N-(3-(p-azido-m-[125I]iodophenyl)propionyl)-succinimide—A heterobifunctional reagent for the synthesis of radioactive photoaffinity ligands: Synthesis of a carrier-free 125I-labeled cardiac glycoside photoaffinity label

Lowndes, Joseph M.; Hokin-Neaverson, Mabel; Ruoho, Arnold E.

doi:10.1016/0003-2697(88)90007-3

Cited by 16 publications

(11 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ability to radioiodinate the photoreactive residue of a peptide ligand provides a distinct advantage over previous photolabeling methods by allowing the identification of the exact amino acid at the photoinsertion site on the receptor as opposed to the mere identification of a fragment or region of the receptor. The strategy of putting 125 I within a photoreactive moiety was introduced by Ruoho and co-workers (Lownder et al, 1988;Morris et al, 1991) for iodoazidophenyl derivatives. HBPA adds to this concept the significant advantages of a photolabile amino acid which can be placed anywhere in a peptide ligand during synthesis and the significant advantage of benzophenone photochemistry, providing an excellent tool for mapping binding sites.…”

Section: Discussionmentioning

confidence: 99%

p-(4-Hydroxybenzoyl)phenylalanine: A Photoreactive Amino Acid Analog Amenable to Radioiodination for Elucidation of Peptide−Protein Interaction Application to Substance P Receptor

et al. 1997

View full text Add to dashboard Cite

Benzoylphenylalanine, a photoreactive phenylalanine analog that can be incorporated into a peptide during solid-phase synthesis, is a useful probe for investigating the interactions of bioactive peptides with their receptors. This probe, however, lacks versatility because it is not detectable by Edman sequencing and because it cannot be labeled with radioiodine, requiring radiolabeling of the peptide ligand at a site distal to the photoreactive amino acid. The separation of the radioisotope and photoaffinity labels along the primary sequence limits identification of the photoinsertion site to a peptide fragment rather than a specific amino acid of the receptor protein. We have now synthesized p-(4-hydroxybenzoyl)phenylalanine by a synthetic route involving reaction of 4-(chloromethyl)benzoic anhydride with phenol in polyphosphoric acid to give the 4-(chloromethyl)benzoyl ester of 4-(chloromethyl)-4'-hydroxybenzophenone followed by reaction of the benzophenone derivative with ethyl acetamidocyanoacetate and subsequent hydrolysis of the product to give p-(4-hydroxybenzoyl)phenylalanine. The novel photolabile amino acid was incorporated into substance P (replacing Phe8 or Lys3) to give 11-mer peptides that bind with high (nM) affinity and specificity to the substance P receptor. Radioiodination of the substance P analogs resulted in the incorporation of 125I at the photoreactive amino acid residue, yielding probes of high (approximately 2000 Ci/mmol) specific activity. Subsequent photolysis of the radiolabeled peptides in the presence of substance P receptor caused covalent attachment of the peptide to the receptor with high photoinsertion yield (approximately 30%); photolabeling was abolished in the presence of excess unlabeled SP. p-(4-Hydroxybenzoyl)phenylalanine retains p-benzoylphenylalanine's high insertion yield and low reactivity with water, but in contrast allows placement of radioiodine and the photoactive moieties within the same residue, providing the ability to identify the specific site(s) of interaction, and identification of the residue by Edman sequencing. This novel amino acid may be useful in the elucidation of the interaction of a variety of peptides with their receptors.

show abstract

Section: Discussionmentioning

confidence: 99%

p-(4-Hydroxybenzoyl)phenylalanine: A Photoreactive Amino Acid Analog Amenable to Radioiodination for Elucidation of Peptide−Protein Interaction Application to Substance P Receptor

et al. 1997

View full text Add to dashboard Cite

show abstract

“…The method for synthesis and purification of N- (3(azido-m-[ 125 I]iodophenyl)propionyl)-succinimide ([ 125 I]AIPPS) was essentially as described by Lowndes et al 16 Photoactivatable Cer ( 125 I-N 3 -Cer) was prepared as described in Zegers et al 17 Briefly, [ 125 I]AIPPS was prepared by radioiodination of (4-aminophenyl)propionic acid, using carrierfree Na 125 I in the presence of thallium trichloride, followed by the synthesis of the azide and conversion to the N-hydroxy-succinimide ester. 125 I-N 3 -Cer was prepared by mixing 300 µCi of [ 125 I]AIPPS (specific activity, 2,000 Ci/mmol) with 1 µmol of D-sphingosine, in 200 µL chloroform containing 2% triethylamine (vol/vol).…”

Section: Methodsmentioning

confidence: 99%

“…For synthesis of nonradioactive I-N 3 -Cer, 100 µmol AIPPS, which was synthesized as described, 16 was incubated with 100 µmol D-sphingosine in 400 µL chloroform containing 2% triethylamine (vol/vol), for 20 hours at 4°C. To allow an easy identification of the reaction products during purification, a trace amount of [ 125 I]AIPPS was added to the reaction mixture.…”

Section: Synthesis Of 125 I-n 3 -Cer I-n 3 -Cer and C 6 -Nbd-cermentioning

confidence: 99%

Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?

