Robin Polt

Interests

Research

Glycopeptide drug development.Glycolipids. Sphingosine derivatives.

Teaching

Yes, I have interests in teaching and mentoring. Besides teaching Synthetic Organic Chemistry (CHEM 446, CHEM 24X, etc), I teach Carbohydrate Chemistry and Biology at the graduate level, and much of this is focused toward glycolipids, glycopeptides and other glycoconjugates of biological interest.

Courses

Scholarly Contributions

Chapters

• Muthu, D., Alves, I. D., Keyari, C. M., Yeomans, L., Egleton, R. D., Bidlack, J. M., Yamamura, H. I., Hruby, V. J., Hruby, V. J., Bilsky, E. J., & Polt, R. L. (2006). Understanding BBB Transport using Glycosylated Enkephalins and Endorphins. In Understanding Biology Using Peptides. American Peptide Symposia, vol 9. Springer, New York, NY. doi:10.1007/978-0-387-26575-9_134
• Egleton, R. D., Bilsky, E. J., Mitchell, S. A., Kriss, C. T., Pratt, M. R., Davis, P., Jones, H., Porrecca, F., Yamamura, H. I., Hruby, V. J., Hruby, V. J., Pratt, M. R., & Polt, R. L. (2002). Practical glycopeptide analgesics: Blood-brain barrier transport and binding of glycosylated enkephalin analogs. In INRC. Springer Netherlands. doi:10.1007/0-306-46881-6_307
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  Robin Polt, Richard D. Egleton, Edward J. Bilsky, Scott A. Mitchell, Caroline T. Kriss, Matt R. Pratt, Peg Davis, Heather Jones, Frank Porrecca, Henry I. Yamamura, and Victor J. Hruby Department of Chemistry, University of Arizona, Tucson, AZ 85721, U.S.A.; Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724, U.S.A.; and Department of Biological Sciences, University of Northern Colorado, CO 80639, U.S.A.
• Mitchell, S. A., Pratt, M. R., Hruby, V. J., Hruby, V. J., Pratt, M. R., & Polt, R. L. (2002). Synthesis of several O -glycopeptide analogs of enkephalin. In Peptides for the New Millennium. American Peptide Symposia. Springer, Dordrecht. doi:10.1007/0-306-46881-6_8
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  Work within the Polt laboratory has focussed on the exploration and exploitation of carbohydrate moieties in the design and synthesis of opiate glycopeptides. These glycopeptides have been synthesized based on the pharmacophore D-Cys -enkephalin (DCDCE) [1]. A -D-glucose-containing analog of DCDCE, LSZ-1025, displayed potent analgesia when administered peripherally [2,3]. In an effort to understand the role of the amino acid glycoside in both penetration of the blood-brain barrier (BBB) and opiate receptor binding and stimulation, a series of glycopeptides were synthesized. This work reflects the optimized synthesis of several enkephalin glycopeptides, represented by several structural modifications. Incorporation of D-amino acid glucosides to test the environment of the amino acid-glycoside linkage, variance of the glycan moiety on serine to verify the impact of different sugars, and implementation of several different pharmacophores, such as in SAM-1095 where a linear pharmacophore [4] was used, highlight our design rationale. The impact of these changes was assessed after detailed pharmacological testing.
• Dangel, B. D., Tadikonda, B., Kelly, B. D., & Polt, R. L. (2001). Bifurcated Dipeptide Schiff Bases as Ligands for Enantioselective Catalysis: Micro-Metalloenzymes. In ACS Meeting Abstract. Springer, Dordrecht. doi:10.1007/978-94-010-0464-0_124
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  Enantioselective catalysis mediated by transition metals complexed to enantio-merically pure ligands has become an important feature in organic synthesis. While numerous ligands have been designed for this purpose, and many have used amino acids as a source of chirality, only a very few (c.f. Belokon [1]) have used amino acids or peptides directly for this purpose. We have discovered that the use of highly hindered benzophenone imines as N-terminal capping groups imparts a chiral “twist” to the resulting transition metal complexes [2], and that this feature can be exploited to produce enantioselective catalysts. Use of Merrifield- or Wang-type resins and solid-phase peptide synthesis allows for the facile construction of ligand arrays, which facilitates “catalyst discovery” via parallel or combinatorial approaches.
• Palian, M. M., Elmagbari, N., Elmagbari, N. O., Davis, P., Wei, H., Egleton, R., Porecca, F., Yamamura, H. I., Hruby, V. J., Hruby, V. J., Bilsky, E. J., & Polt, R. L. (2001). α-Helical Glycopeptide Analgesics: β-Endorphin Mimics with Good in vivo Potency. In Peptides: The Wave of the Future. American Peptide Symposia, vol 7. Springer, Dordrecht. doi:10.1007/978-94-010-0464-0_231
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  Glycopeptide enkephalins [1] show potent analgesic activity in vivo [2], and are capable of penetrating the blood-brain barrier [3]. The opioids themselves represent a broad class of centrally acting ligands that may target the δ, κ, or μ opioid receptors in the brain or the spinal column in order to produce pain relief, or other effects.
• Polt, R. L. (2001). 9‐Fluorenylmethyl Chloroformate. In Encyclopedia of Reagents for Organic Synthesis. John Wiley & Sons, Ltd. doi:10.1002/047084289X.RF002
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  [28920-43-6] C15H11ClO2 (MW 258.71) InChI = 1S/C15H11ClO2/c16-15(17)18-9-14-12-7-3-1-5-10(12)11-6-2-4-8-13(11)14/h1-8,14H,9H2 InChIKey = IRXSLJNXXZKURP-UHFFFAOYSA-N (base-labile protecting group for primary and secondary amines;3 used in peptide synthesis;5 used for derivatization of amines and amino acids prior to HPLC analysis and fluorescence detection13) Alternate Names: 9-fluorenylmethoxycarbonyl chloride; FMOC-Cl. Physical Data: mp 62–64 °C. Solubility: sol organics (CH2Cl2, THF, dioxane); reacts with alcohols, amines, and water. Form Supplied in: white crystalline solid; widely available. Handling, Storage, and Precautions: FMOC-Cl is an acid chloride, and should be protected from moisture and heat. The reagent can evolve CO2 (pressure) upon prolonged storage; therefore bottles should be opened carefully. FMOC-Cl is harmful if swallowed, inhaled, or absorbed through skin, is corrosive, and is a strong lachrymator. Inhalation may be fatal. Use in a fume hood.
• Williams, S. A., Abbruscato, T. J., Szabo, L., Hruby, V. J., Hruby, V. J., Davis, T. P., & Polt, R. L. (1996). THE EFFECT OF GLYCOSYLATION ON THE UPTAKE OF AN ENKEPHALIN ANALOGUE INTO THE CENTRAL NERVOUS SYSTEM. In Biology and Physiology of the Blood-Brain Barrier. Advances in Behavioral Biology, vol 46. Springer, Boston, MA. doi:10.1007/978-1-4757-9489-2_13
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  In contrast to unglycosylated controls, glycosylated [D-Cys2,5]enkephalin-ser-gly (glycosylated DCDCE-ser-gly) elicits analgesia after intraperitoneal administration. This was postulated to be due to the presence of the glucose moiety allowing the analogue to cross the BBB via the glucose carrier. To test this hypothesis, the present study investigated the biological stability and the CNS uptake ofunglycosylated and glycosylated DCDCE-ser-gly. Interestingly, the metabolic half-lives and ability to cross the in vitro BBB was found to be similar for both analogues. In situ brain perfusion indicated that the brain uptake of glycosylated DCDCE-ser-gly was greater than that for the vascular marker, [14C]sucrose, but similar to the CSF uptake of the peptide. CNS uptake of glycosylated DCDCE-ser-gly was not affected by replacing D- with L- glucose, nor with the addition of 10 µM unlabelled glycosylated DCDCE-ser-gly. In summary, the difference in analgesic response of glycosylated compared to unglycosylated DCDCE-ser-gly, is not related to either differing metabolic profiles, nor the ability of the glycosylated analogue to use the glucose carrier to enter the CNS. However, this study does not eliminate the involvement of a different low affinity, saturable uptake system taking the glycosylated, but not the unglycosylated form.

Journals/Publications

• Al-Obeidi, F., Bartlett, M. J., Falk, T., Goodman, H. J., Heien, M. L., Kumirov, V. K., Liu, C., Polt, R., Sprőber, S., Streicher, J. M., Szabó, L. Z., & Tanguturi, P. (2023).

  Structure-Based Design of Glycosylated Oxytocin Analogues with Improved Selectivity and Antinociceptive Activity

