甲酸铵催化转移氢化还原肽链中的芳香硝基——对氨基苯丙氨酸的间接引入

研究了用甲酸铵催化转移氢化法(AF-CTH)对不同类型肽中的芳香硝基的还原行为, 这些肽类化合物包括促黑激素(MSH: 四肽)、促黄体素释放激素(LHRH: 十肽)和强腓肽(十七肽)的类似物. 用HPLC对还原过程进行了跟踪监测, 结果显示, 除含对氯苯丙氨酸残基的LHRH类似物因发生脱氯副反应不适合用AF-CTH还原外, 其余序列还原过程中均无明显副反应发生, 硝基几乎定量地转化成为相应的氨基, 实现了对氨基苯丙氨酸向肽链的间接引入. 另外发现, 硝基还原所需的时间与肽链长度有关, 肽链越长, 还原所需时间越长, 但与其在序列中的位置关系不明显.     

Tricarbonylchromium tryptophan derivatives and their use in peptide synthesis

Incorporation of a Cr(CO)₃ ligand into the indole ring of N-α-t-butoxycarbonyl-tryptophan methyl ester was achieved in 47% yield. The corresponding para-nitrophenyl ester was used in the solid phase synthesis of a peptidic hormone (LHRH) analogue with the aim of decreasing tryptophan alkylation. No improvement was observed.     

含有络合功能基的非天然氨基酸的设计、合成及在生物活性肽中的应用

设计合成了一类侧链带有络合基团的非天然氨基酸, 即侧链带有N,N-二羧甲基氨甲基、N,N-二酰胺甲基氨甲基和N,N-二羟乙基氨甲基的苯丙氨酸衍生物, 并将这类非天然氨基酸用于促性腺激素释放激素(LHRH)类似物的固相合成. 高效液相色谱分析结果表明, 粗肽的纯度较好, 易于纯化; 用电喷雾质谱测定了多肽的分子量. 这些非天然氨基酸可作为其它肽类药物合成的构建单元.     

Neuroendocrine Peptides - Analysis by Reversed Phase High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) method is described for determining the biologically active neuroendocrine peptides thyrotropin releasing hormone (TRH), leucine (leu) and methionine (met) enkephalin, angiotensin II, delta sleep inducing peptide (DSIP), luteinizing hormone releasing hormone (LHRH), substance P and growth hormone release inhibiting factor (somatostatin). The selection of mobile phases was limited to these systems that do not exhibit strong absorbtion at 215 nm and 254 nm. Under isocratic conditions at room temperatures with the appropriate selection of mobile phase it was possible by reversed phase chromatography to resolve all of the peptides investigated. We can resolve with the systems employed peptides differing by only one amino acid in chain length as well as peptides differing by only one amino acid in the chain sequence. The method is rapid, does not require derivitization, can be used with aqueous matrixes and is sensitive in the nanomolar range.

Our research has shown that most synthetic peptides lack purity and that all of the peptides except LHRH lack stability when stored in aqueous sterile solution at 8°C for four weeks. The implications of this latter finding are under investigation.     

Synthesis and bioactivities of new LHRH antagonists containing a novel unnatural amino acids at position five

Synthesis and bioactivities of new antagonists of luteinizing hormone releasing hormone (LHRH) with novel unnatural amino acids at position five are reported. Most of them showed some antiovulatory activity at 0.5 microgram/rat and two of them inhibited ovulation completely at 1 microgram/rat using saline as vehicle.     

Synthesis of a β-turn mimetic suitable for incorporation in the peptide hormone LHRH

LHRH is a decapeptide hormone which plays a central role in neuroendocrinology. Conformational studies have suggested that LHRH may adopt a β-turn involving residues 5-8 when bound to its receptor. A β-turn mimetic with side chains corresponding to those of a Tyr-Gly-Leu-Orn tetrapeptide has therefore been synthesized for incorporation at positions 5-8 in LHRH. In the turn mimetic, residues i and i+1 are connected by a ψ[CH₂O] isostere instead of an amide bond, while a covalent ethylene bridge replaces the hydrogen bond which is often found between residues i and i+3 in β-turns. The turn mimetic was assembled from three types of building blocks: an azido aldehyde, an Fmoc protected amino acid and a protected dipeptide amine.     

