SOLID PHASE SYNTHESIS OF THE PROTECTED 27 – 42 HEXADECAPEPTIDE OF THE HEAVY CHAIN FROM MYELOMA IMMUNOGLOBULIN M603 Elimination of Side Reactions Associated with Glycyl-2-oxypropionyl-resin

A fully protected 27–42 hexadecapeptide of the variable region of myeloma immunoglobulin M603 was synthesized on a 2-bromopropionyl-resin by the solid phase method. Side reactions due to cyclization of glycyl-2-oxypropionylresin were studied under different reaction conditions. The loss of peptide chains at the dipeptide and tripeptide stages due to diketopiperazine formation was also examined. These side reactions were circumvented by using a combination of fragment and stepwise coupling methods. The synthesized protected peptide was removed from the resin in 85% yield by photolysis, and purified by crystallization and by chromatography on a Sephadex LH-60 column.     

Multiple peptide synthesis using a single support (MPS3)

An automated multiple peptide synthesis method to synthesize, cleave, and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods. The approach relies on coupling of amino acid mixtures to the resin and it has been found that DCC/HOBt gives the best coupling performance. Fast Atom Bombardment Mass Spectrometry (FAB-MS) was used to rapidly and efficiently identify the peptides in each synthetic mixture which significantly assisted the purification process by HPLC. The method has been successfully applied to the synthesis of magainin 2 and angiotensinogen peptides.     

Biologically active conformations of thymopentin Studies with conformationally restricted analogs

Four cyclic analogs of thymopentin were synthesized and evaluated for biological activity on the human T cell line CEM. Three of these conformationally restricted analogs were biologically active. The one analog which most closely mimicked the conformation predicted from NMR and theoretical energy minimization calculations proved to be inactive. These studies establish that the biologically active conformations of thymopentin differ from the most probable conformation predicted from solution NMR and theoretical energy minimization studies.     

Synthesis of the protected tridecapeptide (56–68) of the VH domain of mouse myeloma immunoglobulin M603 and its reattachment to resin supports

A protected tridecapeptide, representing a new peptide corresponding to residues 56–68 of the V_H domain in the mouse M603 myeloma protein, has been prepared by solid phase peptide synthesis. The protected tridecapeptide was prepared using the photolabile 4-bromomethyl-(3-nitro)-benzamidomethyl-resin and the multidetachable 2-[4-bromomethyl)phenylacetoxy] propionyl-resin as solid supports. The synthetic protocol and protecting groups were the same for both syntheses. The protected tridecapeptide was removed photolytically from both supports and the sequence integrity was determined by preview analysis using the solid phase Edman degradation procedure. The protected tridecapeptide-OMPA was purified to homogeneity by DMF/H₂O precipitation and LH-60 chromatography. The purity of the protected peptide was further demonstrated by high pressure liquid chromatography on the free peptide after HF deprotection. The protected tridecapeptide was reattached to 4-bromomethyl-(3-nitro)-benzamidomethyl-resin to give the photolabile Boc-(protected) peptidyl-4 - oxymethyl - (3 - nitro) benzamidomethyl - resin in 25% yield. The protected tridecapeptide-oxymethylphenylacetic acid derivative was reattached to aminomethyl-resin to give Boc -(protected)peptidyl-2-[(4-oxymethyl)phenyl]acet-amidomethyl-resin in 45% yield and to 2-bromopropionyl-resin generating the multidetachable Boc - (protected)peptidyl - 2 - [(4 - oxymethyl) phenylacetoxy] propionyl-resin in 80% yield. The reactivity of these reattached peptides was demonstrated by the quantitative coupling of Boc-leucine to the protected peptide-resin. The advantages and disadvantages of the different resins with respect to solid phase fragment synthesis are discussed.     

Aminopeptidase resistant Arg-Gly-Asp analogs are stable in plasma and inhibit platelet aggregation

Tetrapeptides containing the sequence Arg-Gly-Asp (RGD) antagonize fibrinogen binding to its platelet receptor (gp IIb/IIIa, integrin α_11bβ₃) and inhibit platelet aggregation in vitro. The peptides RGDS and RGDY(Me)-NH₂ were rapidly degraded when incubated in human, rat, and dog plasma. HPLC analysis indicated that amino acids were sequentially removed from the peptide N-terminus, and this degradation was prevented by the aminopeptidase inhibitor bestatin. Analogs of RGDY(Me)-NH₂ with an acetylated or deleted α-amino group were prepared. Both analogs were stable when incubated in plasma, blocked ¹²⁵I-fibrinogen binding to activated platelets (IC₅₀= 10–30μm ) and inhibited ADP induced platelet aggregation (IC₅₀= 10–30μm ). This study concludes that aminopeptidase rapidly degrades RGD peptides in plasma, an important issue for in vivo testing of RGD peptides and analogs. RGD analogs intrinsically stabilized against aminopeptidase are stable in plasma and are important tools for antithrombotic studies involving antagonism of gp IIb/IIIa.