Abstract
To develop affinity labels for δ opioid receptors based on peptide antagonists, the Phe 4 residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib 2,Aib 3]leucine enkephalin (ICI-174,864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH 2Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH 2) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing δ or μ opioid receptors. The δ receptor affinities of the N,N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe 4 decreased binding affinity at both δ and μ receptors in standard radioligand binding assays; the one exception was N,N-dibenzyl[Phe(p-NCS) 4]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for δ receptors (IC 50 = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC 50 = 78.2 nM). The decreases in μ receptor affinities were greater than in δ receptor affinities so that all of the analogues tested exhibited significantly greater δ receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N,N-dibenzyl[Phe(p-NCS) 4]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to δ receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of δ opioid receptors.
| Original language | English |
|---|---|
| Pages (from-to) | 3941-3948 |
| Number of pages | 8 |
| Journal | Journal of Medicinal Chemistry |
| Volume | 43 |
| Issue number | 21 |
| DOIs | |
| State | Published - Oct 19 2000 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Organic Chemistry
Cite this
Synthesis and evaluation of n,n-dialkyl enkephalin-based affinity labels for δ opioid receptors. / Maeda, D. Y.; Ishmael, J. E.; Murray, Thomas F.; Aldrich, J. V.
In: Journal of Medicinal Chemistry, Vol. 43, No. 21, 19.10.2000, p. 3941-3948.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Synthesis and evaluation of n,n-dialkyl enkephalin-based affinity labels for δ opioid receptors
AU - Maeda, D. Y.
AU - Ishmael, J. E.
AU - Murray, Thomas F.
AU - Aldrich, J. V.
PY - 2000/10/19
Y1 - 2000/10/19
N2 - To develop affinity labels for δ opioid receptors based on peptide antagonists, the Phe 4 residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib 2,Aib 3]leucine enkephalin (ICI-174,864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH 2Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH 2) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing δ or μ opioid receptors. The δ receptor affinities of the N,N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe 4 decreased binding affinity at both δ and μ receptors in standard radioligand binding assays; the one exception was N,N-dibenzyl[Phe(p-NCS) 4]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for δ receptors (IC 50 = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC 50 = 78.2 nM). The decreases in μ receptor affinities were greater than in δ receptor affinities so that all of the analogues tested exhibited significantly greater δ receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N,N-dibenzyl[Phe(p-NCS) 4]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to δ receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of δ opioid receptors.
AB - To develop affinity labels for δ opioid receptors based on peptide antagonists, the Phe 4 residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib 2,Aib 3]leucine enkephalin (ICI-174,864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH 2Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH 2) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing δ or μ opioid receptors. The δ receptor affinities of the N,N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe 4 decreased binding affinity at both δ and μ receptors in standard radioligand binding assays; the one exception was N,N-dibenzyl[Phe(p-NCS) 4]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for δ receptors (IC 50 = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC 50 = 78.2 nM). The decreases in μ receptor affinities were greater than in δ receptor affinities so that all of the analogues tested exhibited significantly greater δ receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N,N-dibenzyl[Phe(p-NCS) 4]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to δ receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of δ opioid receptors.
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U2 - 10.1021/jm000123u
DO - 10.1021/jm000123u
M3 - Article
VL - 43
SP - 3941
EP - 3948
JO - Journal of Medicinal Chemistry
JF - Journal of Medicinal Chemistry
SN - 0022-2623
IS - 21
ER -