Solid-phase Chemistry Research Articles

A High-Throughput O-Glycopeptide Discovery Platform for Seromic Profiling

Biomarker microarrays are becoming valuable tools for serological screening of disease-associated autoantibodies. Post-translational modifications (PTMs) such as glycosylation extend the range of protein function, and a variety of glycosylated proteins are known to be altered in disease progression. Here, we have developed a synthetic screening microarray platform for facile display of O-glycosylated peptides (O-PTMs). By introduction of a capping step during chemical solid-phase glycopeptide synthesis, selective enrichment of N-terminal glycopeptide end products was achieved on an amine-reactive hydrogel-coated microarray glass surface, allowing high-throughput display of large numbers of glycopeptides. Utilizing a repertoire of recombinant glycosyltransferases enabled further diversification of the array libraries in situ and display of a new level of potential biomarker candidates for serological screening. As proof-of-concept, we have demonstrated that MUC1 glycopeptides could be assembled and used to detect autoantibodies in vaccine-induced disease-free breast cancer patients and in patients with confirmed disease at time of diagnosis.

X-ray Crystal Structures of Monomeric and Dimeric Peptide Inhibitors in Complex with the Human Neonatal Fc Receptor, FcRn

The neonatal Fc receptor, FcRn, is responsible for the long half-life of IgG molecules in vivo and is a potential therapeutic target for the treatment of autoimmune diseases. A family of peptides comprising the consensus motif GHFGGXY, where X is preferably a hydrophobic amino acid, was shown previously to inhibit the human IgG:human FcRn protein-protein interaction (Mezo, A. R., McDonnell, K. A., Tan Hehir, C. A., Low, S. C., Palombella, V. J., Stattel, J. M., Kamphaus, G. D., Fraley, C., Zhang, Y., Dumont, J. A., and Bitonti, A. J. (2008) Proc. Natl. Acad. Sci. U.S.A., 105, 2337-2342). Herein, the x-ray crystal structure of a representative monomeric peptide in complex with human FcRn was solved to 2.6 A resolution. The structure shows that the peptide binds to human FcRn at the same general binding site as does the Fc domain of IgG. The data correlate well with structure-activity relationship data relating to how the peptide family binds to human FcRn. In addition, the x-ray crystal structure of a representative dimeric peptide in complex with human FcRn shows how the bivalent ligand can bridge two FcRn molecules, which may be relevant to the mechanism by which the dimeric peptides inhibit FcRn and increase IgG catabolism in vivo. Modeling of the peptide:FcRn structure as compared with available structural data on Fc and FcRn suggest that the His-6 and Phe-7 (peptide) partially mimic the interaction of His-310 and Ile-253 (Fc) in binding to FcRn, but using a different backbone topology.

Thermodynamic model for estimating the composition of passive films and Flade potential on Fe-Cr alloys in aqueous solutions

A thermodynamic model of a passive film is developed, in which the formation of the film on the surface of an Fe-Cr alloy in an aqueous solution is considered to be a result of the stable solid-phase chemical and adsorption equilibrium at the alloy-inner passive film layer interface. In the calculations, the Cr2O3 content in the passive film is determined by both the Gibbs energy change (ΔG < 0) in the chemical oxidation of the alloy components by the water oxygen and the change in the surface Gibbs energy (ΔG S > 0) of the alloy. The ΔG S change results in the negative adsorption of chromium atoms, which shifts the 3Fe + Cr2O3 ↔ 3FeO + 2Cr equilibrium toward the FeO formation in the passive film. Calculations showed that the enrichment of the passive film in chromium oxide should sharply increase in a chromium content range of 10–20% in the alloy, which agrees with the known data of X-ray photoelectron spectroscopy of the passive films. A formula is derived for estimating the Flade potential of Fe-Cr alloys, which relates the Flade potentials of individual Fe and Cr components to the FeO and Cr2O3 contents in the passive film.