Abundant <Emphasis Type="Italic">b</Emphasis>-type ions produced in electron capture dissociation of peptides without basic amino acid residues

We have investigated electron capture dissociation (ECD) of doubly protonated peptides with few or no basic amino acid residues (BAARs). For peptides containing one His, abundant b-type ions were only found when His was located adjacent to the N-terminus. Interestingly, b-type ions, particularly b(5)(+), were found to be the dominant product ions in ECD of peptides without BAARs. Fragmentation patterns of luteinizing hormone releasing hormone (LHRH) and vasopressin (VP), containing one Arg and one His, respectively, were compared to those of Q(8)-LHRH and oxytocin (OT) in which the BAAR is replaced with a non-BAAR. More b-type ions were found for Q(8)-LHRH and OT than for LHRH and VP. We also performed ECD of melittin and found no b-type ions from ECD of the 4+ charge state; however, many low abundance b-type ions were produced in ECD of the 5+ charge state. Possible mechanisms for the formation of b-type ions are discussed and we propose that such ions are formed as a consequence of protons being located at backbone amide nitrogens.     

Benzimidazole-5-sulfonamides as novel nonpeptide luteinizing hormone releasing hormone (LHRH) antagonists: minimization of mechanism-based CYP3A4 inhibition

Herein we report the development of novel, potent and non-peptide luteinizing hormone releasing hormone (LHRH) antagonists. The optimization towards derivatives free from mechanism-based CYP3A4 inhibition is described. The identification of a main metabolite guided us towards structural modifications of the benzyl moiety, which resulted in significant improvements of the CYP3A4 profile, while maintaining potent LHRH antagonist activity.     

Expression of Luteinizing Hormone-Releasing Hormone (LHRH) and Type-I LHRH Receptor in Transitional Cell Carcinoma Type of Human Bladder Cancer

Bladder cancer (BC) is the tenth most frequently detected cancer in both sexes. Type-I luteinizing hormone-releasing hormone (LHRH) receptor (LHRH-R-I) is expressed not only in the pituitary, but also in several types of cancer disease. There are few data about LHRH-R-I expression in human BC. This study aimed to investigate the expression of LHRH and LHRH-R-I in the transitional cell carcinoma (TCC) type of human BC. RNA was extracted from 24 human bladder tumor specimens and three BC cell lines. RT-PCR was performed to detect mRNA for LHRH and LHRH-R-I. The protein of LHRH-R-I was further studied by immunohistochemistry (IHC), ligand competition assay, and Western Blot. PCR products of LHRH were found in 19 of 24 (79%) specimens and mRNA of LHRH-R-I was detected in 20 of 24 specimens (83%). Positive immunostaining for LHRH-R-I with different expression intensity was found in all samples examined, showing negative correlation with TCC grade. Radioligand binding studies also showed the presence of specific LHRH-R-I and high affinity binding of LHRH analogs. The high incidence of LHRH-R in BC suggests that it could serve as a molecular target for therapy of human BC with cytotoxic LHRH analogs or modern powerful antagonistic analogs of LHRH.     

Convergent solid phase peptide synthesis. I. Synthesis of protected segments on a hydroxymethylphenyloxymethyl resin using the base labile FMOC α-amine protection. Model synthesis of LHRH.

Convergent solid phase peptide synthesis has been applied to yield LHRH. The segments 1–6 and 7–10 of LHRH were synthesized on a hydroxymethylphenyloxymethyl resin using the base labile Fmoc protecting group on the α-amines. The side chains were protected by HF labile groups. Purification of the segments was performed on Sephadex LH-20 columns and by HPLC on Silica Gel 60 columns. The two segments were then assembled on an α-aminobenzyl resin to yield entire sequence of LHRH. After HF treatment and standard purification on Sephadex G-15 and carboxymethylcellulose CM-52 the desired LHRH was obtained. Synthesis of the segments by the same strategy on carbazoyloxymethylphenyloxymethyl resin showed up unexpected difficulties.     