  . ACS Medicinal Chemistry Letters, 14(2), 163-170. doi:10.1021/acsmedchemlett.2c00455
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  This is the beginning of a new chapter in the glycoside drug arena.  Prof. C. Sue Carter, a giant in the field of oxytocin reasearch has been promoting this paper and Hannah Goodman's dissertation to the field.
• Polt, R. L., Falk, T., Heien, M. L., Streicher, J. M., Rowe, R. K., Madhavan, L., Morrison, H. W., Melvin, J., Green, T. R., Giordano, K. R., Saber, M., Ortiz, J. B., Liu, C., Szabò, L., Bartlett, M. J., Molnar, G., Tanguturi, P., Bernard, K., & Apostol, C. R. (2022). Design and Synthesis of Novel Brain Penetrant Glycopeptide Analogues of PACAP with Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism.. Frontiers in drug discovery, Vol. 1:(818003). doi:https://doi.org/10.3389/fddsv.2021.818003
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  There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects via the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP’s poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues (2LS80Mel and 2LS98Lac) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson’s disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates.
• Polt, R., Mansour, H. M., Hay, M., Alabsi, W., Al-obeidi, F. A., & Acosta, M. F. (2021). Synthesis, Physicochemical Characterization, In Vitro 2D/3D Human Cell Culture, and In Vitro Aerosol Dispersion Performance of Advanced Spray Dried and Co-Spray Dried Angiotensin (1-7) Peptide and PNA5 with Trehalose as Microparticles/Nanoparticles for Targeted Respiratory Delivery as Dry Powder Inhalers.. Pharmaceutics, 13(8), 1278. doi:10.3390/pharmaceutics13081278
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  The peptide hormone Angiotensin (1-7), Ang (1-7) or (Asp-Arg-Val-Tyr-Ile-His-Pro), is an essential component of the renin-angiotensin system (RAS) peripherally and is an agonist of the Mas receptor centrally. Activation of this receptor in the CNS stimulates various biological activities that make the Ang (1-7)/MAS axis a novel therapeutic approach for the treatment of many diseases. The related O-linked glycopeptide, Asp-Arg-Val-Tyr-Ile-His-Ser-(O-β-D-Glc)-amide (PNA5), is a biousian revision of the native peptide hormone Ang (1-7) and shows enhanced stability in vivo and greater levels of brain penetration. We have synthesized the native Ang (1-7) peptide and the glycopeptide, PNA5, and have formulated them for targeted respiratory delivery as inhalable dry powders. Solid phase peptide synthesis (SPPS) successfully produced Ang (1-7) and PNA5. Measurements of solubility and lipophilicity of raw Ang (1-7) and raw PNA5 using experimental and computational approaches confirmed that both the peptide and glycopeptide have high-water solubility and are amphipathic. Advanced organic solution spray drying was used to engineer the particles and produce spray-dried powders (SD) of both the peptide and the glycopeptide, as well as co-spray-dried powders (co-SD) with the non-reducing sugar and pharmaceutical excipient, trehalose. The native peptide, glycopeptide, SD, and co-SD powders were comprehensively characterized, and exhibited distinct glass transitions (Tg) consistent with the amorphous glassy state formation with Tgs that are compatible with use in vivo. The homogeneous particles displayed small sizes in the nanometer size range and low residual water content in the solid-state. Excellent aerosol dispersion performance with a human DPI device was demonstrated. In vitro human cell viability assays showed that Ang (1-7) and PNA5 are biocompatible and safe for different human respiratory and brain cells.
• Polt, R., Mansour, H. M., Polt, R., Mansour, H. M., Encinas-basurto, D., Eedara, B. B., & Alabsi, W. (2021). Spray-Dried Inhalable Powder Formulations of Therapeutic Proteins and Peptides.. AAPS PharmSciTech, 22(5), 185.
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  Respiratory diseases are among the leading causes of morbidity and mortality worldwide. Innovations in biochemical engineering and understanding of the pathophysiology of respiratory diseases resulted in the development of many therapeutic proteins and peptide drugs with high specificity and potency. Currently, protein and peptide drugs are mostly administered by injections due to their large molecular size, poor oral absorption, and labile physicochemical properties. However, parenteral administration has several limitations such as frequent dosing due to the short half-life of protein and peptide in blood, pain on administration, sterility requirement, and poor patient compliance. Among various noninvasive routes of administrations, the pulmonary route has received a great deal of attention and is a better alternative to deliver protein and peptide drugs for treating respiratory diseases and systemic diseases. Among the various aerosol dosage forms, dry powder inhaler (DPI) systems appear to be promising for inhalation delivery of proteins and peptides due to their improved stability in solid state. This review focuses on the development of DPI formulations of protein and peptide drugs using advanced spray drying. An overview of the challenges in maintaining protein stability during the drying process and stabilizing excipients used in spray drying of proteins and peptide drugs is discussed. Finally, a summary of spray-dried DPI formulations of protein and peptide drugs, their characterization, various DPI devices used to deliver protein and peptide drugs, and current clinical status are discussed.
• Falk, T., Sherman, S. J., Polt, R. L., Kennedy, R. T., Heien, M. L., Parent, K. L., Bidlack, J. M., Flores, A. J., Szabo, L., Mabrouk, O. S., & Bartlett, M. J. (2020). The Delta-Specific Opioid Glycopeptide BBI-11008: CNS Penetration and Behavioral Analysis in a Preclinical Model of Levodopa-Induced Dyskinesia. International Journal of Molecular Sciences.
• Falk, T., Vanderah, T. W., Szabo, L., So, L. Y., Skinner, D. P., Sherman, S. J., Polt, R., Parent, K. L., Heien, M. L., Falk, T., & Bartlett, M. J. (2020). Highly-selective µ-opioid receptor antagonism does not block L-DOPA-induced dyskinesia in a rodent model.. BMC research notes, 13(1), 149. doi:10.1186/s13104-020-04994-7
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  Dopamine-replacement utilizing L-DOPA is still the mainstay treatment for Parkinson's disease (PD), but often leads to development of L-DOPA-induced dyskinesia (LID), which can be as debilitating as the motor deficits. There is currently no satisfactory pharmacological adjunct therapy. The endogenous opioid peptides enkephalin and dynorphin are important co-transmitters in the direct and indirect striatofugal pathways and have been implicated in genesis and expression of LID. Opioid receptor antagonists and agonists with different selectivity profiles have been investigated for anti-dyskinetic potential in preclinical models. In this study we investigated effects of the highly-selective μ-opioid receptor antagonist CTAP (> 1200-fold selectivity for μ- over δ-opioid receptors) and a novel glycopeptide congener (gCTAP5) that was glycosylated to increase stability, in the standard rat LID model..Intraperitoneal administration (i.p.) of either 0.5 mg/kg or 1 mg/kg CTAP and gCTAP5 completely blocked morphine's antinociceptive effect (10 mg/kg; i.p.) in the warm water tail-flick test, showing in vivo activity in rats after systemic injection. Neither treatment with CTAP (10 mg/kg; i.p.), nor gCTAP5 (5 mg/kg; i.p.) had any effect on L-DOPA-induced limb, axial, orolingual, or locomotor abnormal involuntary movements. The data indicate that highly-selective μ-opioid receptor antagonism alone might not be sufficient to be anti-dyskinetic.
• Maier, R. M., Hogan, D. E., Tian, F., Polt, R. L., Klimecki, W., Malm, S. W., Kegel, L. L., Pemberton, J. E., Szabo, L. Z., Hunjan, A. S., Hunjan, A. S., Szabo, L. Z., Kegel, L. L., Pemberton, J. E., Malm, S. W., Klimecki, W., Tian, F., Polt, R. L., Hogan, D. E., & Maier, R. M. (2020). Biodegradability and toxicity of cellobiosides and melibiosides. Journal of Surfactants and Detergents. doi:10.1002/jsde.12421
• Maier, R. M., Polt, R. L., Klimecki, W., Pemberton, J. E., Hunjan, A. S., Szabo, Z., Kegel, L. L., Malm, S. W., Tian, F., & Hogan, D. E. (2020). Biodegradability and Toxicity of Cellobiosides and Melibiosides. Journal of Surfactants and Detergents, 23, 715-724.
• Marciniak, A., Polt, R., Marciniak, A., Garcia, J. G., & Camp, S. M. (2020). In silico Docking Studies of Fingolimod and S1P1 Agonists.. Frontiers in pharmacology, 11, 247. doi:10.3389/fphar.2020.00247
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  The sphingosine-1-phosphate receptor 1 (S1P1), originally the endothelial differentiation gene 1 receptor (EDG-1), is one of five G protein-coupled receptors (GPCRs) S1P1 - 5 that bind to and are activated by sphingosine-1-phosphate (S1P). The lipid S1P is an intermediate in sphingolipid homeostasis, and S1P1 is a major medical target for immune system modulation; agonism of the receptor produces a myriad of biological responses, including endothelial cell barrier integrity, chemotaxis, lymphocyte trafficking/targeting, angiogenesis, as well as regulation of the cardiovascular system. Use of in silico docking simulations on the crystal structure of S1P1 allows for pinpointing the residues within the receptor's active site that actively contribute to the binding of S1P, and point to how these specific interactions can be exploited to design more effective synthetic analogs to specifically target S1P1 in the presence of the closely related receptors S1P2, S1P3, S1P4, and S1P5. We examined the binding properties of the endogenous substrate as well as a selection of synthetic sphingosine-derived S1P1 modulators of S1P1 with in silico docking simulations using the software package Molecular Operating Environment® (MOE®). The modeling studies reveal the relevance of phosphorylation, i.e., the presence of a phosphate or phosphonate moiety within the substrate for successful binding to occur, and indicate which residues are responsible for S1P1 binding of the most prominent sphingosine-1-phosphate receptor (S1PR) modulators, including fingolimod and its structural relatives. Furthermore, trends in steric preferences as for the binding of enantiomers to S1P1 could be observed, facilitating future design of receptor-specific substrates to precisely target the active site of S1P1.
• Marciniak, A., Polt, R., Perez, R. G., Marciniak, A., Garcia, J. G., Garcia, A. N., Dudek, S. M., Chiang, E. T., Camp, S. M., & Bittman, R. (2020). Sphingosine-1-phosphate receptor-independent lung endothelial cell barrier disruption induced by FTY720 regioisomers.. Pulmonary circulation, 10(1), 10.1177_2045894. doi:10.1177/2045894020905521
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  RationaleVascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregul...
• Polt, R. L. (2020). Lessons in Stereochemistry: Resolution, Synthesis, and Characterization of Chiral Compounds. J. Chemical Education, Do you REALLY think I have the time to type all this stuff? This would be SO EASY with Web of Science.....
• Polt, R., Hay, M., Apostol, C. R., Hay, M., Apostol, C. R., Polt, R., Hay, M., & Apostol, C. R. (2020). Glycopeptide drugs: A pharmacological dimension between "Small Molecules" and "Biologics".. Peptides, 131, 170369. doi:10.1016/j.peptides.2020.170369
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  Peptides are an important class of molecules with diverse biological activities. Many endogenous peptides, especially neuropeptides and peptide hormones, play critical roles in development and regulating homeostasis. Furthermore, as drug candidates their high receptor selectivity and potent binding leads to reduced off-target interactions and potential negative side effects. However, the therapeutic potential of peptides is severely hampered by their poor stability in vivo and low permeability across biological membranes. Several strategies have been successfully employed over the decades to address these concerns, and one of the most promising strategies is glycosylation. It has been demonstrated in numerous cases that glycosylation is an effective synthetic approach to improve the pharmacokinetic profiles and membrane permeability of peptides. The effects of glycosylation on peptide stability and peptide-membrane interactions in the context of blood-brain barrier penetration will be explored. Numerous examples of glycosylated analogues of endogenous peptides targeting class A and B G-protein coupled receptors (GPCRs) with an emphasis on O-linked glycopeptides will be reviewed. Notable examples of N-, S-, and C-linked glycopeptides will also be discussed. A small section is devoted to synthetic methods for the preparation of glycopeptides and requisite amino acid glycoside building blocks.
• Polt, R., Mansour, H. M., Alabsi, W., & Al-obeidi, F. A. (2020). Organic Solution Advanced Spray-Dried Microparticulate/Nanoparticulate Dry Powders of Lactomorphin for Respiratory Delivery: Physicochemical Characterization, In Vitro Aerosol Dispersion, and Cellular Studies.. Pharmaceutics, 13(1), 26. doi:10.3390/pharmaceutics13010026
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  The purpose of this study was to formulate Lactomorphin (MMP2200) in its pure state as spray-dried(SD) powders, and with the excipient Trehalose as co-spray-dried(co-SD) powders; for intranasal and deep lung administration with Dry Powder Inhalers (DPI). Lactomorphin is a glycopeptide which was developed for the control of moderate to severe pain. Particles were rationally designed and produced by advanced spray drying particle engineering in a closed mode from a dilute organic solution. Comprehensive physicochemical characterization using different analytical techniques was carried out to analyze the particle size, particle morphology, particle surface morphology, solid-state transitions, crystallinity/non-crystallinity, and residual water content. The particle chemical composition was confirmed using attenuated total reflectance-Fourier-transform infrared (ATR-FTIR), and Confocal Raman Microscopy (CRM) confirmed the particles' chemical homogeneity. The solubility and Partition coefficient (LogP) of Lactomorphin were determined by the analytical and computational methodology and revealed the hydrophilicity of Lactomorphin. A thermal degradation study was performed by exposing samples of solid-state Lactomorphin to a high temperature (62 °C) combined with zero relative humidity (RH) and to a high temperature (62 °C) combined with a high RH (75%) to evaluate the stability of Lactomorphin under these two different conditions. The solid-state processed particles exhibited excellent aerosol dispersion performance with an FDA-approved human DPI device to reach lower airways. The cell viability resazurin assay showed that Lactomorphin is safe up to 1000 μg/mL on nasal epithelium cells, lung cells, endothelial, and astrocyte brain cells.
• Polt, R., Mansour, H. M., Alabsi, W., & Al-obeidi, F. A. (2020). Organic Solution Advanced Spray-Dried Microparticulate/Nanoparticulate Dry Powders of Lactomorphin for Respiratory Delivery: Physicochemical Characterization, In Vitro Aerosol Dispersion, and Cellular Studies.. Pharmaceutics, 13(1). doi:10.3390/pharmaceutics13010026
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  The purpose of this study was to formulate Lactomorphin (MMP2200) in its pure state as spray-dried(SD) powders, and with the excipient Trehalose as co-spray-dried(co-SD) powders; for intranasal and deep lung administration with Dry Powder Inhalers (DPI). Lactomorphin is a glycopeptide which was developed for the control of moderate to severe pain. Particles were rationally designed and produced by advanced spray drying particle engineering in a closed mode from a dilute organic solution. Comprehensive physicochemical characterization using different analytical techniques was carried out to analyze the particle size, particle morphology, particle surface morphology, solid-state transitions, crystallinity/non-crystallinity, and residual water content. The particle chemical composition was confirmed using attenuated total reflectance-Fourier-transform infrared (ATR-FTIR), and Confocal Raman Microscopy (CRM) confirmed the particles' chemical homogeneity. The solubility and Partition coefficient (LogP) of Lactomorphin were determined by the analytical and computational methodology and revealed the hydrophilicity of Lactomorphin. A thermal degradation study was performed by exposing samples of solid-state Lactomorphin to a high temperature (62 °C) combined with zero relative humidity (RH) and to a high temperature (62 °C) combined with a high RH (75%) to evaluate the stability of Lactomorphin under these two different conditions. The solid-state processed particles exhibited excellent aerosol dispersion performance with an FDA-approved human DPI device to reach lower airways. The cell viability resazurin assay showed that Lactomorphin is safe up to 1000 μg/mL on nasal epithelium cells, lung cells, endothelial, and astrocyte brain cells.
• Rice, K. C., Polt, R., Jutkiewicz, E. M., & Burgess, G. E. (2020). Characterization of Delta Opioid Receptor Activity of MMP2200, a Mixed Efficacy Mu and Delta Opioid Receptor Agonist. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.05714
• Maier, R. M., Curry, J. E., Pemberton, J. E., Polt, R. L., Klimecki, W., Tanguay, R. L., Hunjan, A. S., Simonich, M., Palos Pacheco, R., Olivares, C., Malm, S. W., Tian, F., & Hogan, D. E. (2019). Biodegradability and toxicity of monorhamnolipid biosurfactant diastereomers. Journal of Hazardous Materials, 364, 600-607. doi:10.1016/j.jhazmat.2018.10.050
• Falk, T., Falk, T., Sherman, S. J., Sherman, S. J., Polt, R. L., Polt, R. L., Porreca, F., Porreca, F., Heien, M. L., Heien, M. L., Parent, K. L., Parent, K. L., Root, B. K., Root, B. K., Bartlett, M. J., Bartlett, M. J., Flores, A. J., & Flores, A. J. (2018). The combination of the opioid glycopeptide MMP-2200 and a NMDA receptor antagonist reduced L-DOPA-induced dyskinesia and MMP-2200 by itself reduced dopamine receptor 2-like agonist-induced dyskinesia. Neuropharmacology, 141, 260-271. doi:doi: 10.1016/j.neuropharm.2018.09.005
• Marciniak, A., Polt, R., Marciniak, A., Garcia, J. G., & Camp, S. M. (2018). An update on sphingosine-1-phosphate receptor 1 modulators.. Bioorganic & medicinal chemistry letters, 28(23-24), 3585-3591. doi:10.1016/j.bmcl.2018.10.042
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  Sphingolipids represent an essential class of lipids found in all eukaryotes, and strongly influence cellular signal transduction. Autoimmune diseases like asthma and multiple sclerosis (MS) are mediated by the sphingosine-1-phosphate receptor 1 (S1P1) to express a variety of symptoms and disease patterns. Inspired by its natural substrate, an array of artificial sphingolipid derivatives has been developed to target this specific G protein-coupled receptor (GPCR) in an attempt to suppress autoimmune disorders. FTY720, also known as fingolimod, is the first oral disease-modifying therapy for MS on the market. In pursuit of improved stability, bioavailability, and efficiency, structural analogues of this initial prodrug have emerged over time. This review covers a brief introduction to the sphingolipid metabolism, the mechanism of action on S1P1, and an updated overview of synthetic sphingosine S1P1 agonists.
• Polt, R., Stevenson, S. W., Denise, G., James, C., Katherine, C., Phillip, A., Rebecca, K., Emily, W., Brooke, S. L., Julio, D., Jean, B. M., Lajos, S., & Edward, B. J. (2020). Behavioral pharmacology of the mixed-action delta-selective opioid receptor agonist BBI-11008: studies on acute, inflammatory and neuropathic pain, respiration, and drug self-administration. Psychopharmacology. doi:https://doi.org/10.1007/s00213-019-05449-z
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  Rationale and objectives The present study characterized the behavioral pharmacology of a novel, mixed-action delta-selective (78:1) opioid receptor agonist, BBI-11008. This glycopeptide drug candidate was tested in assays assessing antinociception (acute, inflammatory, and neuropathic pain-like conditions) and side-effect endpoints (respiratory depression and drug self-administration). Results BBI-11008 had a 78-fold greater affinity for the delta opioid receptor than the mu receptor, and there was no binding to the kappa opioid receptor. BBI-11008 (3.2–100; 10–32 mg kg−1, i.v.) and morphine (1–10; 1–3.2 mg kg−1, i.v.) produced antinociceptive and anti-allodynic effects in assays of acute thermal nociception and complete Freund’s adjuvant (CFA)-induced inflammatory pain, with BBI-11008 being less potent than morphine in both assays. BBI-11008 (1–18 mg kg−1, i.v.) had similar efficacy to gabapentin (10–56 mg kg−1, i.v.) in a spinal nerve ligation (SNL) model of neuropathic pain. In the respiration assay, with increasing %CO2 exposure, BBI-11008 produced an initial increase (32 mg kg−1, s.c.) and then decrease (56 mg kg−1,s.c.) in minute volume (MV) whereas morphine (3.2–32 mg kg−1, s.c.) produced dose-dependent decreases in MV. In the drug self-administration procedure, BBI-11008 did not maintain self-administration at any dose tested.Conclusions These results suggest that the glycopeptide drug candidate possesses broad-spectrum antinociceptive and anti-allodynic activity across a range of pain-like conditions. Relative to morphine or fentanyl, the profile for BBI-11008 in the respiration and drug self-administration assays suggests that BBI-11008 may have less pronounced deleterious side effects. Continued assessment of this compound is warranted.
• Kegel, L. L., Szabo, L. Z., Polt, R. L., & Pemberton, J. E. (2016). Alkyl-melibioside and Alkyl-cellobioside Solution Phase Properties: Effect of Sugar Headgroup and Alkyl Chain Length on Surfactant Performance. Green Chemistry.
• Pemberton, J. E., Polt, R. L., Maier, R. M., Wang, H., Coss, C. S., Eismin, R. J., & Palos Pacheco, R. (2017). Synthesis and Characterization of Four Diastereomers of Monorhamnolipids. Journal of the American Chemical Society, 139, 5125-5132.
• Kegel, L. L., Szabo, L. Z., Polt, R. L., & Pemberton, J. E. (2016). Alkyl-melibioside and Alkyl-cellobioside Solution Phase Properties: Effect of Sugar Headgroup and Alkyl Chain Length on Surfactant Performance. Green Chemistry, 18, 4446-4460. doi:DOI: 10.1039/C6GC00436A
• Szabo, L., Hanrahan, D. J., Jones, E. M., Martin, E., Pemberton, J. E., & Polt, R. L. (2016). Preparation of S-Glycoside Surfactants and Cysteine Thioglycosides. Carbohydrate Research, 422, 1-4.
• Polt, R. (2015). Glycolipids and glycopeptides: Minimally competent Lewis acid catalysis produces surfactants for use ex vivo and in vivo. ACS Meeting Abstract.
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  U of minimally competent main group Lewis acids such as InBr3 and Bi (OSO2CF3)3 permit the formation of glycosides in high yield and purity from simple sugar per-acetates at room temperature or above with remarkable α/β-selectivity. Classical reactions rely on very reactive glycosyl donors in conjunction with metal promoters (Hg++, Ag+ etc). Newer methods have used trichloroacetimidates and strong Bronsted or Lewis acids or thioglycosides in conjunction with oxidative or thiophilic activators. Use of the more stable glycosyl per acetates in conjunction with stoichiometric amounts of strong Lewis acids (BF3.Et2O, FeBr3 etc) has been explored with limited success. The use of “minimally competent” Lewis acids has allowed us to perform glycosidation reactions with catalytic amounts of InBr3 or Bi (OTfl)3 well above room temperature and without significant decomposition of the glycoside products. We will discuss the exploitation of these methods to produce glycolipid surfactants from renewable resources as well as glycopeptide drugs that penetrate the blood-brain barrier (BBB). These “biousian glycopeptides” are not subject to “Lipinski’s rules” that would otherwise eliminate them as CNS drug candidates. Glycosylated amphipathic helices or “address segments” have been used to target G-protein coupled receptors (GPCRs) in the brain after intravenous administration. The addition of glycosides to endogenous peptide neurotransmitters and peptide hormones imparts favorable pharmacokinetic and pharmacodynamics (PK/PD) properties and enables penetration of the BBB which is not constrained by molecular weight.
