Modifications to the N-terminus but not the C-terminus of calcitonin gene-related peptide(8-37) produce antagonists with increased affinity

D. David Smith, Shankar Saha, Guoyong Fang, Courtney Schaffert, David J J Waugh, Wanyun Zeng, Geza Toth, Martin Hulce, Peter W. Abel

Research output: Contribution to journalArticle

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Abstract

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 β-methylphenylalanine (β-MePhe), and L- and D-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the β-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of β-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-α-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-α-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-α-CGRP(8-37) and h-β-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-α-CGRP-(8-37) and h-β-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and 1H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His10. 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-α-CGRP-(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-α-CGRP(8-37) are antagonists that are more potent than h-α-CGRP(8-37). In conclusion, these studies underscore the importance of Phe37 of h-α-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His10 as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.

Original languageEnglish
Pages (from-to)2427-2435
Number of pages9
JournalJournal of Medicinal Chemistry
Volume46
Issue number12
DOIs
StatePublished - Jun 5 2003

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Calcitonin Gene-Related Peptide Receptors
Enantiomers
High Pressure Liquid Chromatography
Derivatives
Isomers
calcitonin gene-related peptide (8-37)
Substitution reactions
Amino Acids
Acetylation
High performance liquid chromatography
Reverse-Phase Chromatography
Crystallization
Byproducts
Cations
Carbon
Resins
Experiments
Nuclear magnetic resonance
Peptides

All Science Journal Classification (ASJC) codes

  • Organic Chemistry

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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. / Smith, D. David; Saha, Shankar; Fang, Guoyong; Schaffert, Courtney; Waugh, David J J; Zeng, Wanyun; Toth, Geza; Hulce, Martin; Abel, Peter W.

In: Journal of Medicinal Chemistry, Vol. 46, No. 12, 05.06.2003, p. 2427-2435.

Research output: Contribution to journalArticle

Smith, D. David ; Saha, Shankar ; Fang, Guoyong ; Schaffert, Courtney ; Waugh, David J J ; Zeng, Wanyun ; Toth, Geza ; Hulce, Martin ; Abel, Peter W. / Modifications to the N-terminus but not the C-terminus of calcitonin gene-related peptide(8-37) produce antagonists with increased affinity. In: Journal of Medicinal Chemistry. 2003 ; Vol. 46, No. 12. pp. 2427-2435.
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abstract = "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 β-methylphenylalanine (β-MePhe), and L- and D-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the β-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of β-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-α-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-α-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-α-CGRP(8-37) and h-β-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-α-CGRP-(8-37) and h-β-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and 1H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His10. 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-α-CGRP-(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-α-CGRP(8-37) are antagonists that are more potent than h-α-CGRP(8-37). In conclusion, these studies underscore the importance of Phe37 of h-α-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His10 as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.",
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TY - JOUR

T1 - Modifications to the N-terminus but not the C-terminus of calcitonin gene-related peptide(8-37) produce antagonists with increased affinity

AU - Smith, D. David

AU - Saha, Shankar

AU - Fang, Guoyong

AU - Schaffert, Courtney

AU - Waugh, David J J

AU - Zeng, Wanyun

AU - Toth, Geza

AU - Hulce, Martin

AU - Abel, Peter W.

PY - 2003/6/5

Y1 - 2003/6/5

N2 - 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 β-methylphenylalanine (β-MePhe), and L- and D-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the β-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of β-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-α-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-α-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-α-CGRP(8-37) and h-β-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-α-CGRP-(8-37) and h-β-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and 1H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His10. 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-α-CGRP-(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-α-CGRP(8-37) are antagonists that are more potent than h-α-CGRP(8-37). In conclusion, these studies underscore the importance of Phe37 of h-α-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His10 as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.

AB - 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 β-methylphenylalanine (β-MePhe), and L- and D-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the β-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of β-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-α-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-α-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-α-CGRP(8-37) and h-β-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-α-CGRP-(8-37) and h-β-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and 1H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His10. 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-α-CGRP-(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-α-CGRP(8-37) are antagonists that are more potent than h-α-CGRP(8-37). In conclusion, these studies underscore the importance of Phe37 of h-α-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His10 as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.

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