Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling

Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid-phase method. The peptides Ac-[Cys(Cam)^14,17, GlyS²³]-apocytochrome c-(1–23) (I), CF₃CO-[GlyS⁶⁰]-apocytochrome c-(24–60) (II), and CF₃CO-apocytochrome c-(61–104) (III) were purified by chromatography on CM-cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61–104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N-hydroxysuccinimide to give the 24–104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM-cellulose and HPLC gave a 0.6% yield of [Cys-(Cam)^14,17]-apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map.     

β-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human β-endorphin (β-EP) which contain cystine bridges, [Cys¹⁵-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (I) and [Cys¹⁶-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2–2.5 times the opiate receptor binding activity of β-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg^{9,19,24,28,29}Cys(Cam)^11,26, Phe²⁷,Gly³¹] and [Arg^{9,19,24,28,29},Cys-(Cam)^12,26, Phe²⁷, Gly³¹], were shown to have approximately the same opiate receptor activity as β-endorphin.     

RETRO ANALOGS RELATED TO OXYTOCIN

[D-alle³]-retro-D-deaminotocinamide (I), retro-L-deaminotocinamide (III) and their respective N-formyl derivatives (II and IV) were synthesized by the stepwise active ester method: deaminotocinamide was prepared by the solid-phase method. The retroanalogs of deaminotocinamide, tested at concentrations up to 10^-5 M, were found to be without activity as agonists or antagonists in the oxytocic assay. At 10^-4 M, [D-alle³]-retro-D-deaminotocinamide is a weak competitive inhibitor of oxytocin. [D-alle³, Gly⁷]-retro-D-deaminooxytocin (V) was synthesized either by the active ester method or by a fragment condensation method employing the retro-D-ring, [D-alle³]-retro-D-deaminotocinamide, and “D-tail”, Boc-Gly-D-Leu-Gly, as the fragments. The N-formyl derivative (VI) of V was prepared by selective N-formylation with p-nitrophenyl formate. Additionally, [D-alle³, Gly⁷, Malonamide⁹]-retro-D-deaminooxytocin (VII) and the model compound [Gly⁷]-retro-L-deaminooxytocin (VIII) were synthesized by the fragment condensation method. In the oxytocic assay, the retro-analogs were found to be neither agonists nor antagonists at concentrations up to 10^-5 M, except for [D-alle³, Gly⁷]-retro-D-deaminooxytocin which showed weak oxytocic activity in the range of 10^-5 to 10^-4 M and may be a partial agonist. It is concluded that the addition of the tripeptide “tail” to the retro-D-ring did not significantly alter the biological activity of the latter, which remains about 10⁵ times lower than that of the active hormone analog [Gly⁷]-oxytocin and about 10⁷ times lower than the hormone itself. The marginal activity of all these retro-D-analogs appears to be directly related to the peptide bond reversal and suggests that the peptide backbone may play an essential role in peptide hormone action.     

Enkephalin analogs: synthesis and properties of analogs with lipophilic or extended carboxyl-terminus. Quantitative structure-activity relationship of analogs modified in residue position 5

Four analogs of enkephalin (EK) have been synthesized by the solid-phase method and their biological activities have also been investigated. All four analogs were less active than Met-enkephalin (Met-EK) as shown by relative potencies in the guinea pig ileum (GPI) assay: Met-EK, 100; [Phe⁵]-EK-NH₂, 59; [Trp⁵]-EK-NH₂, 11; Met-EK-Cys(Cam)-OH, 37; and N, N'-bis(Met-EK)-cystine, 34. Two of the analogs were more potent than Met-EK as shown by relative potencies in the mouse tail-flick assay for analgesia: Met-EK, 100; [Phe⁵]-EK-NH₂, 1340; [Trp⁵]-EK-NH₂, 1640. Quantitative structure-activity relationship calculations were carried out for GPI potencies of analogs substituted in position 5. The calculation indicated that, in this position, the bulkiness had the main influence.     

