Proton NMR investigation of Ln3+ complexes of thymopoietin 32-36.

The pentapeptide Arg-Lys-Asp-Val-Tyr (TP5) is a biologically active fragment of thymopoietin, the thymic hormone that induces selective T-cell differentiation. The formation of lanthanide(III) complexes of TP5 is demonstrated through the observation of Tb3+ fluorescence enhancement. The equilibria, stoichiometry and solution conformation of the La3+, Pr3+ and Yb3+ complexes of TP5 have been investigated using NMR spectroscopy. In addition, the dissociation constants of two methyl ester analogs of TP5 have been studied. Evidence is presented supporting an interaction between the arginine guanidino N epsilon H and the aspartate carboxylate of TP5. Binding of Ln3+ appears to be accompanied by a disruption (or weakening) of this interaction and a concomitant increase in the 180 degrees rotamer population for the aspartate carboxylate group. The observed trends in the magnitudes of the dissociation constants and the rotamer populations appear to suggest that, although a significant amount of monodentate complexes may also exist, the metal ion binds predominantly to both carboxylates in a bidentate fashion.

Synthesis of a proposed thymic factor.

Less than Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn, a proposed serum thymic factor, has been synthesized. The protected precursor, less than Glu-Ala-Lys(i-Noc)-ser(Bzl)-Gln-Gly-Gly-Ser(Bzl)-Asn, was prepared by a combination of solid phase and solution methods. The benzyl blocking groups were removed by HF and the i-Noc blocking group was removed by catalytic hydrogenation.

Biologically active analogs of thymopentin with enhanced enzymatic stability.

Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32-36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro2-TP5-NH2 and Aib2-TP5-NH2 retained activity and were shown to exhibit a high degree of stability when incubated in human serum.

Detection and quantitation of low levels of protected and unprotected (R)-amino acids in the synthesis of thymopentin, an immunoregulatory peptide.

Two chiral chromatographic procedures (HPLC and capillary GC) were developed to monitor the extent of epimerization of protected and unprotected amino acids used in a synthesis of thymopentin, an immunoregulatory peptide currently in clinical trials. The capillary GC method allowed the detection of the (R)-enantiomer in the presence of the (S)-enantiomer at levels of greater than or equal to 0.2%, while the HPLC method allowed similar detection at levels of greater than or equal to 0.1%.

Synthetic immunostimulants (excluding sulfur-containing compounds).

Many different approaches have been used, over the last 15 years, for the design of potential immunostimulating drugs: Fractionation of crude natural substances (of eukaryotic or prokaryotic origin) already known to enhance immune functions, followed by chemical characterization and, in many cases, full synthesis of the active moiety: examples are provided by thymic hormones and the muramyldipeptide (MDP); Chemical modification of natural substances of known chemical structure in order to potentiate or change their biological activities or reduce their toxicity: murabutide, lipophilic MDP derivatives, lipopeptides (such as pimelautide), tuftsin analogs; Chemical synthesis (often without preconceived ideas about structure-activity relationship) of a great variety of molecules which are then screened in vitro and in vivo for immunopharmacological activity. The chemical structures and the biological profiles (in terms of possible primary cellular targets and mechanisms of immunostimulating activities) of representatives of class (b) and class (c) immunostimulants are reviewed in this paper.

Synthesis of the C-terminal half of thymosin alpha 1 by the polymeric reagent method.

In this report we further show the utility and efficiency of polymer-bound 1-hydroxybenzotriazole (PHBT) as an almost ideal support for the polymeric reagent method of peptide synthesis. This was demonstrated by the synthesis of thymosin alpha 1 (15-28), in which two suitably blocked segments, Boc-Asp (OtBu)-Leu-Lys (2Cz)-Glu (OBzl)-Lys (2Cz)-Lys (2Cz)-OH (3) and Boc-Glu (OBzl)-Val-Val-Glu (OBzl)-Glu (OBzl)-Ala-Glu (OBzl)-Asn-OBzl (2), were prepared entirely by utilizing PHBT activation for each coupling step. After appropriate deblocking of 2, segments 2 and 3 were coupled by the DCC-HOBT method, followed by complete deblocking and ion-exchange chromatographic purification, affording the C-terminal half of thymosin alpha 1, H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (1).