Editorial for the Special Issue on the 2nd Modern Solid Phase Peptide Synthesis and its Applications Symposium

In the Spring of 1962 Professor Bruce Merrifield gave his first public description of the concept of solid phase chemistry for the synthesis of peptides at the Federation of the American Societies for Experimental Biology (FASEB) annual meeting. Fifty years on, some of the latest developments in solid phase peptide chemistry were reported at the 3rd Modern Solid Phase Peptide Synthesis and its Applications Symposium that was held at Daydream Island in Queensland, Australia, from 13th to 16th October, 2011. The symposium, an official satellite meeting of the 9th Australian Peptide Conference, and originally conceived 6 years earlier by Professor John Wade (Symposium Chairperson) has the goal of bringing together academic and industrial scientists to present and discuss the latest developments in solid phase peptide chemistry. The symposium attracted over a hundred delegates from around the world who described the synthesis and use of new and novel resins and reaction parameters to produce peptides of a high yield and purity. The field of solid phase peptide chemistry is ever expanding and the symposium aimed to capture these advances in SPPS and through the invited speakers (Paul Alewood, Brian Chia, Mike Gait, Ralf Hoffman, Knud Jensen, Lei Liu, John McMurray and James Tam) to stimulate interest and discussion in research into the synthesis of post-translationally modified peptides, microwave assisted chemistries, peptidomimetics and therapeutics, oligonucleotide analogues and ligation chemistries for inducing structure and/or protein synthesis. The opening plenary session given by James Tam and Paul Alewood on ‘‘Tandem acyl shifts for preparing peptide thioesters for Fmoc chemistry’’ and ‘‘Folding miniproteins’’, projected the symposium into a series of lively and enthusiastic discussions which continued for the whole symposium and made for new scientific insights and collaborations. The scientific program covered the major themes in SPPS with sessions which included ‘‘SPS of peptidomimetics’’, ‘‘SPPS Methodology’’, ‘‘SPS of antimicrobial peptides’’, ‘‘Microwave assisted SP biomolecule synthesis’’, ‘‘APPS—biological applications’’ and ‘‘Solid phase protein synthesis’’. Once again oral presentations were complemented by an evening poster session and dinner to encourage further discussions and collaborations. The success and continuation of the meeting has many parts and contributions and the organising committee would like to thank the financial support provided by and participation of several major sponsors including the Australian Peptide Association. The previous two Modern Solid Phase Peptide Synthesis and its Applications Symposia generated considerable excitement that resulted in Special Issues of the International Journal of Peptide Research and Therapeutics. The third meeting was no different and after the meeting delegates were invited to submit an article on their work and that that captured the essence of the meeting. This special issue brings together a collection of fully peer-reviewed articles on all of the areas covered in the meeting and demonstrates why the Modern SPPS meeting generates such scientific anticipation and excitement amongst the delegates. We would like to thank each of the contributing authors for their patience and cooperation in making this special issue possible. We are also very grateful to all of N. M. O’Brien-Simpson (&) Melbourne Dental School, Oral Health CRC, The University of Melbourne, Melbourne, VIC, Australia e-mail: neil.obs@unimelb.edu.au

Atmospheric transport of persistent semi-volatile organic chemicals to the Arctic and cold condensation in the mid-troposphere – Part 1: 2-D modeling in mean atmosphere

Abstract. In the first part of this study for revisiting the cold condensation effect on global distribution of semi-volatile organic chemicals (SVOCs), the atmospheric transport of SVOCs to the Arctic in the mid-troposphere in a mean meridional atmospheric circulation over the Northern Hemisphere was simulated by a two-dimensional (2-D) atmospheric transport model. Results show that under the mean meridional atmospheric circulation the long-range atmospheric transport of SVOCs from warm latitudes to the Arctic occurs primarily in the mid-troposphere. Although major sources are in low and mid-latitude soils, the modeled air concentration of SVOCs in the mid-troposphere is of the same order as or higher than that near the surface, demonstrating that the mid-troposphere is an important pathway and reservoir of SVOCs. The cold condensation of the chemicals is also likely to take place in the mid-troposphere over a source region of SVOCs in warm low latitudes through interacting with clouds. We demonstrate that the temperature dependent vapour pressure and atmospheric degradation rate of SVOCs exhibit similarities between lower atmosphere over the Arctic and the mid-troposphere over a tropical region. Frequent occurrence of atmospheric ascending motion and convection over warm latitudes carry the chemicals to a higher altitude where some of these chemicals may partition onto solid or aqueous phase through interaction with atmospheric aerosols, cloud water droplets and ice particles, and become more persistent at lower temperatures. Stronger winds in the mid-troposphere then convey solid and aqueous phase chemicals to the Arctic where they sink by large-scale descending motion and wet deposition. Using calculated water droplet-air partitioning coefficient of several persistent organic semi-volatile chemicals under a mean air temperature profile from the equator to the North Pole we propose that clouds are likely important sorbing media for SVOCs and pathway of the cold condensation effect and poleward atmospheric transport. The role of deposition and atmospheric descending motion in the cold condensation effect over the Arctic is also discussed.