HUMAN SOMATOTROPIN

Three analogs of the carboxyl terminal plasmin fragment of human somatotropin, [Nle¹⁷⁰, Ala^165,182,189]-human somatotropin-(145–191), [Nle¹⁷⁰, Ala^165,182,189]-human somatotropin-(140–191), and [Lys^135,136,138, Glu^137,139, Nle¹⁷⁰, Ala^165,182,189]-human somatotropin-(135–191) have been synthesized by the solid-phase method. The synthetic peptides were assayed for growth-promoting activity and their potency was found to be comparable to that of [S-carbamideomethylcysteine^165,182,189]-human somatotropin-(141–191) which was derived from the native hormone.     

Control of peptide disulfide regioisomer formation by mixed cysteine–penicillamine bridges

While incorporation of penicillamine residues (Pen; β,β-dimethyl cysteine) into a peptide can cause dramatic changes in biological activity, the tendency of Pen to form mixed disulfides should also allow the exploitation of the steric bulk of the β-methyls as a synthetic device to control the production of disulfide isomers. That is, oxidation of a peptide containing an equal number of Cys and Pen residues should predominantly form products which contain mixed Cys–Pen disulfides. Endothelin (ET) is a 21 amino acid peptide which contains Cys at positions 1, 3, 11 and 15. While oxidation of ET tetrathiol has been reported to produce a 3:1 ratio of the natural 1–15, 3–11 to the unnatural 1–11, 3–15 isomers, we show that oxidation of ET analogs containing two cysteines and two penicillamines predominantly formed products containing Cys–Pen disulfides. Random oxidation (air, aqueous NH₄OH) of the tetrathiols of [Pen^1,11, Nle⁷]-ET-1 or [Pen^3,15, Nle⁷]-ET-1 produced the correct 1–15, 3–11 isomer in > 12:1 and > 22:1 ratios, respectively. Oxidation of the tetrathiol of [Pen^1,15, Nle⁷]-ET-1 favored the unnatural 1–11, 3–15 isomer by a 4:1 ratio, indicating that a normally contrathermodynamic disulfide isomer can become the favored product as a result of the driving force for penicillamine mixed disulfide formation. Disulfide isomers were identified using ion-spray mass spectrometry in conjunction with enzymatic and acid hydrolysis. [Pen^1,11, Nle⁷]-ET-1 was a partial agonist at the ETA receptor (EC₅₀= 7.5 nM in rabbit carotid artery rings; K_d= 4.5 nM in rat A10 cell membranes) while [Pen^3,15, Nle⁷]-ET-1 (EC₅₀= 0.9 nM; K_d= 0.7 nM) was a full agonist with similar potency to ET-1.     

Dehydro-enkephalins. III. Synthesis and biological activity of [ΔAla2, Leu5]-enkephalin

[ΔAla², Leu⁵]-enkephalin has been prepared and shown to be more active than the parent saturated enkephalin in a binding assay using rat brain membranes and [³H] dihydromorphine as a tracer. In a comparison of potencies against [³H] dihydromorphine and [³H]-[d -Ala², d -Leu⁵]-enkephalin as tracers, [ΔAla², Leu⁵]-enkephalin showed preference for μ opiate receptors, possibly due to the hydrophobicity of the ΔAla² residue. A synthetic tetrapeptide enkephalin [ΔAla²]-desLeu⁵-enkephalin had weak activity and high selectivity for the μ receptors. O-Acylation of a serine residue in the peptide was achieved by coupling between the peptide and a carboxylic acid using DCC and a catalytic amount of 4-dimethylaminopyridine.     

