531−540. (22) Marqusee, S.; Baldwin, R. L. Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 8898−8902. (23) Schiffer, M.; Edmundson, A. B. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys. J. 1967, 7, 121−135. (24) Richardson, J. S.; Richardson, D. C. Amino acid preferences for specific locations at the ends of alpha helices. Science 1988, 240, 1648− 1652. (25) Abbadi, A.; Mcharifi, M.; Aubry, A.; Premilat, S.; Boussard, G.; Marraud, M. Involvement of side functions in peptide structures - the Asx turn - occurrence and conformational aspects. J. Am. Chem. Soc. 1991, 113, 2729−2735. (26) Bracken, C.; Gulyas, J.; Taylor, J. W.; Baum, J. Synthesis and nuclear-magnetic-resonance structure determination of an alphahelical, bicyclic, lactam-bridged hexapeptide. J. Am. Chem. Soc. 1994, 116, 6431−6432. (27) Gacel, G.; Zajac, J. M.; Delay-Goyet, P.; Dauge, V.; Roques, B. P. Investigation of the structural parameters involved in the mu and delta opioid receptor discrimination of linear enkephalin-related peptides. J. Med. Chem. 1988, 31, 374−383. (28) Egleton, R. D.; Bilsky, R. J.; Tollin, G.; Dhanasekaran, M.; Lowery, J.; Alves, I.; Davis, P.; Porreca, F.; Yamamura, H. I.; Yeomans, L.; Keyari, C. M.; Polt, R. Biousian glycopeptides penetrate the bloodbrain barrier. Tetrahedron: Asymmetry 2005, 16, 65−75. (29) Lowery, J. J.; Yeomans, L.; Keyari, C. M.; Davis, P.; Porreca, F.; Knapp, B. I.; Bidlack, J. M.; Bilsky, E. J.; Polt, R. Glycosylation improves the central effects of DAMGO. Chem. Biol. Drug Des. 2007, 69, 41−47. (30) Lowery, J. J.; Raymond, T. J.; Giuvelis, D.; Bidlack, J. M.; Polt, R.; Bilsky, E. J. In vivo characterization of MMP-2200, a mixed delta/ mu opioid agonist, in mice. J. Pharmacol. Exp. Ther. 2011, 336, 767− 778. (31) Woody, R. W. Circular dichroism. Meth. Enzymol. 1995, 246, 34−71.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (520) 370-2654. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Office of Naval Research (Grants N00014-05-10807 & N00014-02-1-0471), the National Science Foundation (Grant CHE-9526909), the National Institute of Neurological Disorders and Stroke (NINDS, Grant R01NS52727) and the National Institute of General Medical Sciences (Grant P20GM103643) for support. We also thank Dr. Chad Park for his help with CD measurements, and Dr. Linda Breci for help with MS spectra.

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Structural Requirements for CNS Active Opioid Glycopeptides

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