Synthesis of a new biological response modifier thyrnosin β4 analogue and its restorative effect on depressed

Thymosin β4 is a polypeptide isolated from thymosin fraction 5. This peptide exhibits important activities in the regulation and differentiation of thymus-dependent lymphocytes. An analogue of thymosin β4, [Phe(4F)12] deacetyl- thymosin β4, was synthesized by a solution method, followed by deprotection with 1 M trifluoromethanesulphonic acid (TFMSA)-thioanisole (molar ratio, 1:1) in trifluoroacetic acid (TFA) in the presence of dimethlselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulphoxide on the methionine side chain. The synthetic [Phe(4F)12]deacetyl-thymosin β4 was found to have a restoring effect on the impaired blastogenic response of T-lymphocytes isolated from uraemic patients with recurrent infectious diseases. This analogue exhibited stronger restorative activity than that of our synthetic deacetyl-thymosin β4.


Introduction
The impairment of immunological responsiveness in uraemic patients is well documented. 1'2 All aspects of the immune response appear to be affected by the uraemic state. The numbers, subpopulations and reactivities of circulating lymphocytes may be altered by uraemia. 2 '3 This impairment has been implicated in easy susceptibility to infections and increased incidence of malignancy. In addition, various investigators have reported severe structural changes in the lymph nodes 4 and thymus glands of uraemic patients and animals.
The thymus plays an essential role in the development and maintenance of cellular immune competence. 6 Recent evidence suggests that the thymus produces biologically active peptides which are responsible for the differentiation and functional maturation of precursor T-lymphocytes. 7 Thymosin f14, an N-terminal acetylated peptide containing 43 amino acid residues, was first isolated from calf thymus by Low et al. 8
In our previous papers, 11 we reported syntheses of deacetyl-thymosin f14 and its fragments, and showed that the synthetic deacetyl-thymosin f14 and some of the fragments could have restorative effects on the impaired cell-mediated immunological functions.
So other portions of thymosin f14 may also be necessary for the full activity.
In 1989, Maeda et a/. 12  Boc-Phe(4F)-NHNH-Troc (1.9 g) was treated with TFA-anisole (19 ml-3.8 ml) in an ice bath for 40 min, and TFA was then removed by evaporation. The residue was washed with n-hexane, dried over KOH pellets in vacuo for 2 h, and then dissolved in DMF (10 ml) containing NMM (0.6ml). To this solution, Boc-Lys(Z)-OSu (2.1 g) was added, and the mixture was stirred at room temperature for 7h. The mixture was extracted with EtOAc and then washed successively with 5% NaHCO, H20 5O/o citric acid and H20 dried over MgSO4, and concentrated in vacuo. The residue was reprecipitated from EtOAc with n-hexane. This compound was prepared essentially in the same manner as described for the preparation of I using I (2.5 g) and Boc-Glu(OBzl)-OSu (1.7 g). The product was reprecipitated from EtOAc with ether. Compound (1 g) was treated with TFA-anisole (10 ml-2 ml) as described above. Boc-Met(O)-Su (272 mg) was added to a solution of this product in DMF (10 ml), followed by NMM to keep the solution slightly alkaline.
After 8 h at room temperature, the reaction mixture was poured into 5% NaHCO3 with stirring. The precipitate thereby formed was washed successively with 5% NaHCO3, H20, citric acid and H20. The compound was prepared from VI (798 mg) and Boc-Asp(OBzl)-OSu (232 mg) essentially as described for the preparation of VI. The dried product was reprecipitated from hot EtOAc. Yield (3 eq)] in DMF-DMSO (1:1, 2 ml) and NMM (0.042 ml) were added to the above ice-chilled solution and the mixture was stirred at -10C for 36 h until the solution became ninhydrin-negative. After addition of a few drops of AcOH, the mixture was poured into 5% citric acid with stirring. The resulting powder was washed with 5% citric acid, H20 and MeOH and purified by gel filtration on Sephadex LH-60 using DMSO containing 3% H20 as an eluent. The desired fractions (each 5 ml, tube nos 45-56) were combined, the solvent was evaporated off, and the residue was treated with MeOH to afford a powder.  H-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe(4F)-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser-OH (corresponding to [Phe(4F)12]deacetyl-thymosin f14 [Xl]. The protected tritetracontapeptide ester X (50 mg) was treated with 1 M TFMSA-thioanisole in TFA (2 ml) in the presence of MeiSe (50/,tl) in an ice bath for 120 min, then dry ether was added. The resulting powder was collected by centrifugation, dried over KOH pellets for 2 h and dissolved in 1 N AcOH (5 ml). After being stirred with Amberlite IRA-400 (acetate form, approximately 1 g) for 30 min, the solution was adjusted to pH 6.0 with 1 N AcOH and the solution was lyophilized. The residue was dissolved in H20 (5 ml). The solution, after addition of dithiothreitol (10 mg), was incubated at 60C for 36 h. The solvent was evaporated in vacuo and the residue was dissolved in a small amount of Sephadex G-25 (2.4 x 93 cm), which was eluted with 1% AcOH. Individual fractions (4 ml each) were collected and the absorbance at 260 nm was determined for each fraction. The fractions corresponding to the first main peak (tube nos 52-61) were combined and the solvent was removed by lyophilization. The residue was dissolved in H20 (2 ml) and the solution was applied to a column of DEAE-cellulose (Brown, 2.4 x 50cm), eluted with a linear gradient of 400 ml each of H20 and 0.08 M NH4HCO3 buffer at pH 7.8. Individual fractions (4 ml each) were collected and the absorbance at 260nm was determined. The main peak present in the gradient eluates (tube nos 69-77) were combined and the solvent was condensed to approximately 2 ml. This solution was then applied to a column of Sephadex G-25 (2.4 x 93 cm), which was eluted with 1% AcOH. The desired fractions (4 ml each, tube nos 50-59) were collected and the solvent was removed by lyophilization to give a fluffy powder. Yield tolidine-positive spot. The synthetic peptide exhibited a single spot on a paper electrophoresis: Toyo Roshi No. 51 (2 x 40 cm) at pH 7.1 pyridiniumacetate buffer; mobility, 1.5 cm from the origin toward the anode, after running at 2 mA, 600 V for 80 min. The synthetic peptide exhibited a single peak on HPLC using an analytical Nucleosil 5C18 column (4 x 150mm) at a retention time of 15.48 min, when eluted with a gradient of acetonitrile (20-45%) in 0.1% TFA at a flow rate of 1 ml/min (Fig. 3). Amino