Characterization of a B2-bradykinin receptor in rat renal mesangial cells

The specific binding of bradykinin (BK) was investigated using membrane fractions from mesangial cells in primary culture, a cloned cell line, and in intact adherent cells with three different radiolabelled BK analogues: ¹²⁵I-[Tyr⁰]BK, ¹²⁵I-[Tyr⁵]BK and ¹²⁵I-[Tyr⁸]BK. The best radioligand was ¹²⁵I-[Tyr⁰]BK, and assay conditions were determined to ensure maximal stable binding. Binding appeared to be reversible and not to be inhibited by a wide variety of protease inhibitors including converting enzyme inhibitor and phosphoramidon. The maximum density of binding sites (B_max) was about 88 ± 18 fmol/mg protein, which is equivalent to about 6000 sites/cell, and the dissociation constant averaged 2 nM. No significant difference in B_max was observed between membranes from cells in primary culture and those from cloned cells. Of the BK analogues tested, unmodified BK exhibited the highest inhibition constant (close to 10⁻¹⁰ M). No displacement of ¹²⁵I-[Tyr⁰]BK was observed in the presence of the B₁ agonist des-Arg⁹-BK or several unrelated peptides, including atrial natriuretic factor and angiotensin I and II, whereas 50% inhibition of binding was achieved with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK (10⁻⁹ M). Addition of BK for 3 min to the incubation medium of cloned mesangial cells induced a dose- and time-dependent increase in PGE₂ unlike des-Arg⁹-BK, which showed no such effect. The secretion was strongly inhibited by prior incubation with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK. The pharmacological profile of the binding site determined with various BK agonists and antagonists, and the stimulating effect of binding site activation on prostaglandin release strongly suggest that B₂-kinin-like receptors are present in rat mesangial cells.     

New synthesis and biological activity of the cyclic tetrapeptides tentoxin and [Pro 1] tentoxin

The cyclic tetrapeptide tentoxin and the conformationally related analog [Pro¹] tentoxin have been synthesized by a new solution methodology. D, L-3-phenyl-serine was employed as a synthetic precursor to the Z-dehydrophenylalanine substituent. Increased yields of both compounds were obtained when a modified cyclization procedure was employed through ring closure from the N-terminal substituents of N-methylalanine and proline to a C-terminal glycine. Biological activities were determined in a seedling assay used to measure chlorosis induction. Both tentoxin and [Pro¹] tentoxin exhibited similar chlorosis inducing activity. Whole leaf extracts of chlorotic, [Pro¹] tentoxin-treated seedling leaves lacked active polyphenoloxidase when subjected to electrophoretic analysis. Coupling factor₁ (CF₁) ATPase isozymes were assayed for ATPase activity in 10μuM-100 μuM solutions of tentoxin and [Pro¹] tentoxin. CF₁ ATPase inhibition was observed for both tentoxin and [Pro¹] tentoxin. Inhibition of a single ATPase isozyme by tentoxin was alleviated at or above 50μuM while [Pro¹] tentoxin inhibited two CF₁ ATPases at concentrations up to 110μuM. No alleviation of ATPase inhibition was noted for higher concentrations of [Pro¹] tentoxin.     

Photolabeling identifies transmembrane domain 4 of CXCR4 as a T140 binding site

CXCR4, a G-protein-coupled receptor, which binds the chemokine stromal cell-derived factor 1 alpha (SDF-1α, CXCL12), is one of two co-receptors most frequently used by HIV-1 to infect CD4+ lymphocytes. The SDF-1α/CXCR4 axis is also involved in angiogenesis, in stem cell homing to bone marrow, in rheumatoid arthritis and in cancer. Here, we directly determined the binding site of the inverse agonist T140 on CXCR4 using photoaffinity labeling. Two T140 photoanalogs were synthesized containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in positions 5 or 10, yielding [Bpa⁵]T140 and [Bpa¹⁰]T140. Binding experiments on HEK293 cells stably expressing the wild-type CXCR4 receptor using ¹²⁵I-SDF-1α demonstrated that T140 and both photoanalogs had affinities in the nanomolar range, similar to SDF-1α. Photolabeling led to the formation of specific, covalent 42 kDa T140-CXCR4 complexes. V8 protease digestion of both CXCR4/¹²⁵I-[Bpa⁵]T140 and CXCR4/¹²⁵I-[Bpa¹⁰]T140 adducts generated a fragment of 6 kDa suggesting that the T140 photoanalogs labeled a fragment corresponding to Lys¹⁵⁴-Glu¹⁷⁹ of the receptor's 4th transmembrane domain. Further digestion of this 6 kDa fragment with endo Asp-N led to the generation of a shorter fragment validating the photolabeled region. Our results demonstrate that T140 interacts with residues of the fourth transmembrane domain of the CXCR4 receptor and provide new structural constraints enabling us to model the complex between T140 and CXCR4.     