• Polt, R., & Jones, E. M. (2015). CNS active O-linked glycopeptides.. Frontiers in chemistry, 3, 40. doi:10.3389/fchem.2015.00040
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  Naturally occurring glycopeptides and glycoproteins play important roles in biological processes. Glycosylation is one of the most common post-translational modifications in vivo. Glycopeptides are involved in cell signaling and sorting, providing cell surface markers for recognition. From the drug design and synthesis perspective, modification of a peptide through glycosylation results in increased bioavailability and bioactivity of glycopeptides in living systems with negligible toxicity of degradation products. Glycopeptide synthesis can be accomplished through incorporation of a glycosylated amino acid in solid phase peptide synthesis (SPPS) to form the desired peptide, or via incorporation of sugar-amino acid moieties. Additionally, research indicates that glycosylation increases penetration of the blood-brain barrier (BBB) by peptides, which may lead to novel therapeutics for neurological disorders. Recent applications of glycopeptides have focused on the in vivo central nervous system (CNS) effects after peripheral administration of centrally active peptides modified with various carbohydrates.
• Stevenson, G. W., Luginbuhl, A., Dunbar, C., LaVigne, J., Dutra, J., Atherton, P., Bell, B., Cone, K., Giuvelis, D., Polt, R., Streicher, J. M., & Bilsky, E. J. (2015). The mixed-action delta/mu opioid agonist MMP-2200 does not produce conditioned place preference but does maintain drug self-administration in rats, and induces in vitro markers of tolerance and dependence. PHARMACOLOGY BIOCHEMISTRY AND BEHAVIOR, 132, 49-55.
• Yingxue, L. i., Louis, L. S., Knapp, B. I., Muthu, D., Anglin, B., Giuvelis, D., Bidlack, J. M., Bilsky, E. J., & Polt, R. (2014). Can amphipathic helices influence the CNS antinociceptive activity of glycopeptides related to β-endorphin?. JOURNAL OF MEDICINAL CHEMISTRY, 57(6), 2237-2246.
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  Abstract: Glycosylated β-endorphin analogues of various amphipathicity were studied in vitro and in vivo in mice. Opioid binding affinities of the O-linked glycopeptides (mono- or disaccharides) and unglycosylated peptide controls were measured in human receptors expressed in CHO cells. All were pan-agonists, binding to μ-, δ-, or κ-opioid receptors in the low nanomolar range (2.2-35 nM Ki's). The glycoside moiety was required for intravenous (i.v.) but not for intracerebroventricular (i.c.v.) activity. Circular dichroism and NMR indicated the degree of helicity in H2O, aqueous trifluoroethanol, or micelles. Glycosylation was essential for activity after i.v. administration. It was possible to manipulate the degree of helicity by the alteration of only two amino acid residues in the helical address region of the β-endorphin analogues without destroying μ-, δ-, or κ-agonism, but the antinociceptive activity after i.v. administration could not be directly correlated to the degree of helicity in micelles. © 2014 American Chemical Society.
• Polt, R. L., Coss, C. S., Wang, H., Mudalige, A., & Pemberton, J. E. (2013). A PM-IRRAS Investigation of Monorhamnolipid Orientation at the Air-Water Interface. Langmuir, 29(14), 4441-4450.
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  The rhamnolipid biosurfactants have been considered as possible "green" alternatives to synthetic surfactants due to their greater compatibility with the environment and excellent surface active properties. In order to understand the molecular orientation of rhamnolipids at the air-water interface, a new monorhamnolipid with two octadecyl chains, Rha-C18-C18, has been studied at the air-water interface with polarization modulated-infrared reflection absorption spectroscopy (PM-IRRAS). Since rhamnolipids possess a carboxylic acid, and hence exhibit pH-dependent properties, their water surface orientation is studied in solutions of pH 2, 5, and 8. Rhamnolipids have also been reported to form strong complexes with Pb2+; thus, the effect of the presence of Pb2+ on molecular orientation at the interface is also investigated. PM-IRRA spectra indicate an increase in alkyl chain order and a decrease in alkyl chain tilt angle as the surface pressure of the monolayer increases, with pH-independent tilt angles ranging from 63 degrees to 45 degrees. Molecular modeling using Spartan provides insight into the cause of this large tilt angle as being due to the nature of the monorhamnolipid packing at the air-water interface as dictated by its large hydrophilic headgroup.
• Lefever, M. R., Szab, L. Z., Anglin, B., Ferracane, M., Hogan, J., Cooney, L., & Polt, R. (2012). Glycosylation of α-amino acids by sugar acetate donors with InBr ₃. Minimally competent Lewis acids. Carbohydrate Research, 351, 121-125.
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  PMID: 22342206;PMCID: PMC3296895;Abstract: A simplified method for the preparation of Fmoc-serine and Fmoc-threonine glycosides for use in O-linked glycopeptide synthesis is described. Lewis acids promote glycoside formation, but also promote undesired reactions of the glycoside products. Use of 'minimally competent' Lewis acids such as InBr 3 promotes the desired activation catalytically, and with greatly reduced side products from sugar peracetates. © 2012 Elsevier Ltd. All rights reserved.
• Lefever, M., Li, Y., Anglin, B., Muthu, D., Giuvelis, D., Lowery, J. J., Knapp, B. I., Bidlack, J. M., Bilsky, E. J., & Polt, R. (2015). Structural Requirements for CNS Active Opioid Glycopeptides. JOURNAL OF MEDICINAL CHEMISTRY, 58(15), 5728-5741.
• Mabrouk, O. S., Falk, T., Sherman, S. J., Kennedy, R. T., & Polt, R. (2012). CNS penetration of the opioid glycopeptide MMP-2200: A microdialysis study. Neuroscience Letters, 531(2), 99-103.
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  PMID: 23127847;PMCID: PMC3539793;Abstract: Endogenous opioid peptides enkephalin and dynorphin are major co-transmitters of striatofugal pathways of the basal ganglia. They are involved in the genesis of levodopa-induced dyskinesia and in the modulation of direct and indirect striatal output pathways that are disrupted in Parkinson's disease. One pharmacologic approach is to develop synthetic glycopeptides closely resembling endogenous peptides to restore their normal functions. Glycosylation promotes penetration of the blood-brain barrier. We investigated CNS penetration of the opioid glycopeptide MMP-2200, a mixed δ/μ-agonist based on leu-enkephalin, as measured by in vivo microdialysis and subsequent mass spectrometric analysis in awake, freely moving rats. The glycopeptide (10. mg/kg) reaches the dorsolateral striatum (DLS) rapidly after systemic (i.p.) administration and is stably detectable for the duration of the experiment (80. min). The detected level at the end of the experiment (around 250. pM) is about 10-fold higher than the level of the endogenous leu-enkephalin, measured simultaneously. This is one of the first studies to directly prove that glycosylation of an endogenous opioid peptide leads to excellent blood-brain barrier penetration after systemic injection, and explains robust behavioral effects seen in previous studies by measuring how much glycopeptide reaches the target structure, in this case the DLS. © 2012 Elsevier Ireland Ltd.
• Pemberton, J. E., Coss, C. S., Polt, R., Pemberton, J. E., Maier, R. M., Coss, C. S., & Carrocci, T. (2012). Minimally Competent Lewis Acid Catalysts: Indium(III) and Bismuth(III) Salts Produce Rhamnosides (=6‐Deoxymannosides) in High Yield and Purity. Helvetica Chimica Acta, 95(12), 2652-2659. doi:10.1002/hlca.201200528
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  Glycosylation of decan-1-ol (2), (±)-decan-2-ol (3), and (±)-methyl 3-hydroxydecanoate (4) with L-rhamnose peracetate 5 to produce rhamnosides (=6-deoxymannosides) 6, 7, and 8 in the presence of Lewis acids BF3⋅Et2O, Sc(OTf)3, InBr3, and Bi(OTf)3 was studied (Table 1). While the strong Lewis acids BF3⋅Et2O and Sc(OTf)3 were effective as glycosylation promoters, they had to be used in excess; however, glycosylation required careful control of reaction times and temperatures, and these Lewis acids produced impurities in addition to the desired glycosides. Enantiomerically pure rhamnosides (R)-1 and (S)-1 (Fig.) were obtained from L-rhamnose peracetate 5 and (±)-benzyl 3-hydroxydecanoate (9) via the diastereoisomeric rhamnosides 10 (Table 2; Scheme 3). The much weaker Lewis acids InBr3 and Bi(OTfl)3 produced purer products in high yield under a wider range of conditions (higher temperatures), and were effective glycosylation promoters even when used catalytically (
• Polt, R., Kennedy, R. T., Sherman, S. J., Falk, T., & Mabrouk, O. S. (2012). CNS penetration of the opioid glycopeptide MMP-2200: a microdialysis study.. Neuroscience letters, 531(2), 99-103. doi:10.1016/j.neulet.2012.10.029
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  Endogenous opioid peptides enkephalin and dynorphin are major co-transmitters of striatofugal pathways of the basal ganglia. They are involved in the genesis of levodopa-induced dyskinesia and in the modulation of direct and indirect striatal output pathways that are disrupted in Parkinson's disease. One pharmacologic approach is to develop synthetic glycopeptides closely resembling endogenous peptides to restore their normal functions. Glycosylation promotes penetration of the blood-brain barrier. We investigated CNS penetration of the opioid glycopeptide MMP-2200, a mixed δ/μ-agonist based on leu-enkephalin, as measured by in vivo microdialysis and subsequent mass spectrometric analysis in awake, freely moving rats. The glycopeptide (10 mg/kg) reaches the dorsolateral striatum (DLS) rapidly after systemic (i.p.) administration and is stably detectable for the duration of the experiment (80 min). The detected level at the end of the experiment (around 250 pM) is about 10-fold higher than the level of the endogenous leu-enkephalin, measured simultaneously. This is one of the first studies to directly prove that glycosylation of an endogenous opioid peptide leads to excellent blood-brain barrier penetration after systemic injection, and explains robust behavioral effects seen in previous studies by measuring how much glycopeptide reaches the target structure, in this case the DLS.
• Polt, R., Lefever, M. R., Szabò, L. Z., Anglin, B., Ferracane, M., Hogan, J., Cooney, L., & Polt, R. L. (2012). Glycosylation of α-amino acids by sugar acetate donors with InBr3. Minimally competent Lewis acids. Carbohydrate research, 351.
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  A simplified method for the preparation of Fmoc-serine and Fmoc-threonine glycosides for use in O-linked glycopeptide synthesis is described. Lewis acids promote glycoside formation, but also promote undesired reactions of the glycoside products. Use of 'minimally competent' Lewis acids such as InBr(3) promotes the desired activation catalytically, and with greatly reduced side products from sugar peracetates.
• Polt, R., Li, Y., Lefever, M. R., Muthu, D., Bidlack, J. M., Bilsky, E. J., & Polt, R. L. (2012). Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins. Future medicinal chemistry, 4(2).
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  Over the past two decades, potent and selective analgesics have been developed from endogenous opioid peptides. Glycosylation provides an important means of modulating interaction with biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows penetration of cellular barriers that block efficient drug distribution, including the blood-brain barrier. Extremely potent and selective opiate agonists have been developed from endogenous peptides, some of which show great promise as drug candidates.
• Yingxue, L. i., Lefever, M. R., Muthu, D., Bidlack, J. M., Bilsky, E. J., & Polt, R. (2012). Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins. Future Medicinal Chemistry, 4(2), 205-226.
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  PMID: 22300099;PMCID: PMC3306179;Abstract: Over the past two decades, potent and selective analgesics have been developed from endogenous opioid peptides. Glycosylation provides an important means of modulating interaction with biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows penetration of cellular barriers that block efficient drug distribution, including the blood-brain barrier. Extremely potent and selective opiate agonists have been developed from endogenous peptides, some of which show great promise as drug candidates. © 2012 Future Science Ltd.
• Keyari, C. M., Polt, R., & Nichol, G. S. (2011). (S)-Methyl 2-{(S)-2-[bis(4-methoxyphenyl)methylideneamino]-3- hydroxypropanamido}-3-methylbutanoate. Acta Crystallographica Section E: Structure Reports Online, 67(1), o13-o14.
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  PMID: 21522640;PMCID: PMC3050411;Abstract: The title compound, C24H30N2O6, a Schiff base, adopts an extended conformation in which the methoxy groups are essentially coplanar with the aromatic ring to which they are bonded (mean planes fitted through the non-H atoms of each methoxyphenyl group have r.m.s. deviations of 0.078 and 0.044 Å) and the angle between mean planes fitted through the aromatic rings is 87.57 (10)°. An intramolecular N - H⋯N hydrogen bond keeps the imine and amide groups essentially coplanar. A mean plane fitted through these groups has an r.m.s. deviation of 0.0545 Å. Additional O - H⋯O hydrogen bonding parallel with the a axis links the molecules into a hydrogen-bonded chain in the crystal. C - H⋯O and C - H⋯π interactions are found within the crystal packing. The compound has been assigned the S,S configuration on the basis of the chemical synthesis, which used pure homotopic lamino acids, and we have no reason to believe that the compound has epimerized.
• Lowery, J. J., Raymond, T. J., Giuvelis, D., Bidlack, J. M., Polt, R., & Bilsky, E. J. (2011). In vivo characterization of MMP-2200, a mixed δ/μ opioid agonist, in mice. Journal of Pharmacology and Experimental Therapeutics, 336(3), 767-778.
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  PMID: 21118955;PMCID: PMC3061528;Abstract: We have previously reported the chemistry and antinociceptive properties of a series of glycosylated enkephalin analogs (glycopeptides) exhibiting approximately equal affinity and efficacy at δ opioid receptors (DORs) and μ opioid receptors (MORs). More detailed pharmacology of the lead glycopeptide MMP-2200 [H2N-Tyr-D-Thr-Gly-Phe-Leu-Ser-(O-β-D- lactose)-CONH2] is presented. MMP-2200 produced dose-related antinociception in the 55°C tail-flick assay after various routes of administration. The antinociceptive effects of MMP-2200 were blocked by pretreatment with the general opioid antagonist naloxone and partially blocked by the MOR-selective antagonist β-funaltrexamine and the DOR-selective antagonist naltrindole. The κ opioid receptor antagonist nor-binaltorphimine and the peripherally active opioid antagonist naloxone-methiodide were ineffective in blocking the antinociceptive effects of MMP-2200. At equi-antinociceptive doses, MMP-2200 produced significantly less stimulation of locomotor activity compared with morphine. Repeated administration of equivalent doses of morphine and MMP-2200 (twice daily for 3 days) produced antinociceptive tolerance (∼13- and 5-fold right-ward shifts, respectively). In acute and chronic physical dependence assays, naloxone precipitated a more severe withdrawal in mice receiving morphine compared with equivalent doses of the glycopeptide. Both morphine and MMP-2200 inhibited respiration and gastrointestinal transit. In summary, MMP-2200 acts as a mixed DOR/MOR agonist in vivo, which may in part account for its high antinociceptive potency after systemic administration, as well as its decreased propensity to produce locomotor stimulation, tolerance, and physical dependence in mice, compared with the MOR-selective agonist morphine. For other measures (e.g., gastrointestinal transit and respiration), the significant MOR component may not allow differentiation from morphine. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.
• Polt, R., Yeomans, L., Muthu, D., Lowery, J. J., Martinez, H. N., Abrell, L., Lin, G., Strom, K., Knapp, B. I., Bidlack, J. M., Bilsky, E. J., & Polt, R. L. (2011). Phosphorylation of enkephalins: NMR and CD studies in aqueous and membrane-mimicking environments. Chemical biology & drug design, 78(5).
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  Phosphorylation of l-serine-containing enkephalin analogs has been explored as an alternative to glycosylation in an effort to increase blood-brain barrier permeability and CNS bioavailability of peptide pharmacophores. Two enkephalin-based peptides were modified for these studies, a set related to DTLES, a mixed μ/δ-agonist, and one related to DAMGO, a highly selective μ-agonist. Each unglycosylated peptide was compared to its phosphate, its mono-benzylphosphate ester, and its β-d-glucoside. Binding was characterized in membrane preparations from Chinese hamster ovary cells expressing human μ, δ and κ-opiate receptors. Antinociception was measured in mice using the 55 °C tail-flick assay. To estimate bioavailability, the antinociceptive effect of each opioid agonist was evaluated after intracerebroventricular (i.c.v.) or intravenous administration (i.v.) of the peptides. Circular dichroism methods and high-field nuclear magnetic resonance were used in the presence and absence of sodium dodecylsulfate to understand how the presence of a membrane might influence the peptide conformations.
• Sherman, S. J., Polt, R., Porreca, F., Szabo, L., Zuniga, L. A., Falk, T., & Yue, X. (2011). Effects of the novel glycopeptide opioid agonist MMP-2200 in preclinical models of Parkinson's disease.. Brain research, 1413, 72-83. doi:10.1016/j.brainres.2011.07.038
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  In Parkinson's disease (PD), the consequence of dopaminergic denervation is an imbalance in the activity of the direct and indirect striatofugal pathways, which include potentially important changes in opioid peptide expression and/or activity. The systemic administration of a novel glycosylated opioid peptide MMP-2200 (a.k.a. lactomorphin) was shown to have potent effects in two standard models of PD: 1) amphetamine-induced rotations in the hemi-Parkinsonian 6-hydroxydopamine (6-OHDA)-treated rat and 2) locomotion in the reserpine-treated rat. MMP-2200, an opioid mu and delta receptor agonist, reduced amphetamine-induced rotations in severely-lesioned hemi-Parkinsonian rats; this effect was fully blocked by naloxone, an opioid receptor antagonist. The selective δ-opioid receptor antagonist naltrindole only partially blocked the effect of MMP-2200. MMP-2200 alone did not induce rotations. This effect was also observed in a mild progressive rat 6-OHDA-lesion model. In animals treated with reserpine, profound akinesia was induced that was reversed with apomorphine. There was a prominent overshoot in animals that received apomorphine compared to non-reserpine treated animals, reflecting the well described phenomenon of dopamine supersensitivity indicating that apomorphine not only reversed akinesia but also induced hyper-kinesia. The opioid peptide MMP-2200 blocked the apomorphine-induced hyper-kinesia. This effect of MMP-2200 was prevented by pre-administration of naloxone. MMP-2200 had no effect in preventing the reserpine-induced akinesia, nor did it affect locomotion in control animals. Taken together, the results from these two models are consistent with the glycopeptide opioid agonist MMP-2200 having a potent effect on movements related to dopaminergic hyper-stimulation following striatal dopamine depletion that are best explained by a reduction in the downstream effects of dopamine agonists in these models.
• Yeomans, L., Muthu, D., Lowery, J. J., Martinez, H. N., Abrell, L., Lin, G., Strom, K., Knapp, B. I., Bidlack, J. M., Bilsky, E. J., & Polt, R. (2011). Phosphorylation of enkephalins: NMR and CD studies in aqueous and membrane-mimicking environments. Chemical Biology and Drug Design, 78(5), 749-756.
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  PMID: 21801311;PMCID: PMC3193572;Abstract: Phosphorylation of l-serine-containing enkephalin analogs has been explored as an alternative to glycosylation in an effort to increase blood-brain barrier permeability and CNS bioavailability of peptide pharmacophores. Two enkephalin-based peptides were modified for these studies, a set related to DTLES, a mixed μ/δ-agonist, and one related to DAMGO, a highly selective μ-agonist. Each unglycosylated peptide was compared to its phosphate, its mono-benzylphosphate ester, and its β-d-glucoside. Binding was characterized in membrane preparations from Chinese hamster ovary cells expressing human μ, δ and κ-opiate receptors. Antinociception was measured in mice using the 55°C tail-flick assay. To estimate bioavailability, the antinociceptive effect of each opioid agonist was evaluated after intracerebroventricular (i.c.v.) or intravenous administration (i.v.) of the peptides. Circular dichroism methods and high-field nuclear magnetic resonance were used in the presence and absence of sodium dodecylsulfate to understand how the presence of a membrane might influence the peptide conformations. © 2011 John Wiley & Sons A/S.
• Yue, X., Falk, T., Zuniga, L. A., Szabò, L., Porreca, F., Polt, R., & Sherman, S. J. (2011). Effects of the novel glycopeptide opioid agonist MMP-2200 in preclinical models of Parkinson's disease. Brain Research, 1413, 72-83.
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  PMID: 21840512;PMCID: PMC3167001;Abstract: In Parkinson's disease (PD), the consequence of dopaminergic denervation is an imbalance in the activity of the direct and indirect striatofugal pathways, which include potentially important changes in opioid peptide expression and/or activity. The systemic administration of a novel glycosylated opioid peptide MMP-2200 (a.k.a. lactomorphin) was shown to have potent effects in two standard models of PD: 1) amphetamine-induced rotations in the hemi-Parkinsonian 6-hydroxydopamine (6-OHDA)-treated rat and 2) locomotion in the reserpine-treated rat. MMP-2200, an opioid mu and delta receptor agonist, reduced amphetamine-induced rotations in severely-lesioned hemi-Parkinsonian rats; this effect was fully blocked by naloxone, an opioid receptor antagonist. The selective δ-opioid receptor antagonist naltrindole only partially blocked the effect of MMP-2200. MMP-2200 alone did not induce rotations. This effect was also observed in a mild progressive rat 6-OHDA-lesion model. In animals treated with reserpine, profound akinesia was induced that was reversed with apomorphine. There was a prominent overshoot in animals that received apomorphine compared to non-reserpine treated animals, reflecting the well described phenomenon of dopamine supersensitivity indicating that apomorphine not only reversed akinesia but also induced hyper-kinesia. The opioid peptide MMP-2200 blocked the apomorphine-induced hyper-kinesia. This effect of MMP-2200 was prevented by pre-administration of naloxone. MMP-2200 had no effect in preventing the reserpine-induced akinesia, nor did it affect locomotion in control animals. Taken together, the results from these two models are consistent with the glycopeptide opioid agonist MMP-2200 having a potent effect on movements related to dopaminergic hyper-stimulation following striatal dopamine depletion that are best explained by a reduction in the downstream effects of dopamine agonists in these models. © 2011 Elsevier B.V. All rights reserved.
• Coss, C. S., Pemberton, J. E., Polt, R., Pemberton, J. E., Maier, R. M., & Coss, C. S. (2010). Synthetic Method for the Development of Monorhamnolipids. The FASEB Journal, 24.
• Keyari, C. M., & Polt, R. (2010). Serine and threonine Schiff base esters react with β-anomeric peracetates in the presence of BF₃·Et₂O to produce β-glycosides. Journal of Carbohydrate Chemistry, 29(4), 181-206.