Haematological measurements for some new erythropoietin hormone analogues synthesized by use of a modified method

A series of erythropoietin hormone analogues (1–9) were synthesized by a modern method, microwave-assisted modified solid-phase peptide synthesis, on polystyrene polyethylene glycol graft copolymer. Peptide 9 was cleaved from the polymer to give peptide 10. Measurement of haematological data for analogue 10 gave good results comparable with those of the original hormone. The structures of the new peptides were confirmed by amino acid analysis and use of spectroscopic evidence.     

Diastereoselective synthesis of β-aminocyclopentene sulfonic acid via hetero Diels-Alder reaction

A new cyclopentene GABA analogue was synthesized as a conformationally rigid analogue of the epilepsy drug vigabatrin. N-Sulfinyl dienophile Diels-Alder methodology, followed by alkaline hydrolysis of the corresponding dihydrothiazine oxide, oxidation and deprotection of the amino group gave cis-4-aminocyclopent-2-ene-1-sulfonic acid. The corresponding N,N-dimethylsulfinamide was also obtained.     

Sequential synthesis of a new analogue of amlodipine bearing a short amino polyethyleneglycol chain

3-Ethyl 5-methyl 2-[(2-(2-(2-aminoethoxy)ethoxy)ethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate as new analogue of amlodipine was prepared in five steps with an overall yield of 22%. The 1,4-dihydropyridine nucleus was built in two steps via Knoevenagel reaction and the amino group of this analogue has been prepared in good yield by Staudinger reduction of the azido 1,4-dihydropyridine precursor in the last step.     

Structural Basis for Recognition of Guanosine by a Synthetic Tricyclic Cytosine Analogue: Guanidinium G‐Clamp

An oligonucleotide analogue containing a novel heterocyclic analogue, the guanidinium G‐clamp, was designed to allow formation of five H‐bonds to guanosine. The guanidinium group was introduced postsynthetically by treatment of the deprotected oligonucleotide containing a free amino group with a solution of 1H‐pyrazole‐1‐carboxamidine and purified by a combination of size‐exclusion chromatography and reversed‐phase HPLC. A single incorporation of this modification into an oligodeoxynucleotide sequence was found to increase duplex stability by 13° and 16° per modification to RNA and DNA, respectively. Crystals of a self‐complementary decamer sequence containing this modification were grown and diffracted to 1‐Å resolution. The structure was solved by molecular replacement and revealed that the modification forms additional H‐bonds to O(6) and N(7) of guanosine through the amino and imino N‐atoms, respectively. The origins of enhanced duplex stability are also attributed to increased stacking interactions mediated by the phenoxazine moiety of the G‐clamp and formation of H‐bond networks between the positively charged guanidinium group, H₂O molecules, and negatively charged O‐atoms from phosphates on the adjacent strand.     

Total synthesis of Trichosanthes trypsin inhibitor and its analogue

Trichosanthes trypsin inhibitor (TTI) is a peptide consisting of 27 amino acid residues with three pairs of disulfide bonds. This paper reports the total synthesis and disulfide bond refolding of this inhibitor and its analogue. After purification, the amino acid sequence and stoichiometrical inhibitory activity against trypsin of the synthetic inhibitor were compatible with those of the natural inhibitor. The analogue of this inhibitor in which residue Met in position 6 was replaced by Ala was also synthesized. The antitrypsin activity of this synthetic analogue was also approximate to that of the natural inhibitor.     

TOTAL SYNTHESIS OF Trichosanthes TRYPSIN INHIBITOR AND ITS ANALOGUE

Trichosanthes trypsin inhibitor (TTI) is a peptide consisting of 27 amino acid residues with three pairs of disulfide bonds. This paper reports the total synthesis and disulfide bond refolding of this inhibitor and its analogue. After purification, the amino acid sequence and stoichiometrical inhibitory activity against trypsin of the synthetic inhibitor were compatible with those of the natural inhibitor. The analogue of this inhibitor in which residue Met in position 6 was replaced by Ala was also synthesized. The antitrypsin activity of this synthetic analogue was also approximate to that of the natural inhibitor.     

