Choice of the optimal synthetic approach for the polypeptide ligands of prostatic specific membrane antigen preparation

Two alternative synthetic schemes involving solid-phase peptide synthesis steps for the preparation of prostatic specific membrane antigen ligands based on Glu-Urea-Lys with peptide fragments in the linker structure are compared. In the first approach, the amino acid key intermediate is attached to the 2-CTC resin by the carboxy group while in the second approach, by the amino one. The preference for each approach is dependent on the particular target molecule     

阿基瑞林制备方法的改进研究

阿基瑞林,又称乙酰基六肽-8、六胜肽等。它作为肉毒毒素的安全替代品,具有显著的抗皱活性,是美妆市场上需求潜力很大的抗皱多肽原料。当前阿基瑞林主要是通过固相合成方法得到的,由于所使用的Rink Amide AM树脂单价高、上载量低,造成了阿基瑞林原料价格高昂,不利于其大规模生产及推广应用。本文中使用固-液相结合的方法,首先用高上载量、单价低的2-CTC树脂合成六肽片段,六肽片段再经酰胺化处理得阿基瑞林粗品,粗品经纯化后得纯度95%以上的阿基瑞林,收率59.77%。本方法成本低、收率高,为C端酰胺化多肽的合成提供了一种新思路。     

胡蜂蜂毒肽的固相合成

采用Fmoc固相合成策略,合成了胡蜂蜂毒肽(COOH-Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu-NH₂)。以Wang树脂为载体,HBTU-HOBt为缩合剂,按照其氨基酸序列依次缩合,最终用切割试剂将其从树脂上切割下来,得到粗肽,经RP-HPLC纯化得到目标肽,纯度97.6%。经HR-MS（EI）分析,确定产物为胡蜂蜂毒肽。     

Facile synthesis of C-terminal peptide hydrazide and thioester of NY-ESO-1 (A39-A68) from an Fmoc-hydrazine 2-chlorotrityl chloride resin

The NY-ESO-1 (A39-A68) peptide hydrazide was prepared through 9-fluorenyl-methoxycarbonyl solid-phase peptide synthesis (Fmoc SPPS) from a new 9-fluorenyl-methoxycarbonyl hydrazine 2-chlorotrityl chloride (Fmoc-hydrazine 2CTC) resin. The new resin was ideal for long-term storage and usage in Fmoc SPPS. Besides, the title peptide hydrazide could be transformed nearly quantitatively into the corresponding peptide thioester, which was both isolable and usable directly in native chemical ligation (NCL).     

抗菌肽IB-367的固相合成与抑菌活性

采用固相合成方法,以Rink Amide树脂为载体,Fmoc保护氨基酸为原料,经苯并三唑-1-四甲基六氟磷酸酯(HBTU)/N,N-二异丙基乙胺(DIEA)缩合,三氟乙酸/苯甲硫醚/乙二硫醇/苯甲醚裂解体系脱除保护基制得IB-367线性肽(4); 4经双氧水氧化制得IB-367一环肽(5); 5经碘乙醇溶液氧化合成抗菌肽IB-367(6),收率34.1%,纯度＞95.0%,其结构经MS(ESI)和氨基酸组成分析确证。抑菌活性研究结果表明：6对大肠杆菌和金黄色葡萄球菌的最小抑菌浓度为5.0 μg·mL^-1。     

Ca-mediated thiolysis of peptide–resin linkage as an option for preparing protected peptide thioesters

A mild new procedure for preparing protected peptide thioesters, based on Ca²⁺-assisted thiolysis of peptide–Kaiser oxime resin (KOR) linkage, is described. Ac-Ile-Ser(Bzl)-Asp(OcHx)-SR (Ac: acetyl; Bzl: benzyl; cHx: cyclohexyl), model peptide, was readily released from the resin by incubating the peptide–KOR at 60 °C in mixtures of DMF with n-butanethiol [R = (CH₂)₃CH₃] or ethyl 3-mercaptopropionate [R = (CH₂)₂COOCH₂CH₃] containing Ca(CH₃COO)₂. After serine and aspartic acid side-chain deprotection under acid conditions, Ac-Ile-Ser-Asp-S(CH₂)₂COOCH₂CH₃ was successfully obtained with good quality and high yield. This type of C-terminal modified peptide may act as an excellent acyl donor in peptide segment condensation by the thioester method, native chemical ligation and enzymatic methods.     

