Further studies into the Boc/solid-phase synthesis of Ser(P)- and Thr(P)-containing peptides

The Ser(P)-containing peptide corresponding to phospholamban 11-19, Ac-Ala-Ile-Arg-Are-Ala-Ser(P)-Thr-Ile-Glu-NH₂, was prepared by the use of Boc-Ser(PO₃Ph₂)-OH in Boc/solid-phase peptide synthesis followed by HF cleavage of the peptide from the polystyrene resin and subsequent platinum-mediated hydrogenolytic cleavage of the phenyl phosphate groups. A study of the HF deprotection step showed that extensive dephosphorylation of the Ser(PO₃Ph₂)-residue occurred using three commonly used HF conditions and gave rise to large quantities of the Ser-containing peptide. The subsequent study of model peptide systems under standard HF conditions established firstly that the extent of dephosphorylation was dependent on the HF-contact time, and secondly that the Ser(PO₃Ph₂) residue underwent dephosphorylation at a slightly higher rate than the Thr(PO₃Ph₂) residue. © Munksgaard 1994.     

SYNTHESIS OF ANALOGS OF THE SERUM THYMIC NONAPEPTIDE, “FACTEUR THYMIQUE SERIQUE” (FTS) Part II

New analogs of FTS (Facteur Thymique Sérique), <Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn · OH, a circulating thymic factor, were prepared by replacing the amino acid residues in positions 1, 3, 4, 5, 6 and 3 and 6 together. Five other analogs of C-terminal heptapeptide were prepared by replacing the amino acid residues in position 3 or 6. These peptides were synthesized using conventional synthesis in solution.     

Acidolytic cleavage of tris(alkoxy)benzylamide (PAL) “internal reference” amino acyl (IRAA) anchoring linkages: validation of accepted procedures in solid-phase peptide synthesis (SPPS)

Under the normal conditions of acidolytic cleavage/deprotection of tris(a1koxy)benzylamide (PAL) anchoring linkages in Fmoc solid-phase peptide synthesis (SPPS), product release occurs by a straightforward single-step pathway. A recently reported cleavage of the NH-VH bond of an amino acyl residue adjacent to PAL [see Int. J. Peptide Protein Res. 38 , 146–153 (1991)] could not be confirmed in novel experiments incorporating a double “internal reference” amino acid (IRAA) design. The results of the present work revalidate the widely accepted application of IRAAs to monitor yields in SPPS, and confirm the reliability of PAL methodology for the preparation of C-terminal peptide amides.     

“Carba” peptide bond surrogates Different approaches to Gly-Ψ(CH2-CH2)-d,l-Xaa pseudo-dipeptide units

Racemic “carba” pseudo-dipeptide units such as Gly-Ψ(CH₂-CH₂)-d,l -Xaa were obtained either through the Horner-Emmons condensation of N-tert.-butyloxycarbonyl-βalaninal with the appropriate substituted triethyl phosphonoacetate, or from commercially available 3-carbethoxy-2-piperidone.     

Efficient solution phase synthesis of [l-(β-mercapto-β, β-cyclopentamethylenepropionic acid)- 2-(O-ethyl-d-tyrosine)-4-valine-9-desglycine]arginine vasopressin

A convergent synthesis of the peptide [1-(β-mercapto-β,β-cyclopentamethylenepropionic acid)-2-(O-ethyl-d -tyrosine)-4-valine-9-desglycine]arginine vasopressin (1), based on the classical solution phase method, was developed. The molecule is assembled by a 3 + 4 coupling via the amide method; then the disulfide bridge is installed by iodine treatment of the bis-acetamidomethyl protected thiols, and the terminal arginine amide added by a 7 + 1 coupling. The method (see Scheme 1) has been used to prepare gram quantities of 1 in more than 98% purity and in 13% yield (based on tetrapeptide intermediate 13) after a single stage purification. The method appears to be particularly suitable for the large scale preparation of 1 and other vasopressin congeners. A novel, albeit low level, transfer of acetamidomethyl group from the sulfur of cysteine to the asparagine amide side-chain was detected following hydrogen chloride treatment of Boc-containing intermediates.     

Applications of BOP reagent in solid phase synthesis Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1–29)-NH2

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219–1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22–29) and (13–29) fragments of [Ala¹⁵]-GRF(1–29)-NH₂. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala¹⁵]-GRF(1–29)-NH₂ was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1–29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (<10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.     

3-(ω-Aminoalkyl)-5,5-dialkyl(or spirocycloalkyl-1′,5-)hydantoins as New 5-HT1A and 5-HT2A Receptor Ligands

A series of new 3-(ω-aminoalkyl)-5,5-disubstituted hydantoins, containing 1-phenylpiperazine, 1-(o-methoxyphenyl)piperazine or 1,2,3,4-tetrahydroisoquinoline fragments, were synthesized by standard alkylation procedures and their 5-HT_1A and 5-HT_2A receptor affinities were determined. It has been shown that the investigated derivatives are recognized by 5-HT_1A and 5-HT_2A receptors due to the presence of a 1-arylpiperazine fragment; however, the terminal hydantoin moiety plays an important role in stabilization of the receptor-ligand complex. It has also been found that the two 1-phenylpiperazine derivatives 32 and 36 are new, selective 5-HT_2A receptor ligands (K_i = 34 and 37 nM, respectively), whereas the derivative of 1-(o-methoxyphenyl)piperazine ( 38 ) is a new, highly potent 5-HT_1A receptor ligand (K_i = 0.51 nM) with a moderate affinity for 5-HT_2A receptors (K_i = 213 nM).     

A convenient general method for synthesis of Nα- or Nω-dithiasuccinoyl (Dts) amino acids and dipeptides: application of polyethylene glycol as a carrier for functional purification*

N ^α-Dithiasuccinoyl (Dts) amino acids ( 1 ) needed for solid-phase peptide synthesis have been prepared in good yields and excellent purities by a new method that exploits the solubility properties of polyethylene glycol (PEG; bifunctional with average molecular weight 2000 was found to be optimal). Suitably side-chain protected amino acid derivatives are first reacted with a polymeric xanthate ( 11 ), following which the free α-carboxyl is blocked by silylation and the Dts heterocycle is elaborated in the same pot by reaction with chlorocarbonylsulfenyl chloride ( 4 ). Upon aqueous workup, the polymeric carrier removes any urethane blocked amino acids which arise during the process. Exaggerated conditions were explored to prove the power of this functional purification approach, and mechanisms of formation of polymer-bound urethanes are proposed and supported by solution model studies. The preparation and characterization of the companion N-(iso-propyldithio)carbonyl derivative of proline is also presented.     

Synthesis of Z- and E-1-Methyl-2-(1-hydroximinoethyl)-6-methoxy-3,4-dihydronaphthalene and Evaluation as Inhibitors of 17α-Hydroxylase-C17,20-Lyase (P450 17)

The synthesis and biological evaluation of Z- and E-1-methyl-2-(1-hydroximinoethyl)-6-methoxy-3,4-dihydro-naphthalene ( Z-1 and E-1 ) as nonsteroidal inhibitors of 17α-hydroxylase-C17,20-lyase (P450 17, CYP17) is described. Z-1 and E-1 were separated by column chromatography and identified by ¹H NMR. The synthesis of the key compound 3 was accomplished by a new reaction acetylating the 1-methyl-6-methoxy-3,4-dihydronaphthalene compound 2 under Friedel-Crafts conditions. Compound 2 was obtained from the 1-tetralone via Wittig reaction. Using a microsomal fraction of human testicular enzyme, Z-1 and E-1 inhibited the target enzyme only marginally.