The Therapeutically Anti-prion Active Antibody-fragment scFv-W226: Paramagnetic Relaxation-Enhanced NMR Spectroscopy aided Structure Elucidation of the Paratope-epitope Interface

Antibodies have become indispensable reagents with numerous applications in biological and biotechnical analysis, in diagnostics as well as in therapy. In all cases, selective interaction with an epitope is crucial and depends on the conformation of the paratope. While epitopes are routinely mapped at high throughput, methods revealing structural insights on a rather short timescale are rare. We here demonstrate paramagnetic relaxation-enhanced (PRE) NMR spectroscopy to be a powerful tool unraveling structural information about epitope-orientation in a groove spanned by the complementary determining regions. In particular, we utilize the spin label TOAC, which is fused to the peptidic epitope using standard solid-phase chemistry and which is characterized by a reduced mobility compared to, e.g., spin labels attached to the side-chain functionalities of cysteine or lysine residues. We apply the method to determine the orientation of helix 1 of the prion protein, which is the epitope for the therapeutically anti-prion active scFv fragment W226.

ChemInform Abstract: Repair and Replication of Oxidized DNA Bases Using Modified Oligodeoxyribonucleotides

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se-Met) strategy). This selenium derivatization strategy via MAD (multi-wavelength anomalous dispersion) phasing has revolutionized protein X-ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X-ray crystal-structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O-atom at the positions 2', 4', 5', and in nucleobases and non-bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid-phase chemical synthesis and enzymatic methods, and the Se-derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein-nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2'-position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom-specific substitution of the nucleotide O-atom, better stability under X-ray radiation, and structure isomorphism. Therefore, our Se-derivatization strategy has great potentials to provide rational solutions for both phase determination and high-quality crystal growth in nucleic-acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site-specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se-derivatization strategy in nucleic acid and protein research are also described in this review.

Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

Chemical synthesis of Ser/Thr AMPylated peptides

Abstract A method for the solid phase chemical synthesis of AMPylated Ser/Thr containing peptides is described. Peptides were phosphonylated using the H-phosphonate method, then the adenosine moiety was introduced using 2′,3′-isopropylidene adenosine and the condensing reagent PyBOP. Oxidation with iodine yielded the desired AMPylated product in good yield following acidolytic cleavage from the resin support.

Aminopeptidase Fingerprints, an Integrated Approach for Identification of Good Substrates and Optimal Inhibitors

Aminopeptidases process the N-terminal amino acids of target substrates by sequential cleavage of one residue at a time. They are found in all cell compartments of prokaryotes and eukaryotes, being implicated in the major proteolytic events of cell survival, defense, growth, and development. We present a new approach for the fast and reliable evaluation of the substrate specificity of individual aminopeptidases. Using solid phase chemistry with the 7-amino-4-carbamoylmethylcoumarin fluorophore, we have synthesized a library of 61 individual natural and unnatural amino acids substrates, chosen to cover a broad spectrum of the possible interactions in the S1 pocket of this type of protease. As proof of concept, we determined the substrate specificity of human, pig, and rat orthologs of aminopeptidase N (CD13), a highly conserved cell surface protease that inactivates enkephalins and other bioactive peptides. Our data reveal a large and hydrophobic character for the S1 pocket of aminopeptidase N that is conserved with aminopeptidase Ns. Our approach, which can be applied in principle to all aminopeptidases, yields useful information for the design of specific inhibitors, and more importantly, reveals a relationship between the kinetics of substrate hydrolysis and the kinetics of enzyme inhibition.

Design and efficient synthesis of a new scaffold based on unsymmetrical protoporphyrin IX derivatives for use in SPPS

Fmoc-protected amine derivatives of Protoporphyrin IX were synthesized by two original methods using solid-phase chemistry. The new compounds represent unsymmetrical scaffolds suitable for the generation of a large range of peptidic porphyrin derivatives through SPPS strategy.