Convenient high yield and stereoselective synthesis of O-glycopeptides using N-α-Fmoc-Tyr/Ser[β-d-Glc(OAc)4]OPfp generated in solution

Fmoc-AA-OPfp (AA=Tyr or Ser) (1 equiv) was reacted with β-d-Glc(OAc)₅ (6 equiv) in the presence of BF₃.Et₂O (6 equiv) in CH₂Cl₂ at room temperature for 2 h, and the glycosylation reaction mixture was used directly to couple to the amino group of the peptide resin without isolation and purification of the Fmoc-AA[β-d-Glc(OAc)₄]-OPfp. Moreover, the -OAc protecting groups of glucose was removed just prior to releasing the peptide from the resin using 6 mM NaOMe in 85% DMF-MeOH. The crude product obtained by TFA cleavage contained >90% of the target O-glycopeptide, and the 500 MHz ¹H NMR analysis revealed that the glycosylation reaction was nearly stereoselective (>97% β-anomer). This method is rapid and stereoselective, and can now be exploited for the routine synthesis of O-glycopeptides.     

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: Part II. Total synthesis of human big gastrin-II and its C-terminal glycine-extended peptide (G34-Gly sulfate) by the solid-phase segment condensation approach

Application of the fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase segment condensation approach to the preparation of sulfated peptides was investigated through the synthesis of human big gastrin-II, a 34-residue sulfated tyrosine [Tyr(SO3H)]-containing peptide. Highly acid-sensitive 2-chlorotrityl resin (Clt resin) was exclusively employed as an anchor-resin for the preparation of the three peptide segments having the C-terminal Pro residue as well as of the Tyr(SO3H)-containing resin-bound segment. By using the PyBOP-mediated coupling protocol [PyBOP=benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphatel, we successively condensed each segment and constructed the 34-residue peptide-resin without any difficulty. The final acid treatment of the fully protected peptide-resin at low temperature (90% aqueous TFA, 0 degree C for 8 h), which can detach a Tyr(SO3H)-containing peptide from the resin and remove the protecting groups concurrently with minimum deterioration of the sulfate, afforded a crude sulfated peptide. After one-step HPLC purification, a highly homogeneous human big gastrin-II was easily obtained in 14% yield from the protected peptide-resin. The sulfate form of the C-terminal glycine-extended gastrin (G34-Gly sulfate), a posttranslational processing intermediate of gastrin-II, was also successfully prepared with the segment condensation approach (11% yield). These results demonstrated the usefulness of the segment condensation protocol for preparing large Tyr(SO3H)-containing peptides.     

Comparative investigation of the cleavage step in the synthesis of model peptide resins: implications for Nalpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nalpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nalpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.     

Synthesis of monofunctionalized gold nanoparticles by fmoc solid-phase reactions

In this communication, solid-phase reactions for the synthesis of Lys-monofunctionalized gold nanoparticles are described. A controlled and selective fabrication of linear nanoparticle arrays can be achieved through peptide linkage systems, and therefore it is essential to prepare Fmoc amino acid nanoparticle building blocks susceptible to Fmoc solid-phase peptide synthesis. Gold nanoparticles containing carboxylic acids (2) in the organic shell were covalently ligated to Lys on solid supports through amide bond coupling reactions. We employed Fmoc-Lys-substituted polymer resins such as Fmoc-Lys-Wang or Fmoc-Lys-HMPA-PEGA. The low density of Lys on the matrix enabled 2 nm-sized gold nanoparticles to react with Lys in a 1:1 ratio. Subsequent cleavage reactions using 60% TFA reagent resulted in Lys transfer from the solid matrix to gold nanoparticles, and the Fmoc-Lys-monofunctionalized gold nanoparticles (5) were obtained with 3-15% yield. Synthesis using HMPA-PEGA resin increased productivity due to the superior swelling properties of PEGA resin in DMF. Monofunctionalization of nanoparticles was microscopically characterized using TEM for the ethylenediamine-bridged nanoparticle dimers (6). By counting the number of 6, we found that at least 60% of cleaved nanoparticles were monofunctionalized by Lys. This method is highly selective and efficient for the preparation of monofunctionalized nanoparticles.     

