Synthesis and biological activity of C-terminally truncated fragments of human-.alpha.-calcitonin gene-related peptide

Smith, David; Li, Jianzhong; Wang, Qiming J.; Murphy, Richard F.; Adrian, Thomas E.; Elias, Yvonne; Bockman, Charles S.; Abel, Peter W.

doi:10.1021/jm00069a012

Cited by 26 publications

(24 citation statements)

References 30 publications

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“…6,17,18 A structure-activity profile for position 37 of h-R-CGRP is emerging in which a L-amino acid residue with a flexible side chain containing a phenyl ring and a C-terminal amide is required for highaffinity binding to CGRP1 receptors. The structureactivity profile is also consistent with structure-activity relationships of the C-terminal position reported for the smaller antagonist [Tyr 0 ]rat-R-CGRP (28)(29)(30)(31)(32)(33)(34)(35)(36)(37) 15 and the endogenous agonist h-R-CGRP. 29 This suggests that CGRP1 receptors have similar requirements of the C-terminal Phe residue for high-affinity binding of agonists and antagonists.…”

Section: Resultssupporting

confidence: 85%

“…It is of note that other workers found that acetylation of the amino terminus of h-R-CGRP (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37), a shorter C-terminal fragment of h-R-CGRP that is also a weak antagonist, caused a 4-fold increase in antagonistic potency. 19 While no explanation was offered, this observation may also be due to increased stability of the N-terminal-acetylated peptide to aminopeptidase degradation.…”

Section: Resultsmentioning

confidence: 94%

“…The tetrapeptides were generated by digestion of h-R-CGRP (1) and analogue 15 with trypsin and purified by RP-HPLC using our previously published methods. 29 Presaturation 1 H NMR spectroscopy of h-R-CGRP (8)(9)(10)(11) in D 2 O unambiguously located the His-imidazole ring hydrogens H2 and H4 at 8.52 and 7.23 ppm, respectively. The upfield H4 appeared as a singlet and H2 appeared as a doublet with J ) 1.20 Hz, both typical in appearance for 5-substituted imidazoles 31 and in coupling constant magnitude for four-bond coupling ( Figure 2A).…”

Section: Resultsmentioning

confidence: 96%

“…26 Conversion of this mixture to the corresponding Fmoc derivatives 28 Solid-phase peptide synthesis using Fmoc-erythro--MePhe-OH yielded a mixture of peptide diastereoisomers 6 and 7 that were readily separated by semipreparative RP-HPLC. A sample of each synthetic product was digested with trypsin, 29 and the C-terminal dipeptide fragment H-Ala--MePhe-NH 2 was isolated by RP-HPLC. After acid hydrolysis of this fragment, the resulting mixture of Ala and -MePhe was subjected to chiral TLC and the -MePhe isomer was identified by comparing its R f value with previously published values for all four isomers of -MePhe.…”

Section: Resultsmentioning

confidence: 99%

“…N-truncated fragments h-R-CGRP (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) and h-R-CGRP (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) are antagonists that bind with low affinity to CGRP receptors on guinea pig atria 19 and [Tyr 0 ]rat-CGRP (28)(29)(30)(31)(32)(33)(34)(35)(36)(37) is a weak antagonist of CGRP receptors in guinea pig pancreatic acini. 20,21 These results suggest that the C-terminal half of CGRP contains structural elements required for it to bind to CGRP receptors and the N-terminal half contains structural elements that are responsible for the highaffinity of CGRP for CGRP receptors.…”