Sequence-Specific Stereomeric Environment in a DNA Duplex Revealed by a Nucleotide-Independent Nitroxide Probe

In site-directed spin labeling, structural and dynamic information on a parent macromolecule is obtained by monitoring a covalently linked nitroxide radical using electron paramagnetic resonance (EPR) spectroscopy. Our group have developed a method of attaching nitroxide species, such as 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), to a specific nucleotide position within a target DNA or RNA sequence. The method relies on site-specific introduction of a phosphorothiate during the solid phase chemical synthesis of nucleic acids, and at each given labeling site the nitroxide is attached to one of two phosphorothioate diastereomers (Rp or Sp) in an approximately 50/50 ratio. We have recently reported that variations in DNA structural and dynamic features at the level of an individual nucleotide can be detected using R5a attached to mixed phosphorothioate diastereomers, in which an observed EPR spectrum is presumably a sum of those obtained from either diastereomer (Popova et al., Biochemistry, 2009, 48, 8540-50). In this work, we report X-band EPR spectra of R5a attached to purified Rp and Sp diastereomers at different sites within a B-form DNA duplex. Results are compared to those obtained with mixed nitroxide diastereomers, and advantages and limitations are discussed regarding the necessity of diastereomer separation when probing DNA local environment. Our work is a further step forward in developing a SDSL methodology that may provide a mean for studying structure and dynamics in large DNA molecules.

Solid-Phase Synthesis of a Biotin Derivative and its Application to the Development of Anti-Biotin Antibodies

A biotin derivative, namely biotin-aminocaproic acid-lysine (BAL), was synthesized with solid-phase chemistry, conjugated to a carrier-protein, and used for rabbit immunization. The aminocaproic acid-lysine "long-arm" was used in order to project the biotin-hapten above the carrier-protein surface. Lysine was selected due to its N(epsilon)-amino group, through which BAL was conjugated to the carrier-protein. BAL was synthesized on a commercially available resin with the Fmoc-solid-phase strategy; this has simplified the experimental procedure, overcome the need for intermediate purification steps, and led to a final product of high purity, with high yield. The anti-BAL antibodies recognized free biotin, as shown with an in-house-developed ELISA, in which biotin conjugated to a synthetic "lysine-dendrimer" was used to coat the ELISA microwells. In immunocytology and Western-blot experiments, the anti-BAL antibodies led to similar results with those obtained with streptavidin. Synthetic derivatives of hapten molecules that can be easily prepared with solid-phase chemistry, such as BAL, may be used for the development of specific antibodies for the corresponding hapten.

Phenols-useful templates for the synthesis of bi-functional orthogonally protected dendron building blocks via solid phase Mitsunobu reaction

Bi-functional dendritic building blocks for convergent dendrimer growth were successfully synthesized from phenolic templates in the solid phase via a Mitsunobu reaction. Each arm of the dendron building block carries an orthogonally protected secondary amine along the arm, and a peripheral primary amine or phenol group (building block type 1) or a tertiary amine junction with orthogonally protected peripheral primary amine or carboxyl groups (building block type 2). The synthetic routes reported in this work are general and applicable for the preparation of diverse building blocks, controlling protection, arm length, and peripheral moieties. These novel dendron units can form unusual dendritic architectures by solid-phase chemistry, which may be incorporated into specific complex structures expanding the scope of dendrimer science.

Solid phase synthesis of novel asymmetric hydrophilic head cholesterol-based cationic lipids with potential DNA delivery

Twenty-four asymmetric divalent head group cholesterol-based cationic lipids were designed and synthesized by parallel solid phase chemistry. These asymmetric head groups composed of amino functionality together with trimethylamino, di(2-hydroxyethyl)amino or guanidinyl groups. Spacers between cationic heads and linker were both equal and unequal in length. These lipids were subjected to evaluation for DNA binding affinities by gel retardation assay and were screened for their transfection efficiency on HEK293 cells. Cationic lipids with equal chain length exhibited high transfection efficiency when polar part contained asymmetric polar heads. In contrast, lipids with unequal chain length exhibited high transfection efficiency when polar part contained symmetric heads. According to the optimal formulation, seven lipids exhibited higher transfection efficiency than the commercially available transfection agents, Effectene™, DOTAP and DC-Chol, to deliver DNA into PC3 human prostate adenocarcinoma cells. 3β-[ N-( N′-Guanidinyl)-2′-aminoethyl)- N-(2-aminoethyl)carbamoyl] cholesterol ( 5) bearing amino and guanidinyl polar heads exhibited highest transfection efficiency with minimal toxicity. The morphology of active liposomes was observed by transmission electron microscopy (TEM) and size of liposomes were around 200–700 nm.