Modifications of the cyclic mu receptor selective tetrapeptide Tyr‐c[d‐Cys‐Phe‐d‐Pen]NH2 (Et): effects on opioid receptor binding and activation

Abstract: The previously described cyclic mu opioid receptor‐selective tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Pen]NH₂ (Et) (JOM‐6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [³⁵S]‐GTPγS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr¹ with conformationally restricted phenolic amino acids. In the [³⁵S]‐GTPγS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range0.4–9 nm . However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC₅₀ value of less than 100 nm . Lastly, we have identified a peptide, d ‐Hat‐c[d ‐Cys‐Phe‐d ‐Pen]NH₂ (Et), with high potency and > 1000‐fold functional selectivity for the mu over delta opioid receptor as measured by the [³⁵S]‐GTPγS assay.     

Synthesis of 14C‐ and 14C3‐labelled Org 37462

Org 37462 (1) is the active ingredient in Orgalutran^®, an innovative product that reduces the time of treatment in in vitro fertilization from four to less than two weeks. Org 37462 is a synthetic decapeptide containing several amino acids that are unnatural in stereochemistry and/or in structure. The synthesis, starting with a ProtectingGroup‐D‐Ala‐resin, is a typical solid state synthesis. For the conduction of several metabolism studies, Org 37462 had to be labelled with carbon 14. It was decided to label D‐3‐(2‐naphthyl)alanine, the last amino acid to be coupled to the resin. We report the synthesis of [¹⁴C]‐ and [¹⁴C₃]‐Org 37462, starting from 2‐bromo‐[¹⁴C‐methyl]‐naphthalene and [¹⁴C₂]‐tert‐butyl glycinate. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of [2H4]oxymetazoline and [14C]oxymetazoline

An efficient synthesis of [²H₄] and [¹⁴C]oxymetazoline has been developed. Both compounds follow the same synthetic route with the introduction of the label occurring at different synthetic steps. The synthesis of [²H₄]oxymetazoline from [²H₄]ethylene diamine was achieved in one step with a 40% yield. The synthesis of [¹⁴C]oxymetazoline from potassium [¹⁴C]cyanide was achieved in two steps with an overall radiochemical yield of 67%. Copyright © 2010 John Wiley & Sons, Ltd.     

β-ENDORPHIN: SYNTHESIS AND ANALGESIC ACTIVITY OF SEVERAL ANALOGS MODIFIED IN POSITIONS 2 AND 5

The solid-phase syntheses of [Sar²]-, [Ala²]-, [D-Leu²]-, [D-Lys²]-β- endorphins and [Pro⁵]-, [Leu⁵]-, [D-Leu⁵]-, [D-Ala², D-Leu⁵]-β-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu² analog to be 48% as potent as β_h-endorphin while the Ala², D-Lys², Leu⁵, and [D-Ala², D-Leu⁵] analogs were 8 to 17% as active. The Sar², D-Leu⁵, and Pro⁵ analogs were less than 1% as potent.     