Adsorption and isotope effects by ion exchange with aqueous-2-aminomethyl-18-crown-6 bonded merrifield resin

Magnesium isotopes effects were investigated by chemical ion exchange using synthesized 2-aminomethyl-18-crown-6 (AM18C6) bonded Merrifield peptide resin. It was found that separation factors larger those reported previously were obtained, and the hydration and isotope mass effects are more significant than that of the complexation. The capacity of the crown ion exchanger was 2.3 meq/g dry resin. The adsorption capacity of the resin for magnesium ion was 26.8 mg/g dry resin at pH 7. The heavier isotopes of magnesium were enriched in the solution phase, while the lighter isotopes were enriched in the resin phase. The separation factors of (24)Mg-(25)Mg, (24)Mg-(26)Mg, and (25)Mg-(26)Mg were 1.0085, 1.0162, and 1.0081, respectively.     

New cyclic RGD peptides: synthesis,characterization, and theoretical activity towards αvβ3 integrin

Two new cyclic RGD peptides were prepared using a click chemistry approach. The linear RGDfV peptide was synthesized by solid-phase peptide synthesis using a 9-fluorenylmetoxicarbonyl (Fmoc) strategy and a 2-chlorotrityl chloride resin. After coupling 5-hexynoic acid the peptide was cleaved from the resin and linked to propargylamine. The bis-alkynyl RGDfV peptide was then reacted with two different bis-azides by treatment with copper iodide and triethylamine. These two cyclic RGD peptides were characterized by NMR and HRMS. In order to evaluate the interaction of these new compounds with integrin α_vβ₃ docking experiments were carried out and the results compared with those obtained with cyclo(RGDf[N–Me]V) (Cilengitide). The two new cyclic RGD peptides showed a higher affinity to the α_vβ₃ integrin when compared with Cilengitide thus representing two new potential integrin α_vβ₃ antagonists.     

Physalaemin-analoga-synthesen am festen träger

The solid phase synthesis of 3 peptide amides (H-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH₂ 1b, H-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH₂ 2b, H-Lys-Phe-Tyr-Gly-Leu-Met-NH₂ 3b with the natural sequences of physalaemin is described. Starting with methionine resin the t-butyloxycarbonyl (t-BOC) amino acids are coupled stepwise to the peptides bound to the resin. The desired compounds were separated by DMF/NH₃ as amides from the resin, 2b and 3b have the same biological activity as peptides synthesized by classical methods.     

Solid-phase synthesis using a new polyacrylic resin : Synthesis of the fragment 14–21 of the amino acid sequence of histone H4

The solid-phase synthesis of the octapeptide 1 AcGly-Ala-Lys-Arg-His-Arg-Lys-ValOMe, which represents the fragment 14-21 of the amino acid sequence of the chromosomal histone H4, as well as of the structurally related nonapeptide 2 AcGly-Ala-Lys-Leu-Arg-His-Arg-Lys-ValOMe, is described using a new polyacrylic resin containing a glycolamide ester linkage(resin-NHCO-CH₂-OCO-peptide) acting as a labile anchoring moiety between the resin and the peptide.After elongation of the polypeptide chain using classical protecting groups, i.e. t-butyloxycarbonyl for the α-NH₂ function, benzyloxycarbonyl, nitro and 2,4-dinitrophenyl groups for the side-chains of Lys, Arg and His respectively, both peptides 1 and 2 were obtained in good yields and with a high purity as shown by high-pressure liquid chromatography, by amino-acid analysis and by high-field proton NMR spectroscopy.This work demonstrates the ability of the newly introduced polyacrylic resin to act as a convenient support for solid-phase peptide synthesis.     