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  Abstract: Improved procedures are reported for the glycosylation of L-serine and L-threonine utilizing activated Schiff base glycosyl acceptors, which are less expensive and more efficient alternatives to published methods. L-serine or L-threonine benzyl ester hydrochloride salts were reacted with the diarylketimine bis-(4-methoxyphenyl)-methanimine in CH3CN at rt to form the more nucleophilic Schiff bases 3a and 3b in excellent yield. These Schiff bases exhibited ring-chain tautomerism in CDCl3 as shown by 1H NMR. Schiff bases 3a and 3b, acting as glycosyl acceptors, reacted at rt with simple sugar peracetate donors with BF3·OEt 2 promotion to provide the corresponding L-serine and L-threonine O-linked glycosides in excellent yields and purities. The dipeptide ester Schiff base Ar2C = N-Ser-Val-OCH3 3e also reacted to provide β-glycosides in excellent yields, and without epimerization. With microwave irradiation the reactions were complete in 2 to 5 min. To investigate this reaction further, classical AgOTf-promoted Koenigs-Knorr reaction of D-glucopyranosyl, lactosyl, and maltosyl bromides were examined, providing the β-glycosides with yields ranging from 35% to 68%. The difference in reactivity between α- and β-carbohydrate peracetate donors was remarkable. The less configurationally stable D-xylopyranosyl tetra-acetate (a pentose) showed no selectivity (αvsβ-configuration) toward the Schiff bases. Copyright © Taylor & Francis Group, LLC.
• Polt, R., Nichol, G. S., & Keyari, C. M. (2010). (S)-Methyl 2-{(S)-2-[bis-(4-meth-oxy-phen-yl)methyl-idene-amino]-3-hy-droxy-propanamido}-3-methyl-butano-ate.. Acta crystallographica. Section E, Structure reports online, 67(Pt 1), o13-4. doi:10.1107/s1600536810049032
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  The title compound, C(24)H(30)N(2)O(6), a Schiff base, adopts an extended conformation in which the meth-oxy groups are essentially coplanar with the aromatic ring to which they are bonded (mean planes fitted through the non-H atoms of each methoxyphenyl group have r.m.s. deviations of 0.078 and 0.044 Å) and the angle between mean planes fitted through the aromatic rings is 87.57 (10)°. An intra-molecular N-H⋯N hydrogen bond keeps the imine and amide groups essentially coplanar. A mean plane fitted through these groups has an r.m.s. deviation of 0.0545 Å. Additional O-H⋯O hydrogen bonding parallel with the a axis links the mol-ecules into a hydrogen-bonded chain in the crystal. C-H⋯O and C-H⋯π inter-actions are found within the crystal packing. The compound has been assigned the S,S configuration on the basis of the chemical synthesis, which used pure homotopic l-amino acids, and we have no reason to believe that the compound has epimerized.
• Keyari, C. M., Knapp, B. I., Bidlack, J. M., Lowey, J., Bilsky, E. J., & Polt, R. (2009). Glycosyl-enkephalins: synthesis and binding at the mu, delta & kappa opioid receptors. Antinociception in mice.. Advances in experimental medicine and biology, 611, 495-496.
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  PMID: 19400281;
• Polt, R., Keyari, C. M., Knapp, B. I., Bidlack, J. M., Lowey, J., Bilsky, E. J., & Polt, R. L. (2009). Glycosyl-enkephalins: synthesis and binding at the mu, delta & kappa opioid receptors. Antinociception in mice. Advances in experimental medicine and biology, 611.
• Porreca, F., Polt, R., Lowery, J. J., Giuvelis, D., Felice, M. D., & Bilsky, E. J. (2009). Assessment of the efficacy and side effect profile of morphine and MMP2200, a mixed delta/mu agonist. The FASEB Journal, 23.
• Oland, L. A., Somogyi, A., Polt, R., Oland, L. A., & Abeytunga, D. T. (2008). Structural studies on the neutral glycosphingolipids of Manduca sexta.. Bioorganic chemistry, 36(2), 70-6. doi:10.1016/j.bioorg.2007.10.002
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  Glycosphingolipids (GSLs) have been implicated as playing major roles in cellular interactions and control of cell proliferation in muticellular organisms. Moreover GSLs and other sphingolipids such as sphingomyelins, ceramides and sphingosines serve a variety of roles in signal transduction. Hence, identification of structures of GSLs in different biota will shed light in understanding their physiological role. During this study, the major glycosphingolipid component present in the extracts of stage-12 and stage-17/18 metamorphosing adults of Manduca sexta was identified as mactosyl ceramide. We report the isolation of several ceramide disaccharides, a ceramide trisaccharide and a ceramide tetrasaccharide. The GSL structures were confirmed by high-resolution mass spectrometry and tandem mass spectrometry. The identity of the monosaccharides was proved using exoglycosidases. The predominant sphingosine chain-length varied from C-14 (tetradecasphing-4-enine) to C-16 (hexadecasphing-4-enine) in these GSLs. Sphingosines of both chain lengths were accompanied by their doubly unsaturated counterparts tetradecasphinga-4,6-diene and hexadecasphinga-4,6-diene. It is also interesting to note the presence of tetradecasphinganine and hexadecasphinganine in minute amounts in the form of a GSL in the extracts of M. sexta. The varying degrees of unsaturation in the sphingosine moiety of GSLs in M. sexta may be biologically significant in insect metamorphosis. The ceramide trisaccharides and ceramide tetrasaccharide belong to the arthro-series, The observation of fucose in the M. sexta GSLs is the first report of the presence of fucose in an arthroseries GSL.
• Pereira, G., Polt, R., Bilsky, E. J., Rice, K. C., & Negus, S. S. (2008). Behavioral pharmacology of the μ/δ opioid glycopeptide MMP2200 in rhesus monkeys. Journal of Pharmacology and Experimental Therapeutics, 326(3), 939-948.
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  PMID: 18511649;PMCID: PMC2587285;Abstract: H2N-Tyr-D-Thr-Gly-Phe-Leu-Ser-(O-β-D-lactose)-CONH 2 (MMP2200) is a novel glycopeptide opioid agonist with similar affinities for μ and δ receptors. Glycosylation promoted brain penetration and production of centrally mediated behavioral effects in mice; however, it is unknown whether the magnitude of enhanced brain penetration is sufficient to permit central mediation of drug effects and production of synergistic μ/δ antinociceptive interactions after systemic administration in primates. To address this issue, the present study compared the effects of MMP2200 and the μ-agonist morphine in four behavioral procedures in rhesus monkeys. In an assay of thermal nociception, morphine (1.0 -5.6 mg/kg) produced dose-dependent antinociception, whereas MMP2200 (10-56 mg/kg) was ineffective. In an assay of capsaicin-induced thermal allodynia, both morphine (0.01-1.0 mg/kg) and MMP2200 (0.032-3.2 mg/kg) produced dose-dependent antiallodynic effects. MMP2200-induced antiallodynia was blocked by the moderately μ-selective antagonist naltrexone (0.01 mg/kg), the δ-selective antagonist naltrindole (1.0 mg/kg), and the peripherally selective opioid antagonist quaternary naltrexone (0.32 mg/kg). In an assay of schedule-controlled behavior, both morphine (0.01-1.0 mg/kg) and MMP2200 (10-56 mg/kg) decreased response rates. Morphine effects were antagonized by naltrexone (0.001-0.01 mg/kg); however, the effects of MMP2200 were not antagonized by either naltrexone (0.01 mg/kg) or naltrindole (1.0 mg/kg). In an assay of drug self-administration, morphine (0.0032-0.32 mg/kg/injection) produced reinforcing effects, whereas MMP2200 (0.032-0.32 mg/kg/injection) did not. These results suggest that systemically administered MMP2200 acted as a peripheral, μ/δ-opioid agonist with limited distribution to the central nervous system in rhesus monkeys. These results also suggest the existence of species differences in the pharmacokinetics and brain penetration of glycopeptides.
• Polt, R. (2008). Endoglycosidases: Biochemistry, Biotechnology, Application Edited by Masahiko Endo (Hirosaki University, Aomori, Japan), Sumihiro Hase (Osaka University, Osaka, Japan), Kenji Yamamoto (Kyoto University, Kyoto, Japan), and Keiichi Takagaki (Hirosaki University School of Medicine, Aomori, Japan). Kodansha Ltd: Tokyo and Springer: Berlin, Heidelberg, New York. 2006. xvi + 255 pp. $169.00. ISBN 4-06-212192-2 (Kodansha) and 3-540-34494-2 (Springer).. Journal of the American Chemical Society, 130(9), 2878-2879. doi:10.1021/ja076925x
• Jalbout, A. F., Abrell, L. M., Adamowicz, L. -., Polt, R. L., Apponi, A. J., & Ziurys, L. M. (2007). Sugar synthesis from a gas-phase formose reaction. Astrobiology, 7(3).
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  Prebiotic possibilities for the synthesis of interstellar ribose through a protic variant of the formose reaction under gas-phase conditions were studied in the absence of any known catalyst. The ion-molecule reaction products, diose and triose, were sought by mass spectrometry, and relevant masses were observed. Ab initio calculations were used to evaluate protic formose mechanism possibilities. A bilateral theoretical and experimental effort yielded a physical model for glycoaldehyde generation whereby a hydronium cation can mediate formaldehyde dimerization followed by covalent bond formation leading to diose and water. These results advance the possibility that ion-molecule reactions between formaldehyde (CH(2)O) and H(3)O(+) lead to formose reaction products and inform us about potential sugar formation processes in interstellar space.
• Lowery, J. J., Yeomans, L., Keyari, C. M., Davis, P., Porreca, F., Knapp, B. I., Bidlack, J. M., Bilsky, E. J., & Polt, R. (2007). Glycosylation improves the central effects of DAMGO. Chemical Biology and Drug Design, 69(1), 41-47.
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  PMID: 17313456;Abstract: A series of μ-agonist DAMGO analogs were synthesized and pharmacologically characterized to test the 'biousian' hypothesis of membrane hopping. DAMGO was altered by incorporating moieties of increasing water solubility into the C-terminus via carboxamide and simple glycoside additions. The hydrophilic C-terminal moieties were varied from glycinol in DAMGO (1) to l-serine amide (2), l-serine amide β-d-xyloside (3), l-serine amide β-d-glucoside (4), and finally to l-serine amide β-lactoside (5). Opioid binding and mouse tail-flick studies were performed. Antinociceptive potency (intravenous) increased, passing through a maximum (A50 ≈ 0.2 μmol/kg) for 2 and 3 as membrane affinity versus water solubility became optimal, and dropped off (A50 ≈ 1.0 μmol/kg) for 4 and 5 as water solubility dominated molecular behavior. Intravenous A50 values were plotted versus hydrodynamic values (glucose units, g.u.) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A50 versus e-Awater/Alipid), and provided either a V-shaped or a U-shaped curve, as predicted by the 'biousian' hypothesis. The μ-selective receptor profile was maintained (Ki's = 0.66-1.3 nm) upon modifications at the C-terminus. The optimal 'degree of glycosylation' for the DAMGO peptide message appears to be between 1.25 and 1.75 g.u. (hydrodynamic g.u.), or 0.75 and 0.90 in terms of the surface-derived amphipathicity values. © 2007 The Authors.
• Bilsky, E., Egleton, R., Mitchell, S., Palian, M., Davis, P., Huber, J., Jones, H., Yamamura, H., Janders, J., Davis, T., Porreca, F., Hruby, V., & Polt, R. (2006). Enkephalin glycopeptide analogues produce analgesia with reduced dependence liability. JOURNAL OF MEDICINAL CHEMISTRY, 43(13), 2586-2590.
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  Endogenous peptides (e.g. enkephalins) control many aspects of brain function, cognition, and perception. The use of these neuroactive peptides in diverse studies has led to an increased understanding of brain function. Unfortunately, the use of brain-derived peptides as pharmaceutical agents to alter brain chemistry in vivo has lagged because peptides do not readily penetrate the blood-brain barrier. Attachment of simple sugars to enkephalins increases their penetration of the blood-brain barrier and allows the resulting glycopeptide analogues to function effectively as drugs. The delta-selective glycosylated Leu-enkephalin amide 2, H2N-Tyr-D-Thr-Gly-Phe-Leu-Ser(beta-D-Glc)-CONH2, produces analgesic effects similar to morphine, even when administered peripherally, yet possesses reduced dependence liability as indicated by naloxone-precipitated withdrawal studies. Similar glycopeptide-based pharmaceuticals hold forth the promise of pain relief with improved side-effect profiles over currently available opioid analgesics.
• Halfen, D. T., Apponi, A. J., Woolf, N., Polt, R., & Ziurys, L. M. (2006). A systematic study of glycolaldehyde in sagittarius B2(N) at 2 and 3 mm: Criteria for detecting large interstellar molecules. Astrophysical Journal Letters, 639(1 I), 237-245.
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  Abstract: A comprehensive study of glycolaldehyde (CH2OHCHO) has been conducted at 2 and 3 mm toward Sgr B2(N) using the Arizona Radio Observatory 12 m telescope. Forty favorable transitions of this species were observed in the range 68-169 GHz. Emission on the 20-70 mK level was detected at frequencies of 38 of these lines, including all transitions arising from the Ka = 0, 1, and 2 ladders. The two transitions not detected were weak and originate in the less populated Ka = 3 levels. Twenty-one percent of the detected lines are distinct, individual features. The remaining transitions are either contaminated by emission from abundant molecules or blended with equivalently weak features. The unblended transitions indicate VLSR = 62.3 ± 2.4 km s-1 and ΔV1/2 = 8.3 ± 3.4 km s-1, line parameters characteristic of organic species in Sgr B2(N). A rotational diagram yields a column density of 5.9 × 1013 cm2 for glycolaldehyde, suggesting a fractional abundance of f(H 2) = 5.9 × 10-11. Observations of formaldehyde toward Sgr B2(N) suggest that H2CO and CH2OHCHO arise from the same gas with an abundance ratio of ∼1/27. H2CO may function as the precursor to glycolaldehyde in a gas-phase "formose" reaction. These observations, combined with past results of Hollis et al., provide convincing evidence for the presence of glycolaldehyde in the ISM. This study suggests that an extensive, self-consistent data set is necessary to identify large organic species in interstellar gas. © 2006. The American Astronomical Society. All rights reserved.
• Dhanasekaran, M., & Polt, R. (2005). New prospects for glycopeptide based analgesia: Glycoside-induced penetration of the Blood-Brain Barrier. Current Drug Delivery, 2(1), 59-73.
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  PMID: 16305409;Abstract: Antinociceptive effects and BBB transport properties of glycosylated enkephalin derivatives are reviewed. Previously, the application of enkephalins as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood brain barrier. This shortcoming has been overcome by glycosylation, paradoxically leading to enhanced BBB transport via transcytosis. Principal design considerations required for enhanced binding, stability and transport of opioid peptides are reviewed. Modifications of the peptide backbone and side chains to achieve optimal receptor binding (μ/δ-selectivity) are presented. The importance of reversible binding between the glycopeptide and membranes is emphasized, and several pertinent examples of peptide-membrane interactions are discussed in the light of glycopeptide transport and opioid binding. An "amphipathic hypothesis" is introduced as a rationale for the observed BBB penetration of the opioid glycopeptides. © 2005 Bentham Science Publishers Ltd.
• Egleton, R. D., Bilsky, E. J., Tollin, G., Dhanasekaran, M., Lowery, J., Alves, I., Davis, P., Porreca, F., Yamamura, H. I., Yeomans, L., Keyari, C. M., & Polt, R. (2005). Biousian glycopeptides penetrate the blood-brain barrier. Tetrahedron Asymmetry, 16(1), 65-75.
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  Abstract: Clinicians have long desired the ability to introduce either exogenous or endogenous neuropeptides directly into the brain in order to alter brain chemistry, but have been thwarted by the blood-brain barrier (BBB). The BBB blocks the introduction of most peptides and proteins into the brain. Glycosylation can be employed as an effective and practical strategy that allows the systemic use of neuropeptides in vivo. A series of glycopeptides based on the Leu-enkephalin analogue YtGFS*-CONH2 led to greatly enhanced stability in vivo and effective penetration of the BBB. Transport through the BBB hinges on the biousian nature of the glycopeptides. That is, the amphipathic glycopeptides possess two conflicting solubility states; one state that is completely water soluble, and another at water-membrane phase boundaries. Multiple lines of evidence suggest that the BBB transport is absorptive endocytosis. Several Leu-enkephalin analogues studied showed antinociceptive potencies greater than morphine. Moreover, these δ-selective glycopeptides lacked many of the μ-opioid side effects generally associated with classical opiate analgesics. The biousian design was extended to much larger glycopeptides (16-17 residues) related to β-endorphin, which also penetrated the BBB and produced antinociception in mice. Plasmon-waveguide resonance (PWR) studies showed that the amphipathic helices bound to membrane bilayers with micromolar to low nanomolar K D's. The presence of diverse endogenous neuropeptide transmitters and neuromodulators in the human brain is potentially applicable to the treatment of a wide range of behavioral disorders. © 2004 Elsevier Ltd. All rights reserved.
• Egleton, R., Mitchell, S., Huber, J., Janders, J., Stropova, D., Polt, R., Yamamura, H., Hruby, V., & Davis, T. (2005). Improved bioavailability to the brain of glycosylated Met-enkephalin analogs. BRAIN RESEARCH, 881(1), 37-46.
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  The blood-brain barrier prevents the entry of many potentially therapeutic peptide drugs to the brain. Glycosylation has shown potential as a methodology for improving delivery to the CNS. Previous studies have shown improved bioavailability and improved centrally mediated analgesia of glycosylated opioids. In this study we investigate the effect of glycosylation on the cyclic opioid peptide [D-Cys(2.5),Ser(6),Gly(7)] enkephalin. The peptide was glycosylated on the Ser(6) via an O-linkage with various sugar moieties and alignments. The peptides were then investigated for receptor binding, physiochemical attributes, in situ brain uptake in female Sprague-Dawley rats and antinociception in male ICR mice. Glycosylation resulted in a slight decrease in affinity to the delta -opioid receptor, and mixed effect on binding to the mu -opioid receptor. There was a significant decrease in lipophilicity resulting from glycosylation and a slight reduction in binding to bovine serum albumin. In situ perfusion showed that brain uptake was improved by up to 98% for several of the glycosylated peptides, and the nociceptive profiles of the peptides, in general, followed the rank order of peptide entry to the brain with up to a 39-fold increase in A.U.C. (C) 2000 Elsevier Science B.V. All rights reserved.
• PETERSON, M., & POLT, R. (2005). N-DIPHENYLMETHYLENE-PROTECTED GLYCOSYL ACCEPTORS - SELECTIVE BETA-O-GLYCOSYLATION TO FORM LACTOSYL-THREO-CERAMIDES. JOURNAL OF ORGANIC CHEMISTRY, 58(16), 4309-4314.
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  A series of N-diphenylmethylene-protected sphingosine derivatives was synthesized (e.g 3a,b and 9a,b). These compounds are efficient glycosyl acceptors and were shown to undergo highly beta-selective glycosylation using a modification of the Koenigs-Knorr reaction previously used in this laboratory for the synthesis of O-linked glycopeptides (none of the alpha-anomers were detected by either H-1 or C-13 NMR).1 The N-diphenylmethylene (Schiff base) protection imparts a favorable intramolecular hydrogen-bonding pattern (Ph2C=N:-->H-O:). This intramolecular hydrogen-bonding enhances the nucleophilicity of the glycosyl acceptor relative to glycosyl acceptors with more conventional N-protection (i.e. Cbz, Boc, acyl etc.). The enhanced nucleophilicity allowed the glycosylation to be carried out under mild conditions (AgOTfl, CH2Cl2, rt overnight), and provided the corresponding beta-glycosphingolipids 4a-c and 11a-d in excellent chemical yield (approximately 70%). After glycosylation, selective acid-catalyzed hydrolysis of the Schiff base protecting group was accomplished without cleavage of the glycosidic bond or the carbohydrate acetate protection. N-Acylation with palmitoyl chloride, followed by Zemplen deacetylation (cat. NaOMe in MeOH), provided the two threo-beta-lactosylceramide analogues 7a and 7b. These analogues possess the unnatural threo-configuration in the ceramide moiety and may prove useful in studies of the biosynthesis and cell surface composition of more complex glycosphingolipids.
• Polt, R., Dhanasekaran, M., & Keyari, C. M. (2005). Glycosylated neuropeptides: A new vista for neuropsychopharmacology?. Medicinal Research Reviews, 25(5), 557-585.
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  PMID: 16075406;Abstract: The application of endogenous neuropeptides (e.g., enkephalins) as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood-brain barrier (BBB). Effective BBB transport of glycosylated enkephalins has been demonstrated in several labs now. Analgesia (antinociception) levels greater than morphine, and with reduced side effects have been observed for several glycopeptides related to enkephalin. Somewhat paradoxically, enhanced BBB transport across this lipophilic barrier is achieved by attaching water-soluble carbohydrate groups to the peptide moieties to produce biousian glycopeptides that can be either water-soluble or membrane bound. Transport is believed to rely on an endocytotic mechanism (transcytosis), and allows for systemic delivery and transport of the water-soluble glycopeptides. Much larger endorphin/dynorphin glycopeptide analogs bearing amphipathic helix address regions also have been shown to penetrate the BBB in mice. This holds forth the possibility of transporting much larger neuropeptides across the BBB, which may encompass a wide variety of receptors beyond the opioid receptors. © 2005 Wiley Periodicals, Inc.
• Polt, R., Dhanasekaran, M., & Polt, R. L. (2005). New prospects for glycopeptide based analgesia: glycoside-induced penetration of the blood-brain barrier. Current drug delivery, 2(1).
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  Antinociceptive effects and BBB transport properties of glycosylated enkephalin derivatives are reviewed. Previously, the application of enkephalins as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood brain barrier. This shortcoming has been overcome by glycosylation, paradoxically leading to enhanced BBB transport via transcytosis. Principal design considerations required for enhanced binding, stability and transport of opioid peptides are reviewed. Modifications of the peptide backbone and side chains to achieve optimal receptor binding (micro/delta-selectivity) are presented. The importance of reversible binding between the glycopeptide and membranes is emphasized, and several pertinent examples of peptide-membrane interactions are discussed in the light of glycopeptide transport and opioid binding. An "amphipathic hypothesis" is introduced as a rationale for the observed BBB penetration of the opioid glycopeptides.
• Polt, R., Dhanasekaran, M., Palian, M. M., Alves, I., Yeomans, L., Keyari, C. M., Davis, P., Bilsky, E. J., Egleton, R. D., Yamamura, H. I., Jacobsen, N. E., Tollin, G., Hruby, V. J., Porreca, F., & Polt, R. L. (2005). Glycopeptides related to beta-endorphin adopt helical amphipathic conformations in the presence of lipid bilayers. Journal of the American Chemical Society, 127(15).