Radioiodination,purification, and evaluation of antihuman tumor-derived immunoglobulin G light chain monoclonal antibody in tumor-bearing nude mice

We report the synthesis and biological evaluation of ¹³¹I-labeled antihuman tumor-derived immunoglobulin G (IgG) light chain monoclonal antibody (4E9) ([¹³¹I]I-4E9) as a promising probe for tumor imaging. [¹³¹I]I-4E9 was synthesized in radiochemical yield of 89.9 ± 4.7% with radiochemical purity of more than 99%. [¹³¹I]I-4E9 showed high stability in normal saline and human serum. In cell uptake studies, [¹³¹I]I-4E9 exhibited favorable binding affinity and high specificity in HeLa MR cells. In biodistribution studies, [¹³¹I]I-4E9 showed high tumor uptake, high tumor/non-tumor ratios, and specific binding in BALB/c nu/nu mice bearing human HeLa MR xenografts. Single-photon emission computerized tomography (SPECT) imaging of [¹³¹I]I-4E9 in the HeLa MR xenograft model demonstrated clear visualization of tumor after 48 h and confirmed specific binding in tumor. These findings suggest that [¹³¹I]I-4E9 possesses favorable biological characteristics and warrants further investigation as a prospective probe for imaging and treatment of cancers.     

Comparative biological activities of potent active-site analogues of α-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several α-melanotropin (α-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ and Ac-[Tyr⁴]-α-MSH_4–11-NH₂ were compared with α-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ was found to be less active than Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr⁴]-α-MSH_4–11-NH₂ was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ over Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on frog melanocytes may be related to the fact that the shorter 4–10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of α-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle⁴]-α-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity. Similarly, incorporation of D-Phe-7 into Tyr-4 containing melanotropin fragments produced analogues Ac-[Tyr⁴, D-Phe⁷]-αMSH_4–10-NH₂ and Ac-[Tyr⁴, D-Phe⁷]-α-MSH_4–11-NH₂, which also exhibited greatly increased biological activity in all three assay systems. Both of these analogues were also found to have prolonged activity in the frog skin bioassay but little or no prolonged activity in the lizard skin bioassay. These two analogues turned out to be full agonists in the mouse melanoma adenylate cyclase bioassay and were equipotent to α-MSH. These results demonstrate that substitution of tyrosine for methionine at position-4 dramatically affects the potency and prolonged activity of these melanotropin analogues and the melanotropic activities observed as a result of such substitutions are themselves affected by concomitant substitutions at the 7(Phe) and 11 (Lys) positions of the analogues.     

Enkephalin in bovine adrenal gland: Multiple molecular forms of [met5]-enkephalin immunoreactive peptides

The existence and heterogeneity of enkephalin-like substances in bovine adrenal glands has been demonstrated using enkephalin radioimmunoassays. Both [met⁵]- and [leu⁵]-enkephalins were found to be highly enriched in the medulla i.e. 6.65 pmol [met⁵]-enkephalin/mg protein but only a very small amount, 0.041 pmol [met⁵]-enkephalin/mg protein, was detected in the cortex. In addition to the pentapeptide, [met⁵]-enkephalin, a large proportion of the total [met⁵]-enkephalin immunoreactive substance was found by gel filtration chromatography to be present in two high molecular weight forms. Determination of the true concentration of the high molecular weight forms, however, awaits purification and use of a proper standard. The existence of the different molecular forms of [met⁵]-enkephalin immunoreactive substances was substantiated by immunoabsorption experiment and gel filtration chromatography with 6 M guanidine. The two high molecular weight immunoreactive substances were not assayable by the radioreceptor binding assay; however, when trypsinized, they released a product which was capable of binding to the opiate receptor and was indistinguishable from the pentapeptide. [met⁵]-enkephalin, with respect to its size and thin layer Chromatographic characteristic. Whether the high molecular weight [met⁵]-enkephalin immunoreactive substances can function as precursors of [met⁵]-enkephalin in adrenal glands remains to be determined.     