Synthesis and properties of aminoacylamido-AMP: chemical optimization for the construction of an N-acyl phosphoramidate linkage

This paper describes the design and synthesis of a new type of aminoacyl-adenylate analogue (aa-AMPN) having an N-acyl phosphoramidate linkage where the oxygen atom of the mixed anhydride bond of aminoacyl-adenylate (aa-AMP) is replaced by an amino group. This new type of aa-AMP analogue is expected to be useful as material for studies on the recognition mechanism of the aminoacylation of tRNA and other biochemical reactions. The condensation of phosphoramidite derivatives of carboxamides with nucleoside derivatives failed, because the activated phosphoramidite derivatives reacted with not only the hydroxyl groups but also another reactive species. An alternative approach was examined by the reaction of 5'-O-phosphoramidite adenosine derivatives with carboxamide derivatives. The TBTr and TSE groups were chosen for protection of the amino group of amino acid amides and the phosphate group, respectively. Detailed studies revealed that the use of 5-(3,5-dinitrophenyl)-1H-tetrazole as an activating catalyst of phosphoramidites resulted in rapid condensation within 10 min to give fully protected aa-AMPN derivatives. No side reaction occurred. Deprotection of these products via a two-step procedure gave aa-AMPN derivatives in good yields. It also turned out that aa-AMPNs thus obtained are stable under both acidic and basic conditions, such as 0.1 M HCl (pH 1.0) and 0.1 M NaOH (pH 13.0).     

Effects of changes in pattern of LHRH pulse on LH secretion by perfused anterior pituitary cells of male rats

In the present study, a perfusion system of dispersed cells was used to investigate the effects of LHRH pulse amplitude and frequency, and LHRH continuous stimulation on LH secretion by anterior pituitary cells of adult male rats. The results have shown that, in the range of LHRH concentrations from 1 X 10(-10) to 1 X 10(-6) mol/L, the dose-response curve of LH secretion was linear. LHRH pulse frequency generated a biphasic LH response: increasing LHRH pulse frequency increased the basal LH secretion and decreased LH/pulse. When 1 X 10(-9) mol/L or greater LHRH was given at frequencies of 3 pulses/h or higher, it was observed that a maximal LH peak was induced and then the LH release declined progressively to its LH basal level, i.e. LHRH self-priming effect and LH desensitization occurred. Enhancement of amplitude of LHRH pulses could reduce pulse frequency required for priming. Increases in frequency of LHRH pulses with high amplitude would provoke the priming effect more quickly. In addition, continuous perfusion of LHRH with different concentrations could also elicit the LHRH self-priming effect and lH desensitization. LHRH with low concentration (1 X 10(-10) mol/L) would take much longer to evoke a self-priming effect. These results indicate that the LH secretion pattern is dependent on LHRH pulsatile amplitude and frequency, and will help to clarify the kinetics mechanisms by which LH pulses fluctuate in vivo.     

微通道连续流动高效绿色合成亮丙瑞林

开发了一种高效绿色的连续流动多肽合成方法, 并成功应用于一种含有9个氨基酸的促性腺激素释放激素类似物(亮丙瑞林)的合成. 该方法采用苄氧羰基(Cbz)保护氨基酸, 在微通道反应器中实现高效偶联与水洗萃取除杂, 并通过钯碳催化剂填充柱氢解反应实现快速洁净地脱除Cbz保护基, 使原料和溶剂的消耗量大大减少, 原子经济性大幅提高. 该高效绿色合成方法将在多肽制药工业中得到更多应用.     

Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase

Thymidine analogues bearing a variety of functional groups at the C5-position via an amino-linker arm were prepared and the substrate activity for PCR using thermophilic KOD Dash DNA polymerase was examined. The enzyme accepted the thymidine analogues bearing pyridine, imidazole, biotin, a cationic-charged guanidinium, a cationic-charged amino, mercaptopyridyl and phenanthrolne groups at the C5-position, forming the corresponding PCR product. However, a thymidine analogue bearing a carboxyl group at the C5-position was a poor substrate and the corresponding PCR products could not be obtained. The thymidine analogue bearing a mercapto group was also a poor substrate for the enzyme, because it dimerized by disulfide linkage under PCR conditions. The enzyme hardly accepts the thymidine analogues with a negatively-charged carboxyl group or a bulky group as a substrate. KOD Dash DNA polymerase, having a broader substrate specificity than any other DNA polymerase, will expand the variety of modified DNAs that can be prepared by PCR.