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  A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (BBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics. NOE volumes provided distance constraints for molecular dynamics calculations used to provide detailed backbone conformations. In all cases, the glycopeptides were largely helical in the presence of membrane bilayer models (micelles or bicelles). Plasmon waveguide resonance (PWR) studies showed hen egg phosphatidyl choline (PC) bilayers produce amphipathic helices laying parallel to the membrane surface, with dissociation constants (K(D)) in the low nanomolar to micromolar concentration range. Two low-energy states are suggested for the glycosylated endorphin analogues, a flexible aqueous state and a restricted membrane bound state. Strong interactions between the glycopeptide amphipaths and membranes are crucial for penetration of the BBB via an endocytotic mechanism (transcytosis).
• Abeytunga, D. T., Glick, J. J., Gibson, N. J., Oland, L. A., Somogyi, A., Wysoki, V. H., & Polt, R. (2004). Presence of unsaturated sphingomyelins and changes in their composition during the life cycle of the moth Manduca sexta. Journal of Lipid Research, 45(7), 1221-1231.
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  PMID: 15102888;Abstract: NMR and electrospray ionization tandem mass spectrometry were used to show for the first time the presence of sphingomyelins in extracts of the tobacco hornworm Manduca sexta (Lepidoptera). The sphingosine in the ceramide was identified as tetradecasphing-4-enine, and the fatty acids were C188:0, C20:0, C22:0, and C24:0 (compound 1). Heterogeneity in the ceramide was observed in sphingomyelins from M. sexta. All of the sphingomyelins were associated with their doubly unsaturated sphingosine, tetradecasphing-4,6-dienine (compound 2), which contained the same set of fatty acids as compound I and represents a novel set of sphingomyelins not previously reported in Lepidoptera. Lipid rafts were isolated from brains of M. sexta, and the association of these novel sphingomyelins with rafts was confirmed. The existence of the additional double bond was also observed in ceramide and ceramide phosphoethanolamine isolated from M. sexta. The levels of the doubly unsaturated ceramide showed modest changes during metamorphosis of M. sexta. These results suggest that Manduca sphingomyelins may participate in the formation of lipid rafts, in keeping with their function in vertebrates.
• Hruby, V. J., Yamamura, H. I., Schmid, W. R., Porreca, F., Polt, R., Palian, M. M., Navratilova, E., Lowery, J. J., Keyari, C. M., Hruby, V. J., Elmagbari, N. O., Egleton, R. D., Dhanasekaran, M., Davis, P., & Bilsky, E. J. (2004). Antinociceptive structure-activity studies with enkephalin-based opioid glycopeptides.. The Journal of pharmacology and experimental therapeutics, 311(1), 290-7. doi:10.1124/jpet.104.069393
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  Development of opioid peptides as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability. Glycosylation of opioid peptides can increase peptide serum stability and BBB penetration. To further define the requirements for optimizing in vivo antinociceptive potency following intravenous administration, we synthesized a series of enkephalin-based glycopeptides using solid phase 9-fluorenylmethyloxy carbamate methods. The compounds differed in the sixth and subsequent amino acid residues (Ser or Thr) and in the attached carbohydrate moiety. In vitro binding and functional smooth muscle bioassays indicated that the addition of mono- or disaccharides did not significantly affect the opioid receptor affinity or agonist activity of the glycopeptides compared with their unglycosylated parent peptides. All of the glycopeptides tested produced potent antinociceptive effects in male ICR mice following intracerebroventricular injection in the 55 degrees C tail-flick test. The calculated A(50) values for the Ser/Thr and monosaccharide combinations were all very similar with values ranging from 0.02 to 0.09 nmol. Selected compounds were administered to mice intravenously and tested for antinociception to indirectly assess serum stability and BBB penetration. All compounds tested produced full antinociceptive effects with calculated A (50) values ranging from 2.2 to 46.4 micromol/kg with the disaccharides having potencies that equaled or exceeded that of morphine on a micromoles per kilogram basis. Substitution of a trisaccharide or bis- and tris-monosaccharides resulted in a decrease in antinociceptive potency. These results provide additional support for the utility of glycosylation to increase central nervous system bioavailability of small peptides and compliment our ongoing stability and blood-brain barrier penetration studies.
• POLT, R., & PETERSON, M. (2004). BETA-AMINO ALCOHOLS FROM AMINO-ACIDS - CHELATION CONTROL VIA SCHIFF-BASES. TETRAHEDRON LETTERS, 31(35), 4985-4986.
• Polt, R., Abeytunga, D. T., Glick, J. J., Gibson, N. J., Oland, L. A., Somogyi, A., Wysocki, V. H., & Polt, R. L. (2004). Presence of unsaturated sphingomyelins and changes in their composition during the life cycle of the moth Manduca sexta. Journal of lipid research, 45(7).
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  NMR and electrospray ionization tandem mass spectrometry were used to show for the first time the presence of sphingomyelins in extracts of the tobacco hornworm Manduca sexta (Lepidoptera). The sphingosine in the ceramide was identified as tetradecasphing-4-enine, and the fatty acids were C18:0, C20:0, C22:0, and C24:0 (compound 1). Heterogeneity in the ceramide was observed in sphingomyelins from M. sexta. All of the sphingomyelins were associated with their doubly unsaturated sphingosine, tetradecasphing-4,6-dienine (compound 2), which contained the same set of fatty acids as compound 1 and represents a novel set of sphingomyelins not previously reported in Lepidoptera. Lipid rafts were isolated from brains of M. sexta, and the association of these novel sphingomyelins with rafts was confirmed. The existence of the additional double bond was also observed in ceramide and ceramide phosphoethanolamine isolated from M. sexta. The levels of the doubly unsaturated ceramide showed modest changes during metamorphosis of M. sexta. These results suggest that Manduca sphingomyelins may participate in the formation of lipid rafts, in keeping with their function in vertebrates.
• Polt, R., Slavish, J. P., Friel, D. K., Oland, L. A., & Polt, R. L. (2004). New PDMP analogues inhibit process outgrowth in an insect cell line. Bioorganic & medicinal chemistry letters, 14(6).
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  d-threo-1-Phenyl-2-aminodecanoyl-3-morpholinopropanol (d-threo-PDMP) has previously been shown to inhibit the biosynthesis of glycosphingolipids (GSLs) in mammals and mammalian cell lines by the inhibition of glucosylceramide synthase. New d-threo-PDMP analogues were synthesized from d-serine, and found to suppress neurite extension in an embryonic insect cell line from the moth Manduca sexta, and in explanted neural tissue from insect pupae. Inhibition occurred at lower concentrations than d-threo-PDMP. The observed suppression of neurite formation was found to be reversible after the removal of the compounds. Due to their small size and short life cycle, M. sexta is shown to be an ideal model organism for studies of GSL effects in cellular development, and for drug development studies.
• POLT, R., PORRECA, F., SZABO, L., BILSKY, E., DAVIS, P., ABBRUSCATO, T., DAVIS, T., HORVATH, R., YAMAMURA, H., & HRUBY, V. (2003). GLYCOPEPTIDE ENKEPHALIN ANALOGS PRODUCE ANALGESIA IN MICE - EVIDENCE FOR PENETRATION OF THE BLOOD-BRAIN-BARRIER. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 91(15), 7114-7118.
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  Most peptides have not proved useful as neuroactive drugs because they are blocked by the blood-brain barrier and do not reach their receptors within the brain. Intraperitoneally administered L-serinyl beta-D-glucoside analogues of [Met(5)]enkephalin (glycopeptides) have been shown to be transported across the blood-brain barrier to bind with targeted mu- and delta-opioid receptors in the mouse brain. The opioid nature of the binding has been demonstrated with intracerebroventricularly administered naloxone. Paradoxically, glucosylation decreases the lipophilicity of the peptides while promoting transport across the lipophilic endothelial layer. It is suggested that glucose transporter GLUT-1 is responsible for the transport of the peptide message. Profound and long-lasting analgesia has been observed in mice (tail-flick and hot-plate assays) with two of the glycopeptide analogues when administered intraperitoneally.
• Palian, M. M., Boguslavsky, V. I., O'Brien, D. F., & Polt, R. (2003). Glycopeptide-membrane interactions: Glycosyl enkephalin analogues adopt turn conformations by NMR and CD in amphipathic media. Journal of the American Chemical Society, 125(19), 5823-5831.
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  PMID: 12733923;Abstract: Four enkephalin analogues (Tyr-D-Thr-Gly-Phe-Leu-Ser-CONH2, 1, and the related O-linked glycopeptides bearing the monosaccharide β-glucose, 2, the disaccharide β-maltose, 3, and the trisaccharide β-maltotriose, 4) were synthesized, purified by HPLC, and biophysical studies were conducted to examine their interactions with membrane model systems. Glycopeptide 2 has been previously reported to penetrate the blood-brain barrier (BBB), and produce potent analgesia superior to morphine in mice (J. Med. Chem. 2000, 43, 2586-90 and J. Pharm. Exp. Ther. 2001, 299, 967-972). The parent peptide and its three glycopeptide derivatives were studied in aqueous solution and in the presence of micelles using 2-D NMR, CD, and molecular mechanics (Monte Carlo studies). Consistent with previous conformational studies on cyclic opioid agonist glycopeptides, it was seen that glycosylation did not significantly perturb the peptide backbone in aqueous solution, but all four compounds strongly associated with 5-30 mM SDS or DPC micelles, and underwent profound membrane-induced conformational changes. Interaction was also observed with POPC:POPE:cholesterol lipid vesicles (LUV) in equilibrium dialysis experiments. Although the peptide backbones of 1-4 possessed random coil structures in water, in the presence of the lipid phase they each formed a nearly identical pair of structures, all with a stable β-turn motif at the C-terminus. Use of spin labels (Mn2+ and 5-DOXYL-stearic acid) allowed for the determination of the position and orientation of the compounds relative to the surface of the micelle.
• Polt, R., Kelly, B. D., Dangel, B. D., Tadikonda, U. B., Ross, R. E., Raitsimring, A. M., & Astashkin, A. V. (2003). Optically active 4- and 5-coordinate transition metal complexes of bifurcated dipeptide Schiff bases. Inorganic Chemistry, 42(2), 566-574.
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  PMID: 12693240;Abstract: Symmetrical and unsymmetrical benzophenone Schiff bases of bifurcated dipeptides [e.g., Ar2C=N-CHR1CONH∼HNCO-CHR2-N= CAr2] have been synthesized using Boc methodology. These ligands may be regarded as chiral porphyrin mimics because of the α-carbons of the amino acids. The Schiff bases function as effective ligands for transition metals, particularly the late transition metals Ni(II), Cu(II), and Zn. Upon metal insertion, there is loss of the amide protons, resulting in N4 chelating ligands that retain the amino acid based chirality as well as newly generated metal-centered chirality, which for the Ni(II) complexes have been shown by X-ray analysis to be A (left-handed helix) if the amino acids are S. For Ni(II) and Cu(II), metal insertion results in highly colored complexes and is easily followed by UV-vis spectrophotometry. Several Ni(II) complexes were also characterized by 1H NMR. Co(II) and Mn(II) complexes were characterized by CW EPR. Two Cu(II) complexes, 7f-CuII and 7k-CuII, were characterized by EPR (ENDOR and ESEEM), which clearly showed the pentacoordinate nature of 7k-CurrII.
• Polt, R., Palian, M. M., Boguslavsky, V. I., O'Brien, D. F., & Polt, R. L. (2003). Glycopeptide-membrane interactions: glycosyl enkephalin analogues adopt turn conformations by NMR and CD in amphipathic media. Journal of the American Chemical Society, 125(19).
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  Four enkephalin analogues (Tyr-D-Thr-Gly-Phe-Leu-Ser-CONH(2), 1, and the related O-linked glycopeptides bearing the monosaccharide beta-glucose, 2, the disaccharide beta-maltose, 3, and the trisaccharide beta-maltotriose, 4) were synthesized, purified by HPLC, and biophysical studies were conducted to examine their interactions with membrane model systems. Glycopeptide 2 has been previously reported to penetrate the blood-brain barrier (BBB), and produce potent analgesia superior to morphine in mice (J. Med. Chem.2000, 43, 2586-90 and J. Pharm. Exp. Ther. 2001, 299, 967-972). The parent peptide and its three glycopeptide derivatives were studied in aqueous solution and in the presence of micelles using 2-D NMR, CD, and molecular mechanics (Monte Carlo studies). Consistent with previous conformational studies on cyclic opioid agonist glycopeptides, it was seen that glycosylation did not significantly perturb the peptide backbone in aqueous solution, but all four compounds strongly associated with 5-30 mM SDS or DPC micelles, and underwent profound membrane-induced conformational changes. Interaction was also observed with POPC:POPE:cholesterol lipid vesicles (LUV) in equilibrium dialysis experiments. Although the peptide backbones of 1-4 possessed random coil structures in water, in the presence of the lipid phase they each formed a nearly identical pair of structures, all with a stable beta-turn motif at the C-terminus. Use of spin labels (Mn(2+) and 5-DOXYL-stearic acid) allowed for the determination of the position and orientation of the compounds relative to the surface of the micelle.
• Stork, G., Shiner, C. S., Polt, R. L., & Cheng, C. W. (2002). Deprotonation of Chelating Enamines. Direct Formation of β‐Lithio Enamines.. ChemInform, 17(20). doi:10.1002/chin.198620113
• Dangel, B., Clarke, M., Haley, J., Sames, D., & Polt, R. (2001). Amino acid-derived ligands for transition metals: Catalysis via a minimalist interpretation of a metalloprotein. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 119(44), 10865-10866.
• Egleton, R. D., Mitchell, S. A., Huber, J. D., Palian, M. M., Polt, R., & Davis, T. P. (2001). Improved blood-brain barrier penetration and enhanced analgesia of an opioid peptide by glycosylation. Journal of Pharmacology and Experimental Therapeutics, 299(3), 967-972.
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  PMID: 11714884;Abstract: Neuropeptide pharmaceuticals have potential for the treatment of neurological disorders, but the blood-brain barrier (BBB) limits entry of peptides to the brain. Several strategies to improve brain delivery are currently under investigation, including glycosylation. In this study we investigated the effect of O-linked glycosylation on Ser6 of a linear opioid peptide amide Tyr-D-Thr-Gly-Phe-Leu-Ser-NH2 on metabolic stability, BBB transport, and analgesia. Peptide stability was studied in brain and serum from both rat and mouse by high-performance liquid chromatography. BBB transport properties were investigated by rat in situ perfusion. Tail-flick analgesia studies were performed on male ICR mice, injected i.v. with 100 μg of peptide ligand. Glycosylation of Ser6 of the peptide led to a significant increase in enzymatic stability in both serum and brain. Glycosylation significantly increased the BBB permeability of the peptide from a value of 1.0 ± 0.2 μl·min-1·g-1 to 2.2 ± 0.2 μl·min-1·g-1 (p < 0.05), without significantly altering the initial volume of distribution. Analgesia studies showed that the glycosylated peptide gave a significantly improved analgesia after i.v. administration compared with nonglycosylated peptide. The improved analgesia profile shown by the glycosylated peptide is due in part to an improvement in bioavailability to the central nervous system. The bioavailability is increased by improving stability and transport into the brain.
• Hruby, V. J., Pratt, M. R., Polt, R., Mitchell, S. A., & Hruby, V. J. (2001). Solid-phase synthesis of O-linked glycopeptide analogues of enkephalin.. The Journal of organic chemistry, 66(7), 2327-42. doi:10.1021/jo005712m
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  The synthesis of 18 N-alpha-FMOC-amino acid glycosides for solid-phase glycopeptide assembly is reported. The glycosides were synthesized either from the corresponding O'Donnell Schiff bases or from N-alpha-FMOC-amino protected serine or threonine and the appropriate glycosyl bromide using Hanessian's modification of the Koenigs-Knorr reaction. Reaction rates of D-glycosyl bromides (e.g., acetobromoglucose) with the L- and D-forms of serine and threonine are distinctly different and can be rationalized in terms of the steric interactions within the two types of diastereomeric transition states for the D/L and D/D reactant pairs. The N-alpha-FMOC-protected glycosides [monosaccharides Xyl, Glc, Gal, Man, GlcNAc, and GalNAc; disaccharides Gal-beta(1-4)-Glc (lactose), Glc-beta(1-4)-Glc (cellobiose), and Gal-alpha(1-6)-Glc (melibiose)] were incorporated into 22 enkephalin glycopeptide analogues. These peptide opiates bearing the pharmacophore H-Tyr-c[DCys-Gly-Phe-DCys]- were designed to probe the significance of the glycoside moiety and the carbohydrate-peptide linkage region in blood-brain barrier (BBB) transport, opiate receptor binding, and analgesia.
• Palian, M. M., & Polt, R. (2001). Lipo α-amino-β-hydroxy acids and O-linked glycosides: Building blocks for ceramyl and glycosphingoyl peptides. Journal of Organic Chemistry, 66(21), 7178-7183.
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  PMID: 11597248;
• Palian, M. M., Jacobsen, N. E., & Polt, R. (2001). O-linked glycopeptides retain helicity in water. Journal of Peptide Research, 58(2), 180-189.
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  PMID: 11532077;Abstract: A 17-residue O-linked glycopeptide model incorporating a central α-mannosyl serine residue, and its unglycosylated analog both demonstrate substantial helicity in water. The peptide sequence was derived from previous studies in which differences in overall helicity as a function of single amino acid substitutions were measured by circular dichroism (CD). The helical content was predicted by molecular modeling, and confirmed by CD and NMR. Moreover, the glycopeptide retained its helicity in the presence of SDS micelles, whereas the native peptide lost secondary structure in the presence of micelles. The inference is that the peptide sequence is a more important helix determinant than glycosylation per se.
• Polt, R., & Palian, M. M. (2001). Glycopeptide analgesics. Drugs of the Future, 26(6), 561-576.
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  Abstract: Excluding insulin, the global market for peptide pharmaceuticals is expected to grow from $350 million in 1999 to over $800 million by 2005 (1). Despite their advantages of potency, specificity and low toxicity, peptides have not gained the rapid acceptance by the pharmaceutical industry that one might have predicted in the 1970s, chiefly due to difficulties in peptide manufacture and poor bioavailability. Results from several studies now demonstrate that O-linked glycosylation of peptides can promote the serum stability of several classes of peptides in vivo, and can promote the transport of enkephalin analogs across the blood-brain barrier (BBB). Recent work with glycosylated enkephalin analogs suggests that the incorporation of a glycosylated serine or threonine residue promotes transport across the BBB, which resulted in an AUC increase of over 30-fold in rats. Antinociception assays with mice demonstrate that glycosylated enkephalins can compete with morphine in efficacy, even when administered peripherally. Proper placement of the glycoside moiety is necessary in order to avoid perturbation of the mu/delta selectivity of the parent peptide pharmacophore.
• Dangel, B. D., & Polt, R. (2000). Catalysis by amino acid-derived tetracoordinate complexes: Enantioselective addition of dialkylzincs to aliphatic and aromatic aldehydes. Organic Letters, 2(19), 3003-3006.
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  PMID: 10986093;Abstract: Me2Zn and Et2Zn added to aromatic and aliphatic aldehydes in the presence of 3 mol % of 2. (S)-1-Phenylethanol (91% ee) and (S)-1-phenylpropanol (86% ee) were synthesized from benzaldehyde and (S)-1-furan-2-yl-1-propanol (86% ee) from 2-furaldehyde. Nonanal and 3-phenylpropanal provided (S)-3-undecanol (96% ee) and (S)-1-phenyl-3-pentanol (94% ee). A solid-phase variant was effective with reduced ee's (e.g., 86% ee → 79% ee) for (S)-1-phenylpropanol.
• Elmagbari, N., Egleton, R., Palian, M., Lowery, J., Schmid, W., Davis, P., Navratilova, E., Dhanasekaran, M., Keyari, C., Yamamura, H., Porreca, F., Hruby, V., Polt, R., & Bilsky, E. (2000). Antinociceptive structure-activity studies with enkephalin-based opioid glycopeptides. JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 311(1), 290-297.
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  Development of opioid peptides as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability. Glycosylation of opioid peptides can increase peptide serum stability and BBB penetration. To further define the requirements for optimizing in vivo antinociceptive potency following intravenous administration, we synthesized a series of enkephalin-based glycopeptides using solid phase 9-fluorenylmethyloxy carbamate methods. The compounds differed in the sixth and subsequent amino acid residues (Ser or Thr) and in the attached carbohydrate moiety. In vitro binding and functional smooth muscle bioassays indicated that the addition of mono- or disaccharides did not significantly affect the opioid receptor affinity or agonist activity of the glycopeptides compared with their unglycosylated parent peptides. All of the glycopeptides tested produced potent antinociceptive effects in male ICR mice following intracerebroventricular injection in the 55degreesC tail-flick test. The calculated A(50) values for the Ser/Thr and monosaccharide combinations were all very similar with values ranging from 0.02 to 0.09 nmol. Selected compounds were administered to mice intravenously and tested for antinociception to indirectly assess serum stability and BBB penetration. All compounds tested produced full antinociceptive effects with calculated A(50) values ranging from 2.2 to 46.4 mumol/kg with the disaccharides having potencies that equaled or exceeded that of morphine on a micromoles per kilogram basis. Substitution of a trisaccharide or bis- and tris-monosaccharides resulted in a decrease in antinociceptive potency. These results provide additional support for the utility of glycosylation to increase central nervous system bioavailability of small peptides and compliment our ongoing stability and blood-brain barrier penetration studies.
• Hruby, V. J., Szabo, L. Z., Polt, R., Mitchell, S. A., Lou, B., Kriss, C. T., & Hruby, V. J. (2000). Enkephalin-based drug design: conformational analysis of O-linked glycopeptides by NMR and molecular modeling. Tetrahedron-asymmetry, 11(1), 9-25. doi:10.1016/s0957-4166(99)00544-3
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  Abstract 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.
• Hruby, V. J., Yamamura, H. I., Porreca, F., Polt, R., Palian, M. M., Mitchell, S. A., Jones, H., Janders, J., Huber, J. D., Hruby, V. J., Egleton, R. D., Davis, T. P., Davis, P., & Bilsky, E. J. (2000). Enkephalin glycopeptide analogues produce analgesia with reduced dependence liability.. Journal of medicinal chemistry, 43(13), 2586-90. doi:10.1021/jm000077y
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  Endogenous peptides (e.g. enkephalins) control many aspects of brain function, cognition, and perception. The use of these neuroactive peptides in diverse studies has led to an increased understanding of brain function. Unfortunately, the use of brain-derived peptides as pharmaceutical agents to alter brain chemistry in vivo has lagged because peptides do not readily penetrate the blood-brain barrier. Attachment of simple sugars to enkephalins increases their penetration of the blood-brain barrier and allows the resulting glycopeptide analogues to function effectively as drugs. The delta-selective glycosylated Leu-enkephalin amide 2, H(2)N-Tyr-D-Thr-Gly-Phe-Leu-Ser(beta-D-Glc)-CONH(2), produces analgesic effects similar to morphine, even when administered peripherally, yet possesses reduced dependence liability as indicated by naloxone-precipitated withdrawal studies. Similar glycopeptide-based pharmaceuticals hold forth the promise of pain relief with improved side-effect profiles over currently available opioid analgesics.