Deprotection of the S-trimethylacetamidomethyl (Tacm) group using silver tetrafluoroborate: application to the synthesis of porcine brain natriuretic peptide-32 (pBNP-32)

Silver tetrafluoroborate (AgBF4) in trifluoroacetic acid (TFA) has been found to cleave the S-trimethyl-acetamidomethyl (Tacm) group or the S-acetamidomethyl (Acm) group without affecting other functional groups in a peptide chain. A newly isolated porcine brain natriuretic peptide-32 (pBNP-32) was synthesized by the combined use of the S-Tacm group and AgBF4 deprotection. The synthetic pBNP-32 was obtained in better yield by the AgBF4 procedure than by the standard I2 procedure. The synthetic pBNP-32 has the highest chick rectum relaxant activity among the known members of the atrial natriuretic peptide-brain natriuretic peptide (ANP-BNP) families. Somatostatin was also synthesized by the Fmoc-based solid-phase method using S-Tacm and AgBF4. In this synthesis, the recently developed reagent tetrafluoroboric acid (HBF4) was applied to cleave the peptide from the resin.     

Separation of lithium isotopes with the N3O2 trimerrifield peptide resin

A study on the separation of lithium isotopes was carried out with 1,13-dioxa-4,7,10-triazacyclopentadecane-4,7,10-trimerrifield peptide resin [N₃O₂3M]. The resin having N₃O₂ as an anchor group has a capacity of 0.2 meq/g dry resin. Upon column chromatography [0.1 cm (I.D)×30 cm (height)] using 1.0M NH₄Cl solution as an eluent, a single separation factor of 1.00104 was obtained from the elution curve and isotope ratios based on theGlueckauf theory. The heavier isotope,⁷Li concentrated in the resin phase, while the lighter isotope,⁶Li enriched in the solution phase.     

Separation of lithium isotopes by elution chromatography with an AB18C6 bonded Merrifield peptide resin

Cation exchange chromatographic separation of lithium isotopes was carried out with an 4'-aminobenzo-18-crown-6(AB18C6) bonded Merrifield peptide resin. This resin has a capacity of 2.25 meq/g dry resin. Upon column elution chromatography, a single stage separation factor of 1.0095, was obtained by the Glueckauf theory from the elution curve and isotopic assays. The heavier isotope, ⁷Li, was concentrated in the resin phase, while the lighter one, ⁶Li, concentrated in the solution phase.     

Peptide thioester preparation based on an N-S acyl shift reaction mediated by a thiol ligation auxiliary

Formation of peptide thioesters, based on an N to S acyl shift mediated by an auxiliary, N-4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group, under acidic conditions, is described. The protected peptide was assembled on a hydroxymethylphenylacetamidomethyl resin via an N-Dmmb-amino acid residue according to standard Fmoc solid-phase peptide synthesis following treatment with trifluoroacetic acid. The peptide α-thioester was released from the resin by reaction with 2-mercaptoethanesulfonic acid in the presence of N,N-diisopropylethylamine.     

微波作用下大位阻氨基酸与H-Pro-CTC树脂的高效缩合

微波反应下, 运用新型固相肽合成反应器, 深入研究了五种大位阻氨基酸与H-Pro-CTC树脂(CTC树脂, 2-氯三苯甲基氯树脂)的缩合反应. 使用三次缩合的策略, 分别在DMF/NMP/THF (V∶V∶V＝1∶1∶1), NMP/DMSO/THF (V∶V∶V＝4∶1∶1), DMF/DMSO/THF (V∶V∶V＝4∶1∶1)混合溶剂中缩合一次, 每次缩合反应的最优条件为: 缩合试剂HBTU、氨基酸浓度7 mmol/L、微波辐射3 min、反应温度35 ℃、维持时间3 min, 与传统方法相比, 氨基酸的用量大大减少, 其过量倍数从5倍降低为2倍, 缩合反应速率提高了16倍以上. 五种大位阻氨基酸与H-Pro-CTC树脂的缩合率都提高到80%以上.