Side-reactions in peptide synthesis. 2. Formation of by-products in the large-scale synthesis of THF-gamma 2 by solution synthesis.

The impurities which formed in the large-scale synthesis of THF-gamma 2, an immunomodulatory peptide of formula H-Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu-OH, were identified by a combination of analytical methods, and their structure confirmed by synthesis. Most impurities originated from side-reactions involving the aspartyl residue (cyclization, beta-aspartyl formation and cleavage). Based on this knowledge, modifications were introduced into the work up and the purification procedure which resulted in a very pure final product (> 99% by RP-HPLC).

Synthesis of analogs of the serum thymic nonapeptide, "facteur thymique serique" (FTS). Part I.

Analogs of FTS (Facteur Thymique Sérique), less than :formula: (see text), a circulating thymic factor, were prepared by replacing the amino acid residues in positions 1, 2, 8 and 9, or by shortening the nonapeptide chain at the N- or C-terminal end. These peptides were synthesized by two different schemes using the conventional synthesis in solution.

Synthesis of an immunogenic conjugate and of two 125I-labelled derivatives of the "facteur thymique sérique".

[4-Aminobenzoyl-Gln1]FTS was synthesized from a suitably-protected derivative of the "facteur thymique sérique" (FTS). It was diazotized and coupled to bovine serum albumin, yielding an immunogenic conjugate in which all the functional groups of the peptide remained intact. [4-Aminobenzoyl-Gln1]FTS and another synthetic compound, [3-(4-hydroxyphenyl)propionyl-Gln1]FTS, were 125I-labelled. The binding of these radioactive peptides of the anti-FTS antibodies obtained with our immunogenic conjugate was inhibited by unlabelled FTS.

Synthesis and immunological effect of thymic humoral factor-gamma-2 analogues.

Nine analogues of thymic humoral factor (THF)-gamma-2 were prepared by the solid-phase method, and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin(PHA)-stimulated T-lymphocytes of uremic patients with infectious diseases were examined. The results were as follows: [Arg6]-THF-gamma-2 exhibited higher restoring activity than that of our synthetic THF-gamma-2. [Sar4]-, [Val1]-, [Arg3]-, [Gly5]-, and [Asn3]-THF-gamma-2 were also active but less potent than that of our synthetic THF-gamma-2. Three other peptides, [beta-Ala4]-, [Arg2]-, and [Gln2]-THF-gamma-2, did not show any restoring activity on the impaired blastogenic response of uremic patients with infectious disease.

Solid-phase synthesis of thymosin alpha 1 using tert-butyloxycarbonylaminoacyl-4-(oxymethyl)phenylacetamidomethyl-resin.

Thymosin alpha 1 and its desacetyl analogue were synthesized by the solid-phase method. Use of aminoacyl-4-(oxymethyl)phenylacetamidomethyl-resin resulted in an improved yield and allowed the synthetic products to be purified by simple ion-exchange and gel filtration chromatography. Success of the synthesis was largely due to enhanced stability of the peptide-resin linkage to trifluoroacetic acid and to the elimination of hydroxy functions on the resin. This improved quality of the solid support helps eliminate chain loss and chain termination during the synthesis. The purified synthetic peptides were found to be homogeneous by paper electrophoresis, isoelectric focusing in polyacrylamide gel, and thin-layer chromatography. They also had biological activity in the azathioprine-sensitive rosette assay. Use of the new 9-(2-sulfo)fluorenylmethyloxycarbonyl chloride reagent for purification of protected peptides was also demonstrated and discussed.

Syntheses and effects of human splenin (hSP) fragment 32-48 and an analog on the reduced B-lymphocytes of uremic patients.