• Hruby, V. J., Yamamura, H. I., Stropova, D., Polt, R., Mitchell, S. A., Janders, J., Huber, J. D., Hruby, V. J., Egleton, R. D., & Davis, T. P. (2000). Improved bioavailability to the brain of glycosylated Met-enkephalin analogs.. Brain research, 881(1), 37-46. doi:10.1016/s0006-8993(00)02794-3
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  The blood-brain barrier prevents the entry of many potentially therapeutic peptide drugs to the brain. Glycosylation has shown potential as a methodology for improving delivery to the CNS. Previous studies have shown improved bioavailability and improved centrally mediated analgesia of glycosylated opioids. In this study we investigate the effect of glycosylation on the cyclic opioid peptide [D-Cys(2,5),Ser(6),Gly(7)] enkephalin. The peptide was glycosylated on the Ser(6) via an O-linkage with various sugar moieties and alignments. The peptides were then investigated for receptor binding, physiochemical attributes, in situ brain uptake in female Sprague-Dawley rats and antinociception in male ICR mice. Glycosylation resulted in a slight decrease in affinity to the delta-opioid receptor, and mixed effect on binding to the mu-opioid receptor. There was a significant decrease in lipophilicity resulting from glycosylation and a slight reduction in binding to bovine serum albumin. In situ perfusion showed that brain uptake was improved by up to 98% for several of the glycosylated peptides, and the nociceptive profiles of the peptides, in general, followed the rank order of peptide entry to the brain with up to a 39-fold increase in A.U.C.
• POLT, R., & SEEBACH, D. (2000). ALKYLATION OF IMIDAZOLIDINONE DIPEPTIDE DERIVATIVES - PREPARATION OF ENANTIOMERICALLY PURE DIPEPTIDES AND TRIPEPTIDES BY CHIRALITY TRANSFER VIA A PIVALALDEHYDE N,N-ACETAL CENTER. HELVETICA CHIMICA ACTA, 70(7), 1930-1936.
• Polt, R. (2000). Lipid Synthesis and Manufacture Edited by Frank D. Gunstone (Professor Emeritus, University of St. Andrews, Dundee, Scotland). CRC Press: Boca Raton. 1999. xv + 472 pp. $135.00. ISBN 0-8493-9737-5.. Journal of the American Chemical Society, 122(16), 3985-3985. doi:10.1021/ja995685l
• Razavi, H., & Polt, R. (2000). Asymmetric syntheses of (-)-8-epi-swainsonine triacetate and (+)-1,2-di-epi-swainsonine. Carbonyl addition thwarted by an unprecedented aza-pinacol rearrangement. JOURNAL OF ORGANIC CHEMISTRY, 65(18), 5693-5706.
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  Indolizidines(-)-8-epi-swainsonine triacetate and (+)-1,2-di-epi-swainsonine were synthesized from the O'Donnell Schiff base ester 1 derived from D-serine. Reductive-alkenylation of 1 with Bu-i(5)-Al2H/H2C=CHMgBr followed by substrate-directed dihydroxylation of the pendant allylic group with OsO4, reduction of imine, and cyclization with Ph3P/CCl4 gave the polyhydroxylated pyrrolidines 8a and 8b as advanced intermediates. Efficient protecting group manipulations converted pyrrolidines 8a and 8b to their corresponding partially protected analogues 10a and 10b, which upon Swern oxidation and diastereoselective Keck-type allylation with BF3. Et2O afforded the required three-carbon homologues (10a, >20:1 de; 10b, 3.5:1 de). Use of the chelating Lewis acid MgBr2 instead of BF3. Et2O with 10a led to a novel aza-pinacol rearrangement and allylation at the alpha-carbon to yield amino alcohol 17, which is similar to a hydride migration in the biosynthetic pathway of indolizidine alkaloids. Subsequent hydroboration, cyclization, and deprotection furnished (-)-8-epi-swainsonine triacetate 15a and (+)-1,2-di-epi-swainsonine 16b in good overall yields (6.3% for 1 --> 15a, 13 steps, and 4.0% for 1 --> 16b, 14 steps).
• Razavi, H., & Polt, R. (2000). Asymmetric syntheses of (-)-8-epi-swainsonine triacetate and (+)-1,2-di-epi-swainsonine. Carbonyl addition thwarted by an unprecedented aza-pinacol rearrangement. Journal of Organic Chemistry, 65(18), 5693-5706.
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  PMID: 10970313;Abstract: Indolizidines (-)-8-epi-swainsonine triacetate and (+)-1,2-di-epi-swainsonine were synthesized from the O'Donnell Schiff base ester 1 derived from D-serine. Reductive-alkenylation of 1 with (i)Bu5-Al2H/H2C=CHMgBr followed by substrate-directed dihydroxylation of the pendant allylic group with OsO4, reduction of imine, and cyclization with Ph3P/CCl4 gave the polyhydroxylated pyrrolidines 8a and 8b as advanced intermediates. Efficient protecting group manipulations converted pyrrolidines 8a and 8b to their corresponding partially protected analogues 10a and 10b, which upon Swern oxidation and diastereoselective Keck-type allylation with BF3·Et2O afforded the required three-carbon homologues (10a, > 20:1 de; 10b, 3.5:1 de). Use of the chelating Lewis acid MgBr2 instead of BF3·Et2O with 10a led to a novel aza-pinacol rearrangement and allylation at the α-carbon to yield amino alcohol 17, which is similar to a hydride migration in the biosynthetic pathway of indolizidine alkaloids. Subsequent hydroboration, cyclization, and deprotection furnished (-)-8-epi-swainsonine triacetate 15a and (+)-1,2-di-epi-swainsonine 16b in good overall yields (6.3% for 1 → 15a, 13 steps, and 4.0% for 1 → 16b, 14 steps).
• SAMES, D., & POLT, R. (2000). AN ENANTIOSELECTIVE SYNTHESIS OF N-METHYLFUCOSAMINE VIA TANDEM C-C C-O BOND FORMATION. JOURNAL OF ORGANIC CHEMISTRY, 59(16), 4596-4601.
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  Optically pure D-(+)-N-methylfucosamine (8) has been synthesized from a TBDMS-protected methyl L-serinate benzophenone Schiff base (O'Donnell's Schiff base, 1) in seven steps in 13% overall yield. Efficient construction-of the requisite amino triol acetate 3a with the proper stereochemical configuration is accomplished in two steps with a chelation-controlled reduction-alkylation reaction using \Bu(2)AlH.Al-iBu(3)/E-LiCH=CHCH3, followed by oxidation with OsO4. Conversion of the Schiff base to the N-benzhydryl protecting group and methylation (Eschweiler-Clark) is accomplished in one pot with NaCNBH3 in ti;e presence of H2C=O. Deprotection and oxidation of the primary alcohol, followed by deacetylation with KCN, provided the desired product 8.
• Polt, R., Sames, D., & Chruma, J. (1999). Glycosidase inhibitors: Synthesis of enantiomerically pure aza-sugars from Schiff base amino esters via tandem reduction-alkenylation and osmylation. Journal of Organic Chemistry, 64(17), 6147-6158.
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  Abstract: Nitrogen-in-the-ring 'aza-sugars' have been synthesized in enantiomerically pure form from the amino acid L-alanine in excellent overall yield. The O'Donnell's Schiff base of L-alanine methyl ester 9a was converted to aza-sugar L-fuco-1-deoxy-nojirimycin, 18, and to the epimer L-gulo-1- deoxy-nojirimycin, 20, in eight steps. The overall yields were 20 and 29%, respectively. The methodology for the efficient generation of silyl- and benzyl-protected (E)-3-lithio-2-propen-1-ols, and the use of these alkenyllithiums with iBu5Al2H as nucleophiles in the threo-selective tandem reduction-alkenylation of the Schiff base esters is described. Osmium- catalyzed cis-oxygenation of the resulting olefin products was selective for the galacto (fuco) amino polyols in all cases for the acyclic olefins, and was gulo-selective for the cyclic D-4,5-dihydropyridine pivalate, 17c. TEMPO- NaOCl was selective for oxidation of the primary position of the acyclic Schiff bases, and allowed for minimal protection/deprotection of the intermediates. The resulting N-benzhydryl heterocycles were easily deprotected with H2-Pd at atmospheric pressure.
• Mitchell, S. A., Oates, B. D., Razavi, H., & Polt, R. (1998). Glycosyltransferase inhibitors: Synthesis of D-threo-PDMP, L-threo- PDMP, and other brain glucosylceramide synthase inhibitors from D- or L- serine. Journal of Organic Chemistry, 63(24), 8837-8842.
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  Abstract: The synthesis of enantiomerically pure (1S,2S)-1-phenyl-2- decanoylamino-3-N-morpholino-1-propanol (L-threo-PDMP) (1a) from L-serine, and the enantiomer (LR,2R)-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP] (1b) from D-serine is reported. Reductive alkylation of the fully protected O'Donnell's Schiff base (3b) derived from D-serine provided the β-amino alcohol 5b in high yield and excellent selectivity, which yielded optically pure 1b in high yield after six steps. Three other D- threo-PDMP analogues with various amine groups have been synthesized using the same methodology, including the more potent pyrrolidine compound D- threo-PDPP (1e). A key feature of the synthesis is the isolation of tosylate (8b), which allows for the divergent synthesis of many analogues from a common advanced intermediate. The synthesis is amenable to large-scale production of D-threo-PDMP, L-threo-PDMP, and similar compounds.
• Razavi, H., & Polt, R. (1998). SYNTHESES OF IMINOLYXITOLS VIA TANDEM REDUCTION-ALKENYLATION OF O'DONNELL'S SCHIFF BASES. Tetrahedron Letters, 39(21), 3371-3374. doi:10.1016/s0040-4039(98)00495-x
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  Abstract The concise enantioselective synthesis of iminolyxitol glycosidase inhibitors starting from benzophenone imines of D-serine and L-alanine esters is very efficient. The reductive-alkenylation of the Schiff bases followed by substrate-directed dihydroxylation and amino dehydration gave the polyhydroxylated pyrrolidines in excellent overall yields (19.6% for 3 → 8 , 9.9% for 9 → 13 .)
• Razavi, H., & Polt, R. (1998). Syntheses of iminolyxitols via tandem reduction-alkenylation of O'Donnell's Schiff bases. Tetrahedron Letters, 39(21), 3371-3374.
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  Abstract: The concise enantioselective synthesis of iminolyxitol glycosidase inhibitors starting from benzophenone imines of D-serine and L-alanine esters is very efficient. The reductive-alkenylation of the Schiff bases followed by substrate-directed dihydroxylation and amino dehydration gave the polyhydroxylated pyrrolidines in excellent overall yields (19.6% for 3 → 8. 9.9% for 2 → 13.).
• Chruma, J. J., Sames, D., & Polt, R. (1997). General method for the synthesis of N-methyl amino acids and N-alkyl amino esters from O'Donnell's Schiff bases. Tetrahedron Letters, 38(29), 5085-5086.
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  Abstract: N-methyl amino acids, including L-abrine, and N-alkyl amino esters, were synthesized by reductive amination of O'Donnell's Schiff base amino esters with NaBH3CN and formaldehyde, or the appropriate aldehyde in CH3CN or THF in good to excellent yields, and with high purity.
• Chruma, J., Sames, D., & Polt, R. (1997). General method for the synthesis of N-methyl amino acids and N-alkyl amino esters from O'Donnell's Schiff bases. TETRAHEDRON LETTERS, 38(29), 5085-5086.
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  N-methyl amino acids, including L-abrine, and N-alkyl amino esters, were synthesized by reductive amination of O'Donnell's Schiff base amino esters with NaBH3CN and formaldehyde, or the appropriate aldehyde in CH3CN or THF in good to excellent yields, and with high purity. (C) 1997 Elsevier Science Ltd.
• Dangel, B., Clarke, M., Haley, J., Sames, D., & Polt, R. (1997). Amino acid-derived ligands for transition metals: Catalysis via a minimalist interpretation of a metalloprotein. Journal of the American Chemical Society, 119(44), 10865-10866.
• SZABO, L., RAMZA, J., LANGDON, C., & POLT, R. (1997). STEREOSELECTIVE SYNTHESIS OF O-SERINYL/THREONINYL-2-ACETAMIDO-2-DEOXY-ALPHA-GLYCOSIDES OR O-SERINYL/THREONINYL-2-ACETAMIDO-2-DEOXY-BETA-GLYCOSIDES. CARBOHYDRATE RESEARCH, 274, 11-28.
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  General glycosidation methodology has been developed which can selectively provide 2acetamido-2-deoxy-alpha- or beta-glycosides of beta-hydroxy-alpha-amino acid derivatives [glucopyranoside- (8, 43), galactopyranoside- (9, 13), mannopyranoside- (10), lactoside analogs (11, 38) and 3-O-beta-galactopyranosyl-mannopyranoside (12)] stereoselectively in excellent yield from the highly nucleophilic a-imino esters (Schiff bases) of L-serine and L-threonine. Various glycosides were converted via their amino and acetamido derivatives to Fmoc-protected serinyl- or threoninyl-glycosides (24-28, 37, 41, 46) which are all suitable building blocks for the solid-phase synthesis of O-glycopeptides. Complete H-1- and C-13-NMR data are provided for all compounds.
• Polt, R. L., Szabo, L., & Ramza, J. (1996). New chemical method for synthesis of O-linked glycopeptides. Postpy higieny i medycyny doświadczalnej, 50(5), 493-503.
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  PMID: 9072765;Abstract: The diphenylketone Shiff-base appeared to be a very good protection from amino groups of serine and threonine for glycosylation reaction. O-Glycosides of these amino acids with different monosaccharides, aminosugars and deoxysugars were obtained with excellent yield and very high stereoselectivity. Products of glycosylation reactions were used as building blocks for solid phase glycopeptide synthesis.
• Hruby, V. J., Yamamura, H. I., Williams, S. A., Szabo, L., Qian, X., Porreca, F., Polt, R., Patel, D. V., O'brien, D. F., Misicka, A., Lipkowski, A. W., Li, G., Hruby, V. J., Gillespie, T. J., Davis, T., Bonner, G. G., & Bartosz, H. (1995). DESIGN AND SYNTHESIS OF PEPTIDE LIGANDS WITH UNIQUE BIOCHEMICAL AND BIOLOGICAL PROFILES AT OPIOID RECEPTORS THAT CROSS THE BLOOD BRAIN BARRIER. Analgesia, 1(4), 469-472. doi:10.3727/107156995819562961
• Sames, D., & Polt, R. (1995). Piperidine Triols via Enantioselective Alkylation and Osmylation of Alanine Schiff Base Esters. Synlett, 1995(SI), 552-554. doi:10.1055/s-1995-5282
• Sames, D., Liu, Y., DeYoung, L., & Polt, R. (1995). Preparation of monosilyl acetals from esters via iBu₂AlH reduction and trapping with N-(trimethylsilyl)imidazole. Addition of allyltrimethylsilane to yield homoallylic alcohols or ethers. Journal of Organic Chemistry, 60(7), 2153-2159.
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  Abstract: Alkyl esters were reduced with iBu2AlH or a 1:1 mixture of iBu2AlH and iBu3Al (iBu2AlH·iBu3Al or iBu5Al2H), followed by trapping of the resulting tetrahedral intermediate with TMS-imidazole to produce monosilyl acetals. Reaction of the mixed acetals with allyltrimethylsilane in the presence of Lewis acids (Hosomi-Sakurai reaction) generated homoallylic alcohols or ethers selectively, depending on the substitution of the monosilyl acetal. TMS methoxy acetals (MeO-CH-OTMS) and TMS ethoxy acetals bearing additional complexing groups such as MeO- or Ph2C=N- provided alcohols with 1.5:1-9:1 threoselectivity, while simple TMS ethoxy acetals provided only ethyl ethers as products. The monosilyl acetal configuration was easily epimerized or racemized, and the configuration of the Hosomi-Sakurai product was apparently independent of the initial monosilyl acetal reactant configuration. © 1995 American Chemical Society.
• Szabó, L., Ramza, J., Langdon, C., & Polt, R. (1995). Stereoselective synthesis of O-serinyl/threoninyl-2-acetamido-2-deoxy-α- or β-glycosides. Carbohydrate Research, 274(C), 11-28.
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  PMID: 7585702;Abstract: General glycosidation methodology has been developed which can selectively provide 2-acetamido-2-deoxy-α- or β-glycosides of β-hydroxy-α-amino acid derivatives [glucopyranoside-(8, 43), galactopyranoside- (9, 13), mannopyranoside- (10), lactoside analogs (11, 38) and 3-O-β-galactopyranosyl-mannopyranoside (12)] stereoselectively in excellent yield from the highly nucleophilic α-imino esters (Schiff bases) of l-serine and l-threonine. Various glycosides were converted via their amino and acetamido derivatives to Fmoc-protected serinyl- or threoninyl-glycosides (24-28, 37, 41, 46) which are all suitable building blocks for the solid-phase synthesis of O-glycopeptides. Complete 1H- and 13C-NMR data are provided for all compounds. © 1995.
• Halfen, D., Apponi, A., Woolf, N., Polt, R., & Ziurys, L. (1994). A systematic study of glycolaldehyde in sagittarius B2(N) at 2 and 3 mm: Criteria for detecting large interstellar molecules. ASTROPHYSICAL JOURNAL, 639(1), 237-245.
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  A comprehensive study of glycolaldehyde (CH2OHCHO) has been conducted at 2 and 3 mm toward Sgr B2(N) using the Arizona Radio Observatory 12 m telescope. Forty favorable transitions of this species were observed in the range 68-169 GHz. Emission on the 20-70 mK level was detected at frequencies of 38 of these lines, including all transitions arising from the K-a = 0, 1, and 2 ladders. The two transitions not detected were weak and originate in the less populated K-a = 3 levels. Twenty-one percent of the detected lines are distinct, individual features. The remaining transitions are either contaminated by emission from abundant molecules or blended with equivalently weak features. The unblended transitions indicate V-LSR = 62.3 +/- 2.4 km s(-1) and Delta V-1/2 = 8.3 +/- 3.4 km s(-1), line parameters characteristic of organic species in Sgr B2(N). A rotational diagram yields a column density of 5.9 x 10(13) cm(-2) for glycolaldehyde, suggesting a fractional abundance of f (H-2) 5.9 x 10(-11). Observations of formaldehyde toward Sgr B2(N) suggest that H2CO and CH2OHCHO arise from the same gas with an abundance ratio of similar to 1/27. H2CO may function as the precursor to glycolaldehyde in a gas- phase "formose'' reaction. These observations, combined with past results of Hollis et al., provide convincing evidence for the presence of glycolaldehyde in the ISM. This study suggests that an extensive, self- consistent data set is necessary to identify large organic species in interstellar gas.
• Hruby, V. J., Yamamura, H. I., Szabo, L. Z., Porreca, F., Polt, R., Hruby, V. J., Harvath, R., Davis, T. P., Davis, P., Bilsky, E. J., & Abbruscato, T. J. (1994). Glycopeptide enkephalin analogues produce analgesia in mice: evidence for penetration of the blood-brain barrier.. Proceedings of the National Academy of Sciences of the United States of America, 91(15), 7114-8. doi:10.1073/pnas.91.15.7114
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  Most peptides have not proved useful as neuroactive drugs because they are blocked by the blood-brain barrier and do not reach their receptors within the brain. Intraperitoneally administered L-serinyl beta-D-glucoside analogues of [Met5]enkephalin (glycopeptides) have been shown to be transported across the blood-brain barrier to bind with targeted mu- and delta-opioid receptors in the mouse brain. The opioid nature of the binding has been demonstrated with intracerebroventricularly administered naloxone. Paradoxically, glucosylation decreases the lipophilicity of the peptides while promoting transport across the lipophilic endothelial layer. It is suggested that glucose transporter GLUT-1 is responsible for the transport of the peptide message. Profound and long-lasting analgesia has been observed in mice (tail-flick and hot-plate assays) with two of the glycopeptide analogues when administered intraperitoneally.
• Hruby, V. J., Yamamura, H. I., Szabo, L., Qian, X., Porreca, F., Polt, R., Misicka, A., Meyer, J. P., Lipkowski, A. W., Hruby, V. J., Haaseth, R. C., Davis, T. P., Collins, N., & Bartosz, H. (1994). New opioid compounds in analgesia. Regulatory Peptides, 53, S71-S72. doi:10.1016/0167-0115(94)90246-1
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  Increasing evidence has accumulated that there are subtypes of opioid receptors and that opioid receptors in the brain, spinal column and periphery have different structural requirements for bioactivity. We have sought to design peptide and peptide mimetic analogs that will interact specifically with δ or κ opioid receptor types and subtypes as well as the recently proposed μδ cx receptor. Using computer assisted design, conformational and topographical stereostructural considerations, asymmetric and macrocyclic synthetic chemistry, and multiple assays and binding methods (1), we have designed conformationally and topographically constrained ligands with high potency, selectivity and efficacy at δ 1 , δ 2 , μδ cx , k 1 and other opioid receptors
• Sames, D., & Polt, R. (1994). An enantioselective synthesis of N-methylfucosamine via tandem C-C/C-O bond formation. Journal of Organic Chemistry, 59(16), 4596-4601.
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  Abstract: Optically pure D-(+)-N-methylfucosamine (8) has been synthesized from a TBDMS-protected methyl L-serinate benzophenone Schiff base (O'Donnell's Schiff base, 1) in seven steps in 13% overall yield. Efficient construction of the requisite amino triol acetate 3a with the proper stereochemical configuration is accomplished in two steps with a chelation-controlled reduction - alkylation reaction using |Bu2AlH·Al-iBu3/E-LiCH=CHCH3, followed by oxidation with OsO4. Conversion of the Schiff base to the N-benzhydryl protecting group and methylation (Eschweiler-Clark) is accomplished in one pot with NaCNBH3 in the presence of H2C=O. Deprotection and oxidation of the primary alcohol, followed by deacetylation with KCN, provided the desired product 8. © 1994 American Chemical Society.
• Szabo, L., & Polt, R. (1994). Stereoselective synthesis of β-D-glycopyranosyl-L-serinate or - threoninate derivatives with an unusual migration. Carbohydrate Research, 258, 293-297.
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  PMID: 8039183;Abstract: Steroselective glycosylation is the most important aspect of complex carbohydrate synthesis. Generally, 1,0-acyl glycopyranoses have been used only as starting materials or intermediates for glycosylation reactions. In recent years, however, there has been increased interest in acyl-glycose derivatives with biological activity, i.e. glycossylated opioid peptides and derivatives of glucoronic acids. Our studies show that ZnCl2 can be an efficient promoter in the synthesis of serine or threonine 1,2-trans-O-acyl-derivatives of carbohydrates via migration.
• WEBER, S., ABBRUSCATO, T., BROWNSON, E., LIPKOWSKI, A., POLT, R., MISICKA, A., HAASETH, R., BARTOSZ, H., HRUBY, V., & DAVIS, T. (1994). ASSESSMENT OF AN IN-VITRO BLOOD-BRAIN-BARRIER MODEL USING SEVERAL [MET5]ENKEPHALIN OPIOID ANALOGS. JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 266(3), 1649-1655.
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  Confluent monolayers of primary and continuous passaged cultures of bovine brain microvessel endothelial cells (BMEC) have been suggested to model the blood-brain barrier (BBB). Increased lipophilicity has been previously suggested to increase BBB penetration. The intent of this study was to examine the effect that structural modifications of the [Met5]enkephalin analog DPDPE had on lipophilicity and passage across the BMEC. The BMEC consisted of a monolayer of confluent primary BMEC grown on polycarbonate (10 mum) filters. Permeability coefficients were calculated on the basis of the diffusion of peptides across the BMEC in a Side-Bi-Side(TM) diffusion chamber. Lipophilicity of the peptides examined was determined by using reversed-phase HPLC and calculating the capacity factor (k). Diffusion across the BMEC (for all peptides examined) was linear from 15 to 120 min; therefore, these time points were used to calculate permeability coefficients. Permeability coefficients ranged from 14.34 to 92.00 cm/min (x 10(-4)) , with [rho-ClPhe4,4']biphalin the highest. Analysis of variance coupled with the Newman-Keuls test showed significantly greater (P < .01) passage of select peptide analogs across the BMEC, including [rho-ClPhe4,4']biphalin, [rho-ClPhe4]DPDPE and reduced DPDPE. Interestingly, upon passage across the confluent monolayer, reduced DPDPE was converted to cyclized DPDPE. Calculated HPLC capacity factors ranged from 3.82 to 12.50. The most lipophilic peptide (highest) examined was acetylated Phe0-DPDPE. Analysis of the regression line of permeability coefficients plotted against capacity factors yielded a correlation coefficient of 0.745 (P < .01). The data provided in this study offer strong evidence that increasing peptide lipophilicity enhances passage across the BMEC. The greatest BMEC permeability coefficients, though not the greatest capacity factors, were obtained with peptides having a chlorohalogenation at the Phe4 residue, suggesting that factors other than lipophilicity may play a role in BMEC passage. Comparison of the permeability coefficients obtained from the BMEC system with those obtained from in vivo BBB studies suggest that the BMEC system may be very useful in predicting peptide (analog) passage across the in vivo BBB.