1998

View full text Add to dashboard Cite

A photoreactive sphingolipid precursor was used to investigate the potential involvement of protein-lipid interactions that may convey specificity to sphingolipid transport in the human hepatoma cell line, HepG2. A 125 Ilabeled, photoreactive ceramide, 125 I-N 3 -Cer, was incubated with the cells and became incorporated into two sphingolipid products. The major product was photoreactive sphingomyelin ( 125 I-N 3 -SM) (25% of total radioactivity), while only minor amounts of photoreactive glucosylceramide ( 125 I-N 3 -GlcCer) were formed (F2%). After photoactivation, a restricted number of proteins was labeled. Given the absolute amounts of the newly synthesized, photoreactive lipids and their precursor present in the cells, labeling of the proteins can be assumed to be derived from interaction with either ceramide (Cer) or sphingomyelin (SM), or both. To discriminate between these possibilities, photoactivation and protein analysis was performed in cells treated with D-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol (PDMP), an inhibitor of sphingolipid biosynthesis. In treated cells, the radioactive SM pool was reduced by Ϸ80%. Concomitantly, labeling of a 60-kd protein, seen in control cells, decreased. Furthermore, the 60-kd protein is membrane-associated and insoluble in detergent at low temperature. Moreover, when cells containing photoreactive sphingolipids after a preincubation with the photoreactive Cer were photoactivated and subsequently incubated with fluorescent sphingolipid analogs, transport of the latter to the bile canalicular membrane, as observed in control cells, was inhibited. Taken together, the data suggest that distinct proteins, among them a 60-kd protein, may play a specific and functional role in sphingolipid transport to the bile canalicular membrane. (HEPATOLOGY

show abstract

“…Synthesis of the heterobifunctional photoaffinity ligand N-o3-(pazido-m-["#&I]iodophenyl)propionylqsuccinimide (["#&I]AIPPS) was performed as described [7]. Briefly, ["#&I]AIPPS was prepared by radioiodination of 3-(p-aminophenyl)propionic acid with carrier-free Na"#&I (specific radioactivity approx.…”

Section: Synthesis Of Photoactivatable Ceramide Glccer and Smmentioning

confidence: 99%

Use of photoactivatable sphingolipid analogues to monitor lipid transport in mammalian cells

Zegers

Hoekstra

1997

Biochemical Journal

View full text Add to dashboard Cite

Photoactivatable derivatives of ceramide, glucosylceramide (GlcCer) and sphingomyelin {3-(p-azido-m-[125I]iodophenyl)propionylceramide, 3-(p-azido-m-[125I]iodophenyl)propionyl-GlcCer and 3-(p-azido-m-[125I]iodophenyl)propionylsphingomyelin} were synthesized in an attempt to identify compartment-specific proteins involved in sphingolipid sorting or metabolism. In HT29 and BHK cells the ceramide analogue entered the cell by monomeric diffusion, as evidenced by the probe's efficient internalization at low temperature (4 degrees C). In contrast, the photoactivatable GlcCer was internalized only at elevated temperatures (37 degrees C), presumably reflecting an endocytic mechanism of uptake. The photoactivatable ceramide was mainly metabolized to the corresponding sphingomyelin analogue, but small amounts of GlcCer and galactosylceramide were also synthesized. The newly synthesized photoreactive sphingomyelin was subsequently transported to the cell surface, a process that was effectively inhibited by the presence of brefeldin A. The incubation of cells with photoactivatable analogues at 4 degrees C, followed by illumination, led to the association of sphingolipid with a specific subset of proteins. The protein labelling pattern of ceramide differed from that of glucosylceramide. A further shift in labelling pattern was apparent when the cells were incubated with the lipid analogues at 37 degrees C. Moreover, most of the proteins labelled by photoreactive sphingomyelin seemed to be detergent-insoluble, which is indicative of a location in sphingolipid-rich microdomains at the plasma membrane. The potential of applying photoactivatable sphingolipids to further define and identify the role of distinct proteins in sphingolipid biosynthesis, transport and sorting, is discussed.

show abstract

N-(3-(p-azido-m-[125I]iodophenyl)propionyl)-succinimide—A heterobifunctional reagent for the synthesis of radioactive photoaffinity ligands: Synthesis of a carrier-free 125I-labeled cardiac glycoside photoaffinity label

Cited by 16 publications

References 13 publications

p-(4-Hydroxybenzoyl)phenylalanine: A Photoreactive Amino Acid Analog Amenable to Radioiodination for Elucidation of Peptide−Protein Interaction Application to Substance P Receptor

p-(4-Hydroxybenzoyl)phenylalanine: A Photoreactive Amino Acid Analog Amenable to Radioiodination for Elucidation of Peptide−Protein Interaction Application to Substance P Receptor

Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?

Use of photoactivatable sphingolipid analogues to monitor lipid transport in mammalian cells

Contact Info

Product

Resources

About