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confidence: 99%

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Modifications to the N-Terminus but Not the C-Terminus of Calcitonin Gene-Related Peptide(8-37) Produce Antagonists with Increased Affinity

et al. 2003

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Seventeen novel analogues of human calcitonin gene-related peptide(8-37) (hCGRP(8-37)) were synthesized by solid-phase methods and purified to apparent homogeneity by semipreparative cation exchange and/or reversed-phase high-performance liquid chromatography. The C-terminal Phe was replaced by Gly, cyclohexylalanine (Cha), Tyr, all four isomers of beta-methylphenylalanine (beta-MePhe), and l- and d-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the beta-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of beta-MePhe into the erythro pair of enantiomers (2S,3S, 2R,3R) and the threo pair of enantiomers (2S,3R, 2R,3S), which were then converted to Fmoc derivatives and used in two separate syntheses. Two diastereomeric peptides were obtained from each synthesis and were separated by RP-HPLC to yield enantiomerically pure 6-9. Substitution of Tyr for Phe caused no change in binding affinity at CGRP receptors. All other substitutions for Phe resulted in substantial reductions in binding affinity. Indeed, no binding was observed for analogues 7, 9, and 11, all of which contained a d-amino acid residue in the C-terminal position, and the binding affinities of the remaining analogues were >10-fold lower than that of h-alpha-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-alpha-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) produced analogues 12-14 and 16-18, respectively. A byproduct was isolated by RP-HPLC from the resin-cleaved crude product of each benzylated analogue, which was characterized as the dibenzylated derivative of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and (1)H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His(10). Radioligand binding experiments showed that derivatizing the N-termini substantially increased binding affinities at CGRP receptors. The benzoylated and dibenzylated derivatives had the highest affinities, which were approximately 50-fold greater than those of h-alpha-CGRP(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-alpha-CGRP(8-37) are antagonists that are more potent than h-alpha-CGRP(8-37). In conclusion, these studies underscore the importance of Phe(37) of h-alpha-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His(10) as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Modifications to the N-Terminus but Not the C-Terminus of Calcitonin Gene-Related Peptide(8-37) Produce Antagonists with Increased Affinity

et al. 2003

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Naturally Occurring Peptide Hormones and Neurotransmitters: Synthesis

Liu

Hruby

2008

Wiley Encyclopedia of Chemical Biology

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How to synthesize biologically active polypeptides chemically has long been an important challenge for many fields in chemistry and biology. In the past, the development of solution‐phase and solid‐phase synthesis techniques has made it possible to synthesize large molecules like peptides with molecular weights up to 10,000. The purpose of this review is to provide an overview of how to prepare natural bioactive peptides synthetically. Because of space limitations, this review concentrates on peptide hormones and neurotransmitters.

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Astressin‐amide and astressin‐acid are structurally different in dimethylsulfoxide

et al. 2007

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The C-terminally amidated CRF antagonist astressin binds to CRF-R1 or CRF-R2 receptors with low nanomolar affinity while the corresponding astressin-acid has >100 times less affinity. To understand the role of the amide group in binding, the conformations of astressin-amide and astressin-acid were studied in DMSO using NMR techniques. The 3D NMR structures show that the backbones of both analogs prefer an alpha-helical conformation, with a small kink around Gln(26). However, astressin-amide has a well-defined helical structure from Leu(27) to Ile(41) and a conformation very similar to the bioactive conformation reported by our group (Grace et al., Proc Natl Acad Sci USA 2007, 104, 4858-4863). In contrast, astressin-acid has an irregular helical conformation from Arg(35) onward, including a rearrangement of the side chains in that region. This structural difference highlights the crucial role of the C-terminal amidation for stabilization of astressin's bioactive conformation.

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Synthesis and biological activity of C-terminally truncated fragments of human-.alpha.-calcitonin gene-related peptide

Cited by 26 publications

References 30 publications

Modifications to the N-Terminus but Not the C-Terminus of Calcitonin Gene-Related Peptide(8-37) Produce Antagonists with Increased Affinity

Modifications to the N-Terminus but Not the C-Terminus of Calcitonin Gene-Related Peptide(8-37) Produce Antagonists with Increased Affinity

Naturally Occurring Peptide Hormones and Neurotransmitters: Synthesis

Astressin‐amide and astressin‐acid are structurally different in dimethylsulfoxide

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