• Bruck, M. A., Wijayaratne, T., Polt, R., Li, Y., Collins, N., & Bruck, M. A. (1993). β-Alkoxy Schiff base–oxazolidine tautomerism: solid-state structure of N-diphenylmethylene-l-threonine methyl ester. Acta Crystallographica Section B-structural Science, 49(2), 316-320. doi:10.1107/s0108768192007778
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  The benzophenone Schiff base of methyl L-threonate, C 18 H 19 NO 3 , M r =297.36, crystallized as its cyclic methyl (4S,5R)-S-methyl-2,2-diphenyl-1,3-oxazolidine-4-carboxylate tautomer, orthorhombic, P2 1 2 1 2 1 , a=8.455 (2), b=12.648 (2), e=14.650 (3) A, V=1566.6 (5) A 3 , Z=4, D x =1.26 g cm -3 , λ(Mo Kα)=0.71073 A, μ=0.8 cm -1 , F(000)=632, T=296 K. The structure was solved by direct methods; R=0.040 and wR=0.045 for 1113 observed reflections. H atoms were included in the refinement, but were constrained to their attached atoms. The amine hydrogen (HN) was located from a difference map and was refined with a fixed isotropic thermal parameter
• Hruby, V. J., Weber, S. J., Polt, R., Misicka, A., Lipkowski, A. W., Hruby, V. J., Haaseth, R. C., Davis, T. P., Brownson, E. A., Bartosz, H., & Abbruscato, T. J. (1993). Assessment of an in vitro blood-brain barrier model using several [Met5]enkephalin opioid analogs.. The Journal of pharmacology and experimental therapeutics, 266(3), 1649-55.
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  Confluent monolayers of primary and continuous passaged cultures of bovine brain microvessel endothelial cells (BMEC) have been suggested to model the blood-brain barrier (BBB). Increased lipophilicity has been previously suggested to increase BBB penetration. The intent of this study was to examine the effect that structural modifications of the [Met5]enkephalin analog DPDPE had on lipophilicity and passage across the BMEC. The BMEC consisted of a monolayer of confluent primary BMEC grown on polycarbonate (10 microns) filters. Permeability coefficients were calculated on the basis of the diffusion of peptides across the BMEC in a Side-Bi-Side diffusion chamber. Lipophilicity of the peptides examined was determined by using reversed-phase HPLC and calculating the capacity factor (k). Diffusion across the BMEC (for all peptides examined) was linear from 15 to 120 min; therefore, these time points were used to calculate permeability coefficients. Permeability coefficients ranged from 14.34 to 92.00 cm/min (x 10(-4), with [rho-ClPhe4,4']biphalin the highest. Analysis of variance coupled with the Newman-Keuls test showed significantly greater (P < .01) passage of select peptide analogs across the BMEC, including [rho-ClPhe4,4']biphalin, [rho-ClPhe4]DPDPE and reduced DPDPE. Interestingly, upon passage across the confluent monolayer, reduced DPDPE was converted to cyclized DPDPE. Calculated HPLC capacity factors ranged from 3.82 to 12.50. The most lipophilic peptide (highest) examined was acetylated Phe0-DPDPE. Analysis of the regression line of permeability coefficients plotted against capacity factors yielded a correlation coefficient of 0.745 (P < .01). The data provided in this study offer strong evidence that increasing peptide lipophilicity enhances passage across the BMEC.(ABSTRACT TRUNCATED AT 250 WORDS)
• Maiorino, R. M., Aposhian, M. M., Xu, Z. -., Li, Y., Polt, R. L., & Aposhian, H. V. (1993). Determination and metabolism of dithiol chelating agents. XV. The meso- 2,3-dimercaptosuccinic acid-cysteine (1:2) mixed disulfide, a major urinary metabolite of DMSA in the human, increases the urinary excretion of lead in the rat. Journal of Pharmacology and Experimental Therapeutics, 267(3), 1221-1226.
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  PMID: 8263783;Abstract: Meso-2,3-dimercaptosuccinic acid (DMSA) in humans is an effective p.o. therapeutically useful chelating agent of Pb. In humans given DMSA p.o., the major urinary metabolite is DMSA-cysteine (1:2) mixed disulfide. In order to determine its efficacy in mobilizing Pb and increasing urinary Pb excretion, the mixed disulfide was given to rats treated previously with Pb acetate. The mixed disulfide was as effective as DMSA in increasing the urinary excretion of Pb and mobilizing Pb from the kidney. DMSA, however, appears to be superior for mobilizing Pb from the liver and the brain. After the mixed disulfide was given s.c. to rats, DMSA was found in the blood and urine. Twenty-four hours after administration, 0.7% of the administered mixed disulfide was found in the urine as DMSA, indicating the mixed disulfide can be reduced to DMSA. The mixed disulfide was also reduced in vitro to DMSA during incubation with rat blood. Although in the rat the DMSA-cysteine (1:2) mixed disulfide mobilized Pb from the kidney, increased the urinary excretion of Pb and was to some extent reduced to DMSA, its fate and pharmacological properties in the human, where it is found after DMSA administration, are unknown.
• POLT, R., SZABO, L., TREIBERG, J., LI, Y., & HRUBY, V. (1993). GENERAL-METHODS FOR ALPHA-O-SER/THR OR BETA-O-SER/THR GLYCOSIDES AND GLYCOPEPTIDES - SOLID-PHASE SYNTHESIS OF O-GLYCOSYL CYCLIC ENKEPHALIN ANALOGS. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 114(26), 10249-10258.
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  O-Linked glycopeptides have been efficiently synthesized using the highly nucleophilic alpha-imino esters (O'Donnell's Schiff bases) derived from L-serine (3a-c), L-threonine (4a,b), and a dipeptide ester (5). General methodology has been developed which can provide beta-glycosides of beta-hydroxy-alpha-amino acid derivatives 6-16 in excellent yield (63-94%) and excellent selectivity (>20:1) using Hanessian's modification or Helferich's modification of the Koenigs-Knorr reaction. Likewise, selective alpha-glycosylation has been achieved using the in situ anomerization methodology of Lemieux (28, 30). The increased nucleophilicity of the serine/threonine hydroxyl has been shown to be due to intramolecular hydrogen bonding to the N=CPh2 moiety. Deprotection of the intermediate Schiff bases has been demonstrated, and the glycosides have been incorporated into fully deprotected O-linked glycopeptides in high yield using either solution-phase peptide methods or solid-phase Fmoc-based technology. The potent glycosylenkephalin analogue 38 has been prepared using the solid-phase methodology.
• Peterson, M. A., & Polt, R. (1993). N-diphenylmethylene-protected glycosyl acceptors. Selective β-O-glycosylation to form lactosyl-threo-ceramides. Journal of Organic Chemistry, 58(16), 4309-4314.
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  Abstract: A series of N-diphenylmethylene-protected sphingosine derivatives was synthesized (e.g 3a,b and 9a,b). These compounds are efficient glycosyl acceptors and were shown to undergo highly β-selective glycosylation using a modification of the Koenigs-Knorr reaction previously used in this laboratory for the synthesis of O-linked glycopeptides (none of the α-anomers were detected by either 1H or 13C NMR).1 The N-diphenylmethylene (Schiff base) protection imparts a favorable intramolecular hydrogen-bonding pattern (Ph2C=N:→H-O:). This intramolecular hydrogen-bonding enhances the nucleophilicity of the glycosyl acceptor relative to glycosyl acceptors with more conventional N-protection (i.e. Cbz, Boc, acyl etc.). The enhanced nucleophilicity allowed the glycosylation to be carried out under mild conditions (AgOTfl, CH2Cl2, rt overnight), and provided the corresponding β-glycosphingolipids 4a-c and 11a-d in excellent chemical yield (approximately 70%). After glycosylation, selective acid-catalyzed hydrolysis of the Schiff base protecting group was accomplished without cleavage of the glycosidic bond or the carbohydrate acetate protection. N-Acylation with palmitoyl chloride, followed by Zemplèn deacetylation (cat. NaOMe in MeOH), provided the two threo-β-lactosylcerainide analogues 7a and 7b. These analogues possess the unnatural threo-configuration in the ceramide moiety and may prove useful in studies of the biosynthesis and cell surface composition of more complex glycosphingolipids. © 1993 American Chemical Society.
• SZABO, L., LI, Y., & POLT, R. (1993). O-GLYCOPEPTIDES - A SIMPLE BETA-STEREOSELECTIVE GLYCOSIDATION OF SERINE AND THREONINE VIA A FAVORABLE HYDROGEN-BONDING PATTERN. TETRAHEDRON LETTERS, 32(5), 585-588.
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  Glycosidation of serine and threonine is promoted by using an intramolecular hydrogen bond to the hydroxyl group.
• Dangel, B., & Polt, R. (1992). Catalysis by amino acid-derived tetracoordinate complexes: Enantioselective addition of dialkylzincs to aliphatic and aromatic aldehydes. ORGANIC LETTERS, 2(19), 3003-3006.
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  GRAPHICS
• Hruby, V. J., Treiberg, J., Szabo, L., Polt, R., Li, Y., & Hruby, V. J. (1992). General Methods for α- or β-O-Ser/Thr Glycosides and Glycopeptides. Solid-Phase Synthesis of O-Glycosyl Cyclic Enkephalin Analogues. Journal of the American Chemical Society, 114(26), 10249-10258. doi:10.1021/ja00052a022
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  O-Linked glycopeptides have been efficiently synthesis using the highly nucleophilic α-imino esters (O'Donnell's Schiff bases) derived from L-serine (3a-c), L-threonine (4a,b), and a dipeptide ester (5). General methodology has been developed which can provide β-glycosides of β-hydroxy-α-amino acid derivatives 6-16 in excellent yield (63-94%) and excellent selectivity (>20:1) using Hanessian's modification or Helferich's modification of the Koenigs-Knorr reaction.Likewise,selective α-glycosylation has been achieved using the in situ anomerization methodology of Lemieux.The increased nucleophilicity of the serine/threonine hydroxyl has been shown to be due to intramolecular hydrogen bonding to the N=CPh 2 moiety.Deprotection of the intermediate Schiff bases has been demonstrated ,and the glycosides have been incorporated into fully deprotected O-linked glycopeptides in high yield using either solution-phase peptide methods or solid-phase Fmoc-based technology.An potent glycosylenkephalin analogue has been prepared using the solid-phase methodology
• Peterson, M. A., & Polt, R. (1992). E-1-lithio-1-alkenes in hydrocarbon solvent. Synthetic Communications, 22(3), 477-480.
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  Abstract: Trans-alkenyl iodides are treated with tert-butyllithium in hexane at room temperature to provide pur trans-alkyllithiums in good yield.
• Polt, R., & Peterson, M. A. (1992). E-1-LITHIO-1-ALKENES IN HYDROCARBON SOLVENT. Synthetic Communications, 22(3), 477-480. doi:10.1080/00397919208055426
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  Abstract Trans-alkenyl iodides are treated with tert-butyllithium in hexane at room temperature to provide pure trans-alkenyllithiums in good yield.
• Polt, R., Peterson, M. A., & Deyoung, L. (1992). Aluminoxy acetals from α-amino esters: Chirality transfer via sequential addition of hydride and C-nucleophiles. 2-Amino alcohols and sphingosines. Journal of Organic Chemistry, 57(20), 5469-5480.
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  Abstract: The reaction of α-imino esters (O'Donnell's Schiff bases) with aluminum hydrides to produce acetal-like intermediates and subsequent reaction with carbon nucleophiles has been studied. Treatment of optically pure imine-protected amino esters with iBu2AlH or iBu2AlH·Bu3Al, followed by RMgX or RLi provided threo-2-amino alcohols in high yield (73-85%) and excellent "syn" stereoselectivity (8:1 to >20:1, threo or like product preferred). Use of nonpolar solvents (CH2Cl2-hexane) provided the highest stereoselectivities. Use of the less-reactive iBu2AlH·iBu3Al complex lowered the amount of undesired primary alcohol products observed. Thermally labile aluminoxy acetal intermediates were observed by 1H NMR and were trapped with N-(trimethylsilyl)imidazole to produce relatively stable monosilyl acetals (mixed acetals). Alanine-derived Schiff bases 2a-e showed a correlation between the steric bulk of the ester and threo selectivity. The presence of THF reduced this correlation, suggesting the C-nucleophile addition involves a Lewis acid-assisted SN2-like displacement of the aluminoxy acetal or displacement of a tight-ion pair. In addition to the synthesis of optically pure arylethanolamines 6a-d from representative amino acids, threo-sphingosines 8a-d were synthesized from L-serine-derived Schiff base 4b, and 1-deoxy-threo-sphingosines 9a-d were synthesized from L-alanine in a similar fashion. Experimental details are provided. © 1992 American Chemical Society.
• Polt, R., Yushun, L. i., Fernando, Q., & Rivera, M. (1992). A synthetic method for unsymmetrical disulfides of cysteine: the bis-cysteine disulfide of meso-2,3-dimercaptosuccinic acid.. Tetrahedron Letters, 33(21), 2961-2964.
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  Abstract: meso-Dimercaptosuccinic acid (DMSA) is the drug of choice for the treatment of lead-poisoning. DMSA is excreted in the urine of lead-poisoned rabbits as a conjugate with two molecules of cysteine. To confirm this, and to examine the hypothesis that DMSA may be acting as a pro-drug, we have synthesized the DMSA-bis-disulfide using novel methodology based on the use of sulfenimides derived from cysteine. © 1992.
• Egleton, R., Mitchell, S., Huber, J., Palian, M., Polt, R., & Davis, T. (1991). Improved blood-brain barrier penetration and enhanced analgesia of an opioid peptide by glycosylation. JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 299(3), 967-972.
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  Neuropeptide pharmaceuticals have potential for the treatment of neurological disorders, but the blood-brain barrier (BBB) limits entry of peptides to the brain. Several strategies to improve brain delivery are currently under investigation, including glycosylation. In this study we investigated the effect of O-linked glycosylation on Ser(6) of a linear opioid peptide amide Tyr-D-Thr-Gly-Phe-Leu-Ser-NH2 on metabolic stability, BBB transport, and analgesia. Peptide stability was studied in brain and serum from both rat and mouse by high-performance liquid chromatography. BBB transport properties were investigated by rat in situ perfusion. Tall-flick analgesia studies were performed on male ICR mice, injected i.v. with 100 mug of peptide ligand. Glycosylation of Ser(6) of the peptide led to a significant increase in enzymatic stability in both serum and brain. Glycosylation significantly increased the BBB permeability of the peptide from a value of 1.0 +/- 0.2 mul . min(-1) . g(-1) to 2.2 +/- 0.2 mul . min(-1) . g(-1) (p < 0.05), without significantly altering the initial volume of distribution. Analgesia studies showed that the glycosylated peptide gave a significantly improved analgesia after i.v. administration compared with nonglycosylated peptide. The improved analgesia profile shown by the glycosylated peptide is due in part to an improvement in bioavailability to the central nervous system. The bioavailability is increased by improving stability and transport into the brain.
• Polt, R., & Wijayarathe, T. (1991). Erythrose sesqui-acetals as electrophiles. 2-Deoxy-C-nucleosides from D-glucose.. Tetrahedron Letters, 32(37), 4831-4834.
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  Abstract: Cleavage of glucosides with NalO4 in MeOH furnishes sesqui-acetals (protected dialdehydes). Olefination of these substrates, followed by endocyclic oxymercuration-demercuration furnishes substituted tetrahydrofurans (1,2-dideoxy-1-aryl-D-ribofuranoses). Proper choice of protecting groups can affect the face selectivity of the oxymercuration step to provide only the desired β-C-anomer. © 1991.
• Szabò, L., Yushun, L. i., & Polt, R. (1991). O-glycopeptides: a simple β-stereoselective glycosidation of serine and threonine via a favorable hydrogen bonding pattern.. Tetrahedron Letters, 32(5), 585-588.
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  Abstract: Glycosidation of serine and threonine is promoted by using an intramolecular hydrogen bond to the hydroxyl group. © 1992.
• Polt, R., & Peterson, M. A. (1990). β-Amino alcohols from amino acids: Chelation control via schiff bases.. Tetrahedron Letters, 31(35), 4985-4986.
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  Abstract: Sequential addition of iBu2AIH and RLi or RMgX to Schiff base esters derived from amino acids provides a simple route to β-amino alcohols. The reaction procedes without racemization, and with high threo selectivity. Several representative sphingosines are synthesized. © 1990.
• Polt, R., & Seebach, D. (1989). Stereoselective alkylation of glycine units in dipeptide derivatives: "Chirality transfer" via a pivalaldehyde N,N-acetal center. Journal of the American Chemical Society, 111(7), 2622-2632.
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  Abstract: Dipeptide esters (of glycylglycine, glycylalanine, alanylglycine) and aldehydes (isobutyraldehyde, pivalaldehyde, benzaldehyde) are condensed to (4-oxoimidazolidin-3-yl)acetates and -propionates (1-3). Lithium enolates of these derivatives are generated (deprotonation of the ring and/or side-chain α-carbonyl positions) with LDA, LDA/LiBr, or LHMDS and alkylated with high diastereoselectivity (products 4, 6, 7). Dipeptides of either R,R or S,S configuration can be prepared from the glycine-containing precursors. Surprising proton-transfer effects (Schemes VI-IX, XII) are interpreted as a consequence of "intimate complexation" among LDA, LiBr, lithium enolates, and diisopropylamine. © 1989 American Chemical Society.
• O'Donnell, M. J., Bennett, W. D., Bruder, W. A., Jacobsen, W. N., Knuth, K., LeClef, B., Polt, R. L., Bordwell, F. G., Mrozack, S. R., & Cripe, T. A. (1988). Acidities of glycine Schiff bases and alkylation of their conjugate bases. Journal of the American Chemical Society, 110(25), 8520-8525.
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  Abstract: Equilibrium acidities in Me2SO are reported for six ketimines of the type Ph2C=NCH(R)CO2Et and five aldimines, ArCH=NCH(R)CO2Et. Changing R in the ketimine from H to Ph increased the pKa by 2.2 units. This surprising acidity decrease for Ph substitution points to a substantial increase in steric effect, as do the increases in pKa of 3.8 and 4.2 units observed for the replacement of hydrogen by Me and PhCH2, respectively. Phase-transfer alkylation of the Ph2C=NCH2CO2Et ketimine gave over 90% of monoalkylate whereas, under similar conditions, the aldimine 4-ClC6H4CH=NCH2CO2Et gave a mixture of mono- and dialkylate. The difference is that the pKa of the monoalkylated aldimine is essentially the same as that of the parent, which leads to rapid equilibration with the parent anion and consequent dialkylation. The rates of alkylation in Me2SO of these parent and monoalkylated anions did not differ greatly, showing that the relative pKHAs of the parent acid and its monoalkyl derivative, rather than the relative rates of the mono- and dialkylation reactions, is the principal factor that determines the extent of the competition between monoalkylation and dialkylation. © 1988 American Chemical Society.
• Stork, G., Polt, R. L., Yi, L. i., & Houk, K. N. (1988). Theoretical studies of the lithiation of enamines. Journal of the American Chemical Society, 110(25), 8360-8367.
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  Abstract: In order to probe the origin of regioselectivity of the lithiation of enamines with alkyllithium reagents in hydrocarbon solvents, ab initio calculations were carried out on the reactions of lithium hydride and methyllithium with a series of vinylamines at the 3-21G level. The calculations indicate that the reactions occur via a cyclic transition state in which the base attacks the acidic proton in an almost collinear (anion-proton-base angle = 155-166°) fashion regardless of the ring size. A favorable transition state involves the achievement of both the stereoelectronic requirement for deprotonation and stabilizing coordination of the lithium cation with the base, the nitrogen of the enamine, and the developing anionic center. The rates of deprotonation are dependent upon favorable Li+ coordination, not anion stability. There is good qualitative accord with experimental deprotonation preferences and with 13C spectral changes upon coordination of n-BuLi with the enamines. © 1988 American Chemical Society.
• POLT, R., & SEEBACH, D. (1987). STEREOSELECTIVE ALKYLATION OF GLYCINE UNITS IN DIPEPTIDE DERIVATIVES - CHIRALITY TRANSFER VIA A PIVALALDEHYDE N,N-ACETAL CENTER. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 111(7), 2622-2632.
• Seebach, D., & Polt, R. (1987). Alkylation of Imidazolidinone Dipeptide Derivatives: Preparation of Enantiomerically Pure Di‐ and Tripeptides by ‘Chirality Transfer’ via a Pivalaldehyde N,N‐Acetal Center. Preliminary Communication. Helvetica Chimica Acta, 70(7), 1930-1936. doi:10.1002/hlca.19870700727
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  Glycylglycine, glycyl-(S)-alanine, and (S)-alanylglycine esters are cyclized through pivalaldehyde imines to give dipeptide-derived 3-(benzyloxycarbonyl)-2-(tert-butyl)-5-oxoimidazolidine-1-acetates 1–3. These are alkylated diastereoselectively by Li-enolate formation and addition of alkyl bromides or iodides (products 4–6). Starting from (S)-alanine and glycine, (S)-alanyl-(S)-alanine or (R)-alanyl-(R)-alanine, and (R)-alanyl-(R)alanyl-(S)-alanine- have thus been prepared, with the (tert-butyl)-substituted N,N-acetal center playing the role of a pivot or lever for diastereoselective formation of new stereogenic centers under kinetic or thermodynamic control.
• Stork, G., Shiner, C. S., Cheng, C., & Polt, R. L. (1986). Deprotonation of chelating enamines. Direct formation of β-lithio enamines. Journal of the American Chemical Society, 108(2), 304-305.
• O'Donnell, M. J., Bennett, W. D., & Polt, R. L. (1985). Preparation of an electrophilic glycine cation equivalent and its reaction with heteroatom nucleophiles. Tetrahedron Letters, 26(6), 695-698.
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  Abstract: A variety of heteroatom substituted Schiff base amino esters 4 - 7 are prepared either from the benzophenone imine of glycine ethyl ester (3) or by reaction of acetate 4 with heteroatom nucleophiles. © 1985.
• Polt, R. L., O'donnell, M. J., Falmagne, J. B., & Bennett, W. D. (1985). Preparation of an Electrophilic Glycine Cation Equivalent and Its Reaction with Heteroatom Nucleophiles. The Synthesis of Amino Acids by Reaction of an Electrophilic Glycine Cation Equivalent with Carbon Nucleophiles.. ChemInform, 16(25). doi:10.1002/chin.198525356
• Polt, R. L., Stork, G., Carpenter, G. B., & Williard, P. G. (1984). X-ray crystal structure of N-(2-lithiocyclohexenyl)-N,N′,N′-trimethyl-1,3-propanediamine: A pentavalent lithium?. Journal of the American Chemical Society, 106(15), 4276-4277.
• Williard, P. G., Stork, G., Polt, R. L., & Carpenter, G. B. (1984). X-RAY CRYSTAL STRUCTURE OF N-(2-LITHIOCYCLOHEXENYL)-N,N′,N′-TRIMETHYL-1,3-PROPANEDIAMINE: A PENTAVALENT LITHIUM?. ChemInform, 15(45). doi:10.1002/chin.198445057
• O'Donnell, M. J., & Polt, R. L. (1982). A mild and efficient route to Schiff base derivatives of amino acids. Journal of Organic Chemistry, 47(13), 2663-2666.