A heptadecapeptide, H-Arg-Lys-Ala-Val-Tyr-Val-Glu-Leu-Tyr-Leu-Gln-Ser-Leu-Thr-Ala-Glu-His-OH , corresponding to amino acids 32 to 48 of human splenin (hSP) and an analog in which the amino acid residue at position 34 is changed from Ala to Glu, were synthesized. These peptides were synthesized using conventional solution synthesis and were tested for their effect on reduced B-lymphocytes of uremic patients. Incubation of peripheral lymphocytes isolated from uremic patients with these two synthetic heptadecapeptides, hSP fragment 32-48 and [Glu34]hSP fragment 32-48, had an enhancing effect on the reduced B-lymphocytes, but synthetic bovine thymopoietin II (bTP-II) fragment 32-49 had no effect under the same conditions.

Immuno-regulating peptides, I. Synthesis and structure-activity relationships of thymopentin analogs.

Seventeen peptides, related to thymopoietin pentapeptide, L-arginyl-L-lysyl-L-aspartyl-L-valyl-L-tyrosine (thymopentin) were synthesized by the stepwise strategy in solution. Of these, L-arginyl-L-lysyl-L-aspartic acid and L-arginyl-L-lysyl-L-aspartyl-L-valine, shortened from the C-terminus of the pentapeptide, exhibit significant immuno-stimulating potencies, exceeding those of thymopentin, both in vitro and in vivo immunological tests. Studies on the structure-activity relationships suggest that the potencial active site of thymopoietins is very sensitive to the N- and C-terminal elongations of the peptide chain. For thymic hormones, an active site carrying cumulative chemical signals is proposed instead of well-defined active centers.

Solid-phase synthesis of thymosin beta 4: chemical and biological characterization of the synthetic peptide.

The chemical synthesis of thymosin beta 4 using a solid-phase procedure has been accomplished. The synthetic product was found to be homogeneous on paper electrophoresis at pH 6.5, high-performance liquid chromatography on a reversed-phase column, and isoelectric focusing using polyacrylamide gels. The synthetic material was also shown to be identical with the natural thymosin beta 4 by tryptic peptide mapping, amino acid compositional analyses, and polyacrylamide gel isoelectric focusing. Biologically, synthetic thymosin beta 4 was found to be as active as the natural compound in a terminal deoxynucleotidyltransferase induction assay and in a macrophage migration inhibition assay. The proposed structure of the peptide hormone was thus confirmed by a chemical synthesis.

[Biochemical characterization of a circulating thymic factor]. / Caractérisation biochimique du facteur thymique circulant

A serum thymic factor has been isolated from pig serum. Its amino-acid sequence has been determined by Edman technique, modified by Gray: Gln-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn. A nonapeptide, synthesized on the basis of this sequence is active in the rosette assay used for the isolation of the biological product, both in vitro (10(-15) M) and in vivo (0.1 ng per Mouse).

Synthesis of human splenin (hSP) and examination of its immunological effects on the impaired T- and B-lymphocytes in uremic patients.

Human splenin (hSP) was synthesized by assembling eight peptide fragments followed by deprotection with 1M trifluoromethanesulfonic acid-thioanisole (molar ratios, 1:1) in trifluoroacetic acid in the presence of m-cresol and dimethylselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. Incubation of peripheral lymphocytes isolated from uremic patients with the synthetic hSP showed an enhancing effect on the reduced B-lymphocytes, but had no restoring effect on the impaired blastogenic response of T-lymphocytes.

Synthesis of [Glu34]human splenin (hSP) and examination of its immunological effect on the reduced B-lymphocytes of uremic patients.

[Glu34]human splenin (hSP) was synthesized in a conventional manner by assembling ten peptide fragments followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratio, 1:1) in trifluoroacetic acid in the presence of m-cresol and dimethylselenide. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. Incubation of peripheral lymphocytes isolated from uremic patients with the synthetic [Glu34]hSP showed an enhancing effect on the reduced B-lymphocytes, but synthetic human thymopoietin (hTP) had no effect under the same conditions.