PREPARATION OF A TWO-DISULFIDE BONDED ENZYMICALLY ACTIVE DERIVATIVE FROM HEN EGG LYSOZYME

A method has been developed for preparation of an enzymically active two-disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23° for 40 min. The products are reacted with [1-¹⁴C] iodoacetic acid and then purified by gel filtration and ion-exchange chromatography. An enzymically active derivative containing 4 mol of [1-¹⁴C] carboxymethyl groups and no free sulfhydryl groups is obtained in approximately 18% yield. Examinations of hydrodynamic volume, tryptophan fluorescence, CD and tryptic peptides containing [1-¹⁴C] carboxymethyl cysteine indicate that this derivative contains two presumably native disulfide bonds and two open disulfide bonds between Cys 6 and Cys 127 and between Cys 76 and Cys 94. The rest of the species in the incubation mixture are intact lysozyme. Thus, the species containing two presumably native disulfide bonds and four free sulfhydryl groups at Cys 6, Cys 76, Cys 94 and Cys 127 appears to be only the intermediate accumulating during reduction of lysozyme with dithiothreitol.     

Rapid and efficient synthesis of stable isotope labeled [13C4, D4]‐5‐(hydroxymethyl)thiazole: versatile building block for biologically interesting compounds

5‐(Hydroxymethyl)thiazole is a versatile building block for many biologically active compounds. A rapid and efficient four‐step synthesis of its stable isotope labeled counterpart with four ¹³C and four deuterium atoms in 32% total yield is reported. Condensation of [¹³C₂]‐chloro acetic acid with [¹³C]‐thiourea gave [¹³C₃]‐2,4‐thiazolidinedione. Reaction of [¹³C₃]‐2,4‐thiazolidinedione with phosphorus oxybromide and [¹³C, D]‐DMF (Me₂N¹³CDO) produced [¹³C₄, D]‐2,4‐dibromo‐thiazole‐5‐carboxaldehyde. The resultant aldehyde was then reduced by sodium borodeuteride to [¹³C₄, D₂]‐(2,4‐dibromo‐thiazol‐5‐yl)‐methanol. Catalytic deuteration of [¹³C₄, D₂]‐(2,4‐dibromo‐thiazol‐5‐yl)‐methanol by palladium black with deuterium gas at 1 atm pressure and room temperature produced completely de‐brominated [¹³C₄, D₄]‐5‐(hydroxymethyl)thiazole. De‐bromination of the 2,4‐dibromothiazole by the catalysis of palladium black provides a simple and convenient synthetic method for the stable isotope labeled and potentially radioactive isotope labeled thiazole compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

C‐Terminal 18F‐fluoroethylamidation exemplified on [Gly‐OH9] oxytocin

The no‐carrier‐added (n.c.a.) ¹⁸F‐fluoroethylamidation of the acid function of the protected nonapeptide Boc–Cys–Tyr(tBu)–Ile–Gln(Mtt)–Asn(Mtt)–Cys–Pro–Leu–Gly–OH forming the labelled peptide hormone derivative [Gly‐(2‐[¹⁸F]fluoroethyl)NH⁹]‐oxytocin is described. The labelling conditions were elaborated using a protected tripeptide, identical to the C‐terminal sequence of oxytocin. The prosthetic group n.c.a. 2‐[¹⁸F]fluoroethylamine was synthesised via cryptate mediated n.c.a. ¹⁸F‐fluorination of N‐Boc‐2‐(p‐toluenesulfonyloxy)ethylamine in DMSO (RCY: ca. 60%) and subsequent deprotection with a radiochemical yield of 46±5%. [¹⁸F]Fluoroethylamine was reacted with Z–Pro–Leu–Gly–OH in presence of the coupling reagent TBTU or with activated esters of the model‐tripeptide. The activated ester method as well as the condensation in presence of TBTU yielded ?90% of the ¹⁸F‐fluoroethyl‐amidated tripeptide. TBTU‐mediated condensation of n.c.a. 2‐[¹⁸F]fluoro‐ethylamine with the C‐terminal free acid group of protected oxytocin gave the radiochemical yield of about 75%. Deprotection under acidic conditions led to the formation of [Gly–(2‐[¹⁸F]fluoroethyl)NH⁹]oxytocin within 75 min with a radiochemical yield of about 30% as measured by analytical HPLC. Copyright © 2002 John Wiley & Sons, Ltd.