Proceedings Publications

• Collins, K., Hay, M., Ledford, J., Mansour, H., Polt, R., & Younis, U. (2021).

  Angiotensin (Ang-1-7) and Its Glycopeptide PNA5 Protect Against Overzealous Pulmonary Inflammation to Infectious and Non-Infectious Respiratory Insults

  . In American Journal of Respiratory and Critical Care Medicine.
• Apponi, A. J., Halfen, D. T., Polt, R. L., Abrell, L. M., Ziurys, L. M., Ziurys, L. M., & Halfen, D. T. (2005). INVESTIGATING THE INTERSTELLAR CHEMISTRY LEADING TO SIMPLE SUGARS. In Astrochemistry Conference.

Presentations

• Eismin, R. J., Palos Pacheco, R., Munusamy, E., Hogan, D. E., Maier, R. M., Polt, R. L., Schwartz, S. D., & Pemberton, J. E. (2016, Summer). Microenvironment of Monorhamnolipid Aggregates and their Synthetically Produced Diastereomers as a Function of Solution Conditions. 252nd American Chemical Society National Meeting & Exposition. Philadelphia, PA: American Chemical Society.
• Kegel, L. L., Szabo, L., Polt, R. L., & Pemberton, J. E. (2016, Summer). Alkyl thioglycoside green surfactant properties: Effects of various disaccharide and monosaccharide headgroup and alkyl tail length. 252nd American Chemical Society National Meeting & Exposition. Philadelphia, PA: American Chemical Society.
• Palos Pacheco, R., Kegel, L. L., Coss, C. S., Polt, R. L., & Pemberton, J. E. (2016, Summer). Structure-function relationships of bio-inspired rhamnolipid surfactants. 252nd American Chemical Society National Meeting & Exposition. Philadelphia, PA: American Chemical Society.
• Palos Pacheco, R., Kegel, L. L., Gonzalez, R., Polt, R. L., & Pemberton, J. E. (2016, Summer). Exploitation of chitin as a renewable feedstock for the synthesis of cationic and amphoteric glucosaminoside surfactants and their characterization. 252nd American Chemical Society National Meeting & Exposition. Philadelphia, PA: American Chemical Society.
• Pemberton, J. E., Polt, R. L., Schwartz, S. D., Maier, R. M., & Klimecki, W. (2016, Summer). Biosynthetic and Bio-inspired Glycolipid Surfactants: Properties, Biodegradability, and Toxicity. 20th Annual Green Chemistry & Engineering Conference. Portland, OR: American Chemical Society Green Chemistry Institute.
• Eismin, R. J., Palos-Pacheco, R., Coss, C. S., Polt, R. L., Maier, R. M., Pemberton, J. E., Eismin, R. J., Palos-Pacheco, R., Coss, C. S., Polt, R. L., Maier, R. M., & Pemberton, J. E. (2015, Spring). Aggregation Characteristics of Rhamnolipid Biosurfactants and Their Synthetic Variants. 106th Annual Meeting of the American Oil Chemists Society. Orlando, FL: Americanb Oil Chemists Society.
• Kegel, L. L., Szabo, L., Polt, R. L., Pemberton, J. E., Kegel, L. L., Szabo, L., Polt, R. L., & Pemberton, J. E. (2015, Spring). Properties of Novel Bio-inspired Glycolipid Surfactants: Tailoring Function by Disaccharide Headgroup and Alkyl Tail Length. 106th Annual Meeting of the American Oil Chemists Society. Orlando, FL: American Oil Chemists Society.
• Pemberton, J. E., Palos-Pacheco, R., Coss, C. S., & Polt, R. L. (2015, Spring). Tailoring Rhamnolipid Biosurfactant Properties through Production by Chemical Synthesis. 106th Annual Meeting of the American Oil Chemists Society. Orlando, FL: American Oil Chemists Society.
• Pemberton, J. E., Palos-Pacheco, R., Coss, C. S., Gonzalez, R., Szabo, L., Hogan, D. E., Tian, F., Maier, R. M., & Polt, R. L. (2015, Winter). Tailoring Rhamnolipid and Related Glycolipid Surfactant Properties through Sustainable Production by Green Chemical Synthesis. The International Chemical Congress of Pacific Basin Societies 2015 (PacifiChem). Honolulu, HI.

Poster Presentations

• Madhavan, L., Falk, T., Polt, R. L., Streicher, J. M., Szabò, L. Z., Apostol, C. R., Tanguturi, P., Bartlett, M. J., Saez, J. L., Corenblum, M. J., Lujan, A., & Bernard, K. (2022, Spring). Multimodal effects of systemic PACAP glycopeptide delivery in rodent models of Parkinson’s disease.. 16th International Conference on Alzheimer’s and Parkinson’s Diseases and related neurological disorders, AD/PD™.
• Falk, T., Heien, M. L., Polt, R. L., Streicher, J. M., Madhavan, L., Sherman, S. J., Szabo, L., Apostol, C., Molnar, G., Liu, C., Bartlett, M. J., & Bernard, K. (2021, January). Evaluation of a neuroprotective PACAP glycopeptide as systemically delivered CNS active drug to treat Parkinson’s disease.. Society for Neuroscience Global Connectome Conference.
• Falk, T., Heien, M. L., Polt, R. L., Streicher, J. M., Madhavan, L., Sherman, S. J., Szabo, L., Apostol, C., Molnar, G., Liu, C., Bartlett, M. J., Bernard, K., Falk, T., Heien, M. L., Polt, R. L., Streicher, J. M., Madhavan, L., Sherman, S. J., Szabo, L., , Apostol, C., et al. (2021, Spring). Evaluation of a neuroprotective PACAP glycopeptide as systemically delivered CNS active drug to treat motor and cognitive symptoms in two rodent models of Parkinson’s disease.. 15th International Conference on Alzheimer’s and Parkinson’s Diseases and related neurological disorders, AD/PD™.
• Falk, T., Heien, M. L., Polt, R. L., Streicher, J. M., Madhavan, L., Sherman, S. J., Szabò, L., Apostol, C. R., Molnar, G., Liu, C., Bartlett, M. J., Morrison, H. W., Lujan, A., & Bernard, K. (2021, Fall). Evaluation of a systemically delivered PACAP glycopeptide as a neuroprotective agent in 2 rodent models of Parkinson’s Disease.. Society for Neuroscience Meeting.
• Hay, M., Doyle, K., Ossanna, N., Falk, T., Bartlett, M., Polt, R. L., Mansour, H. M., Konhilas, J. P., Hoyer-Kimura, C., Hay, M., Doyle, K., Ossanna, N., Falk, T., Bartlett, M., Polt, R. L., Mansour, H. M., Konhilas, J. P., & Hoyer-Kimura, C. (2021, March/Spring). Novel Therapeutic and Inflammatory Biomarkers for Vascular Contributions to Cognitive Impairment and Dementia.. AD/PD 2021 15th International Conference on Alzheimer’s and Parkinson’s Diseases Virtual ConferenceAD/PD.
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  Oral e-Poster presentation
• Falk, T., Polt, R. L., Heien, M. L., Streicher, J. M., Ronaldson, P. T., Szabo, L., Molnar, G., Bartlett, M. J., Liu, C., Apostol, C., & Bernard, K. (2020, Summer). Preclinical evaluation of glycosylated PACAP Hormones to treat Parkinson’s disease and Stroke.. Arizona Alzheimer’s Consortium Annual Abstracts, 2020.
• Falk, T., Polt, R. L., Heien, M. L., Streicher, J. M., Ronaldson, P. T., Szabo, L., Molnar, G., Bartlett, M. J., Liu, C., Apostol, C., & Bernard, K. (2020, Summer). Preclinical evaluation of glycosylated PACAP Hormones to treat Parkinson’s disease and Stroke.. Arizona Alzheimer’s Consortium Annual Abstracts.
• Heien, M. L., Falk, T., Polt, R. L., Szabo, L., Apostol, C., Bartlett, M. J., & Liu, C. (2020, Fall). Glycosylation improves stability of neuropeptides and elevates blood brain barrier (BBB) penetration.. ASMS Conference on Mass Spectrometry and Allied Topics Abstracts.
• Polt, R. L., Heien, M. L., Falk, T., Streicher, J. M., Ronaldson, P. T., Rowe, R., Szabo, L., Molnar, G., Bernard, K., Bartlett, M. J., Liu, C., & Apostol, C. (2020, Summer). Glycosylated PACAP Hormones as Potential Therapy for Parkinsonism, Stroke and Traumatic Brain Injury.. American Chemical Society National Meeting.
• Heien, M. L., Polt, R. L., Falk, T., Szabo, L., Hanrahan, D., Smith, C. L., Bartlett, M. J., & Liu, C. (2018, Summer). Blood brain barrier penetration of glycosylated peptides by ‘shotgun microdialysis’ coupled with LC-MS3. ASMS Conference on Mass Spectrometry and Allied Topics Abstracts.
• Polt, R. L., Streicher, J. M., Heien, M. L., Falk, T., Molnar, G., Bartlett, M. J., Szabo, L., Liu, C., & Apostol, C. C. (2018, Fall). Design and Synthesis of Brain Penetrant Glycopeptide Analogues of Pituitary Adenylate Cyclase Activating Peptide (PACAP) for the Treatment of Parkinson’s Disease. Peptide Therapeutic Symposium at the Salk Institute in San Diego.
• Polt, R. L., Szabo, L., Smith, C. L., Liu, C., Molnar, G., Heien, M. L., Bartlett, M. J., Apostol, C. R., Hay, M., Falk, T., & Streicher, J. M. (2018, Fall). Glycopeptides as Systemically Delivered CNS Active Drugs from Endogenous Peptide Hormones. Society for Neuroscience Meeting.
• Polt, R. L., Szabo, L., Smith, C. L., Liu, C., Molnar, G., Heien, M. L., Bartlett, M. J., Apostol, C. R., Hay, M., Falk, T., Streicher, J. M., Polt, R. L., Szabo, L., Smith, C. L., Liu, C., Molnar, G., Heien, M. L., Bartlett, M. J., Apostol, C. R., , Hay, M., et al. (2018, Fall). Glycopeptides as Systemically Delivered CNS Active Drugs from Endogenous Peptide Hormones. Society for Neuroscience.
• Kramer, C., Heien, M. L., Bartlett, M. J., Falk, T., Jones, E. M., Polt, R. L., Stagg, C. J., Stagg, C. J., Jones, E. M., Polt, R. L., Bartlett, M. J., Falk, T., Kramer, C., & Heien, M. L. (2017, Spring). Shotgun microdialysis” with LC-MS3 quantitation for the screening of BBB penetration properties of peptide-based drugs. ASMS Conference on Mass Spectrometry and Allied Topics Abstracts.
• Hanrahan, D. J., Palos-Pacheco, R., Jones, E. M., Szabo, L., Kegel, L. L., Maier, R. M., Pemberton, J. E., Polt, R. L., Hanrahan, D. J., Palos-Pacheco, R., Jones, E. M., Szabo, L., Kegel, L. L., Maier, R. M., Pemberton, J. E., & Polt, R. L. (2015, Spring). Synthesis and Structure-Function Relationships of Glycolipid Surfactants. LIPID MAPS Annual Meeting 2015. La Jolla, CA.
• Hogan, D. E., Palos-Pacheco, R., Polt, R. L., Pemberton, J. E., & Maier, R. M. (2015, Spring). Biosurfactants as a Tool for Metal Removal from Waste Effluents. 249th American Chemical Society National Meeting & Exposition. Denver, CO.
• Polt, R. L., Pemberton, J. E., Coss, C. S., Wang, H., & Palos-Pacheco, R. (2014, 16-20 March). Structure-function relationships of bio-inspired rhamnolipid surfactants. 247th National Meeting of the ACS. Dallas, TX: American Chemical Society.

Reviews

• Li, Y., Lefever, M. R., Muthu, D., Bidlack, J. M., Bilsky, E. J., & Polt, R. (2015. Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins(pp 205-226).
• POLT, R., PETERSON, M., & DEYOUNG, L. (2001. ALUMINOXY ACETALS FROM ALPHA-AMINO ESTERS - CHIRALITY TRANSFER VIA SEQUENTIAL ADDITION OF HYDRIDE AND C-NUCLEOPHILES - 2-AMINO ALCOHOLS AND SPHINGOSINES(pp 5469-5480).
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  The reaction of alpha-imino esters (O'Donnell's Schiff bases) with aluminum hydrides to produce acetal-like intermediates and subsequent reaction with carbon nucleophiles has been studied. Treatment of optically pure imine-protected amino esters with iBu2AlH or iBuAlH.Bu3Al, followed by RMgX or RLi provided threo-2-amino alcohols in high yield (73-85%) and excellent ''syn'' stereoselectivity (8:1 to >20.1, threo or like product preferred). Use of nonpolar solvents (CH2Cl2-hexane) provided the highest stereoselectivities. Use of the less-reactive iBu2AlH.iBu3Al complex lowered the amount of undesired primary alcohol products observed. Thermally labile aluminoxy acetal intermediates were observed by H-1 NMR and were trapped with N-(trimethylsilyl)imidazole to produce relatively stable monosilyl acetals (mixed acetals). Alanine-derived Schiff bases 2a-e showed a correlation between the steric bulk of the ester and threo selectivity The presence of THF reduced this correlation, suggesting the C-nucleophile addition involves a Lewis acid-assisted S(N)2-like displacement of the aluminoxy acetal or displacement of a tight-ion pair. In addition to the synthesis of optically pure arylethanolamines 6a-d from representative amino acids, threo-sphingosines 8a-d were synthesized from L-serine-derived Schiff base 4b, and 1-deoxy-threo-sphingosines 9a-d were synthesized from L-alanine in a similar fashion. Experimental details are provided.
• Dhanasekaran, M., Palian, M., Alves, ., Yeomans, L., Keyari, C., Davis, P., Bilsky, E., Egleton, R., Yamamura, H., Jacobsen, N., Tollin, G., Hruby, V., Porreca, F., & Polt, R. (1999. Glycopeptides related to beta-endorphin adopt helical amphipathic conformations in the presence of lipid bilayers(pp 5435-5448).
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  A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (EBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics. NOE volumes provided distance constraints for molecular dynamics calculations used to provide detailed backbone conformations. In all cases, the glycopeptides were largely helical in the presence of membrane bilayer models (micelles or bicelles). Plasmon waveguide resonance (PWR) studies showed hen egg phosphatidyl choline (PC) bilayers produce amphipathic helices laying parallel to the membrane surface, with dissociation constants (K-D) in the low nanomolar to micromolar concentration range. Two low-energy states are suggested for the glycosylated endorphin analogues, a flexible aqueous state and a restricted membrane bound state. Strong interactions between the glycopeptide amphipaths and membranes are crucial for penetration of the BBB via an endocytotic mechanism (transcytosis).
• Mitchell, S., Pratt, M., Hruby, V., & Polt, R. (1995. Solid-phase synthesis of O-linked glycopeptide analogues of enkephalin(pp 2327-2342).
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  The synthesis of 18 N-alpha -FMOC-amino acid glycosides for solid-phase glycopeptide assembly is reported. The glycosides were synthesized either from the corresponding O'Donnell Schiff bases or from N-alpha -FMOC-amino protected serine or threonine and the appropriate glycosyl bromide using Hanessian's modification of the Koenigs-Knorr reaction. Reaction rates of D-glycosyl bromides (e.g., acetobromoglucose) with the L- and D-forms of serine and threonine are distinctly different and can be rationalized in terms of the steric interactions within the two types of diastereomeric transition states for the D/L and D/D reactant pairs. The N-alpha -FMOC-protected glycosides [monosaccharides Xyl, Glc, Gal, Man, GlcNAc, and GalNAc; disaccharides Gal-beta (1-4)-Gle (lactose), Glc-beta-(1-4)-Glc (cellobiose), and Gal-alpha (1-6)-Glc (melibiose)] were incorporated into 22 enkephalin glycopeptide analogues. These peptide opiates bearing the pharmacophore H-Tyr-c[DCys-Gly-PheDCys]- were designed to probe the significance of the glycoside moiety and the carbohydrate-peptide linkage region in blood-brain barrier (BBB) transport, opiate receptor binding, and analgesia.
• Polt, R., Sames, D., & Chruma, J. (1992. Glycosidase inhibitors: Synthesis of enantiomerically pure aza-sugars from Schiff base amino esters via tandem reduction-alkenylation and osmylation(pp 6147-6158).
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  Nitrogen-in-the-ring "aza-sugars" have been synthesized in enantiomerically pure form from the amino acid L-alanine in excellent overall yield. The O'Donnell's Schiff base of L-alanine methyl ester 9a was converted to ate-sugar L-fuco-1-deoxy-nojirimycin, 18, and to the epimer L-gulo-1-deoxy-nojirimycin, 20, in eight steps. The overall yields were 20 and 29%, respectively. The methodology for the efficient generation of silyl- and benzyl-protected (E)-3-lithio-2-propen-1-ols, and the use of these alkenyllithiums with iBu(5)Al(2)H as nucleophiles in the three-selective tandem reduction-alkenylation of the Schiff base esters is described. Osmium-catalyzed cis-oxygenation of the resulting olefin products was selective for the galacto (fuco) amino polyols in all cases for the acyclic olefins, and was gulo-selective for the cyclic D-4,5-dihydropyridine pivalate, 17c. TEMPO-NaOCl was selective for oxidation of the primary position of the acyclic Schiff bases, and allowed for minimal protection/deprotection of the intermediates. The resulting N-benzhydryl heterocycles were easily deprotected with H-2-Pd at atmospheric pressure.
• Palian, M., Boguslavsky, ., O'Brien, D., & Polt, R. (1990. Glycopeptide-membrane interactions: Glycosyl enkephalin analogues adopt turn conformations by NMR and CD in amphipathic media(pp 5823-5831).
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  Four enkephalin analogues (Tyr-D-Thr-Gly-Phe-Leu-Ser-CONH2, 1, and the related O-linked glycopeptides bearing the monosaccharide beta-glucose, 2, the disaccharide beta-maltose, 3, and the trisaccharide beta-maltotriose, 4) were synthesized, purified by HPLC, and biophysical studies were conducted to examine their interactions with membrane model systems. Glycopeptide 2 has been previously reported to penetrate the blood-brain barrier (BBB), and produce potent analgesia superior to morphine in mice (J. Med. Chem. 2000, 43, 2586-90 and J. Pharm. Exp. Then. 2001, 299, 967-972). The parent peptide and its three glycopeptide derivatives were studied in aqueous solution and in the presence of micelles using 2-D NMR, CD, and molecular mechanics (Monte Carlo studies). Consistent with previous conformational studies on cyclic opioid agonist glycopeptides, it was seen that glycosylation did not significantly perturb the peptide backbone in aqueous solution, but all four compounds strongly associated with 5-30 mM SDS or DPC micelles, and underwent profound membrane-induced conformational changes. Interaction was also observed with POPC:POPE:cholesterol lipid vesicles (LUV) in equilibrium dialysis experiments. Although the peptide backbones of 1-4 possessed random coil structures in water, in the presence of the lipid phase they each formed a nearly identical pair of structures, all with a stable beta-turn motif at the C-terminus. Use of spin labels (Mn2+ and 5-DOXYL-stearic acid) allowed for the determination of the position and orientation of the compounds relative to the surface of the micelle.
• Polt, R., Dhanasekaran, M., & Keyari, C. (1989. Glycosylated neuropeptides: A new vista for neuropsychopharmacology?(pp 557-585).
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  The application of endogenous neuropeptides (e.g., enkephalins) as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood-brain barrier (BBB). Effective BBB transport of glycosylated enkephalins has been demonstrated in several labs now. Analgesia (antinociception) levels greater than morphine, and with reduced side effects have been observed for several glycopeptides related to enkephalin. Somewhat paradoxically, enhanced BBB transport across this lipophilic barrier is achieved by attaching water-soluble carbohydrate groups to the peptide moieties to produce biousian glycopeptides that can be either water-soluble or membrane bound. Transport is believed to rely on an endocytotic mechanism (transcytosis), and allows for systemic delivery and transport of the water-soluble glycopeptides. Much larger endorphin/dynorphin glycopeptide analogs bearing amphipathic helix address regions also have been shown to penetrate the BBB in mice. This holds forth the possibility of transporting much larger neuropeptides across the BBB, which may encompass a wide variety of receptors beyond the opioid receptors. (c) 2005 Wiley Periodicals, Inc.