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 language | English |
|---|---|
| Pages (from-to) | 2427-2435 |
| Number of pages | 9 |
| Journal | Journal of Medicinal Chemistry |
| Volume | 46 |
| Issue number | 12 |
| DOIs | |
| State | Published - Jun 5 2003 |
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All Science Journal Classification (ASJC) codes
- Organic Chemistry
Cite this
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 journal › Article
}
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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U2 - 10.1021/jm020507f
DO - 10.1021/jm020507f
M3 - Article
C2 - 12773046
AN - SCOPUS:0038778313
VL - 46
SP - 2427
EP - 2435
JO - Journal of Medicinal Chemistry
JF - Journal of Medicinal Chemistry
SN - 0022-2623
IS - 12
ER -