Results
From a synthetic viewpoint, compared with our previous syntheses of deacetyl-thymosin/349 and its fragments, 1'11 the thioanisole-mediated TFMSA deprotection procedure 13 was applied in the final step of the present synthesis instead of catalytic hydrogenation or hydrogen fluoride.
The Met residue was reversibly protected as its sulphoxide in order to prevent partial S-alkylation during the N%TFA deprotection as well as partial air oxidation during the synthesis. The Boc group, removable by TFA, was adopted as a temporary N%protecting group for every intermediate. Troc group is known to be cleaved by zinc is in AcOH without affecting other functional groups.
Throughout the synthesis, the purity of every intermediate was checked by thin-layer chromatography, elemental analysis and amino acid analysis of acid hydrolysates. The analytical results were within -t-0.4% of theoretical values in all cases.
The protected octapeptide fragment [VII], Boc-(5-12)-NHNH-Troc, was prepared stepwise by the Su active ester procedure to yield Boc-Asp(OBzl)-Met(O)-Ala-Glu(OBzl)-Ile-Glu(OBzl)-Lys(Z)-Phe(4F)-NHNH-Wroc [VIII, and the Boc groups of intermediates were removed by treatment with TFA-anisole prior to the next coupling reaction. The protected octapeptide fragment [VII] thus obtained was treated with zinc is in AcOH to remove the Troc group, and the zinc acetate was removed by treatment with EDTA to give the required hydrazide, Boc-(5-12)-NHNH2 [VIII], in an analytically pure form. The hydrazine test on the thin-layer chromatogram and elemental analysis data were consistent with homogeneity of the desired product. The three fragments were successively condensed by the azide procedure according to the route shown in Fig.1.
Every reaction was carried out in a mixture of DMF and DMSO and the amount of the acyl component was increased from three to four equivalents as the chain elongation progressed.
Every product was purified either by precipitation from DMSO with MeOH or by gel filtration on Sephadex LH-60. Throughout the synthesis, Gly was used as a diagnostic amino acid in acid hydrolysis. By comparison of recovery of Gly with those newly incorporated amino acids, satisfactory incorporation of each fragment was ascertained.
In the final step of the synthesis, the protected tritetracontapeptide ester was treated with 1 M TFMSA-thioanisole in TFA in the presence of MeiSe, which was employed to facilitate acidic cleavage of protecting groups. The unprotected peptide was next precipitated with peroxide-free ether, converted to the corresponding acetate with Amberlite IRA-400 (acetate form) and then treated with 1 N NH4OH at pH 8.0 to reverse a possible N O shift at the Ser and Thr residues. The Met (O) residue was reduced back to Met in two steps, firstly with thioanisole and MeiSe during the above acid treatment, and secondly with dithiothreitol during incubation of the unprotected peptide. The reduced product was purified by gel filtration on Sephadex G-25, followed by ion-exchange column  Interestingly, our synthetic [Phe(4F)li]deacetyl thymosin f14 showed stronger restoring activity than that of our synthetic deacetyl-thymosin f14. In this study, the strong electron withdrawing fluoride group on the para position of the aromatic ring results in an analogue that possesses stronger activity than that of the parent molecule.
Maeda et al. also reported 12 that [Phe(4F)4] Leuenkephalin showed stronger activity in the guinea-pig ileum and mouse vas deferens assays as compared with Leu-enkephalin. These results might suggest that modification of the Phe residue of thymosin f14 could produce more potent analogues capable of a restorative effect on impaired blastogenic response of T-lymphocytes.

Discussion
We have reported 9-11 evidence of impaired immune function in patients with chronic uraemia. This impairment is reflected in both in vitro and in vivo depressed cell-mediated immune function.
Patients with chronic uraemia may have thymic atrophy. The thymus may show a marked reduction in lymphoid elements and extensive replacement infiltration with fat. Cystic degradation may be seen.
These observations and findings suggested to us that the cell-mediated immune abnormalities seen in chronic uraemia might be attributable to thymic hormone deficiency. The test involves the in vitro incubation of patient lymphocytes with the synthetic peptides and the results of incubations with the [Phe(4F)12]deacetyl-thymosin f14 and deacetyl-thymosin f14 are compared.