PET of Malignant Melanoma Using 18F-Labeled Metallopeptides

Melanocortin type 1 receptor (MC1R), also known as alpha-melanocyte-stimulating hormone (alpha-MSH) receptor, is an attractive molecular target for melanoma imaging and therapy. An (18)F-labeled linear alpha-MSH peptide ((18)F-FB-Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys-NH(2) [NAPamide]) shows promising melanoma imaging properties but with only moderate tumor uptake and retention. A transition metal rhenium-cyclized alpha-MSH peptide, ReO[Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) (ReCCMSH(Arg(11))), has shown high in vitro binding affinity to MC1R and excellent in vivo melanoma-targeting profiles when labeled with radiometals. Therefore, we hypothesized that ReCCMSH(Arg(11)) could be a good platform for the further development of an (18)F-labeled probe for PET of MC1R-positive malignant melanoma. In this study, the metallopeptide Ac-d-Lys-ReCCMSH(Arg(11)) was synthesized using conventional solid-phase peptide synthesis chemistry and a rhenium cyclization reaction. The resulting peptides were then labeled with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB). The (18)F-labeled metallopeptides were further tested for their in vitro receptor binding affinities, in vivo biodistribution, and PET imaging properties. Both isomers of Ac-d-Lys-ReCCMSH(Arg(11)), named as RMSH-1 and RMSH-2, were purified and identified by high-performance liquid chromatography. The binding affinities of RMSH-1 and RMSH-2 and their respective (19)F-SFB-conjugated peptides ((19)F-FB-RMSH-1 and (19)F-FB-RMSH-2) were all determined to be within nanomolar range. Both (18)F-labeled metallopeptides showed good tumor uptake in the B16F10 murine model, with high MC1R expression, but much lower uptake in the A375M human melanoma xenografted in mice, indicating low MC1R expression. (18)F-FB-RMSH-1, when compared with (18)F-FB-RMSH-2, displayed more favorable in vivo performance in terms of slightly higher tumor uptakes and much lower accumulations in the kidney and liver at 2 h after injection. Small-animal PET of (18)F-FB-RMSH-1 and -2 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptakes and poorer tumor-to-normal organ contrasts were observed for the A375M model than for the B16F10 model. (18)F-FB-RMSH-1 and -2 showed higher tumor uptake and better tumor retention than did (18)F-FB-NAPamide. Specific in vivo targeting of (18)F-FB-RMSH-1 to malignant melanoma was successfully achieved in preclinical models with high MC1R expression. Thus, the radiofluorinated metallopeptide (18)F-FB-RMSH-1 is a promising molecular probe for PET of MC1R-positive tumors.

Combined solution-phase and solid-phase synthesis of 2-amino-7,8-dihydropteridin-6(5 H)-ones

An efficient and general synthesis of substituted 2-amino-7,8-dihydropteridin-6(5 H)-ones using a combination of solution-phase and solid-phase chemistry is described. Solution-phase chemistry was used to produce two pyrimidine regioisomers that were separated by flash column chromatography. Utilizing the desired regioisomer, solid-phase chemistry was used to effect the rapid construction of the substituted 2-amino-7,8-dihydropteridin-6(5 H)-one system in high overall yield and purity.

Modifying the conserved C‐terminal tyrosine of the peptide hormone PYY3‐36 to improve Y2 receptor selectivity

The Y2 selective PYY derived peptide PYY3-36 was recently shown to play a role in appetite regulation. Novel PYY3-36 analogs with high selectivity for the Y2 receptor could be potential drug candidates for the treatment of obesity. The C-terminal pentapeptide segment of PYY3-36 is believed to bind to the Y receptors. Tyr-36 is highly conserved across species and only few successful modifications of Tyr-36 have been documented. PYY3-36 analogs were prepared using solid-phase peptide chemistry and tested for binding to the Y1, Y2 and Y4 receptor subtypes by radioligand displacement assay. The Y2 receptor agonists with the best affinity and selectivity were further investigated for activity towards the Y1 and Y2 receptor subtypes. Unexpectedly, modifications of Tyr-36 were well-tolerated, and the analogs of PYY3-36 in which the Tyr-36 hydroxyl group was substituted with a halogen or an amino group were particularly well tolerated and yielded an improved selectivity and approximately equipotent affinity to the Y2 receptor. These modifications could be used to design new potential drug candidates for the treatment of obesity.