Continuous flow solid (gel) phase peptide synthesis using unsupported ultra-high load polymers *

A simple, manually operated, continuous flow apparatus is described for solid (gel) phase peptide synthesis. The approach uses an unsupported phenolic bead form core network at an initial matrix loading of 5 mmol g^-1, the theoretical maximum. The synthesis is performed in a flow reactor under low pressure conditions. “Layered displacement” of reagent solutions and washing solvents is an essential feature that has been developed to facilitate efficient peptide synthesis. The usefulness of the present system in conjunction with N^x Boe protected amino acids is illustrated by the syntheses of [Leu⁵]-enkephalin and dermorphin. The potential for scale up synthesis has also been investigated.     

Solid-phase synthesis of bombesin by continuous flow procedure using Fmoc-amino acids*

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.     

普兰林肽的固相合成

目的:探索普兰林肽的固相合成、氧化条件及纯化方法。方法:采用Fmoc固相多肽合成法,以Rink Amide-AM树脂做载体,HBTU/HOBt/DIEA做缩合剂,逐步缩合得到全保护线性普兰林肽树脂,以TFA/苯甲硫醚/苯酚/H2O/EDT/TIS配比的裂解液脱除保护基团,分别采用空气,二甲基亚砜,双氧水氧化两个半胱氨酸的巯基形成一对二硫键,半制备反相高效液相色谱法纯化。结果:合成含37个氨基酸以及一对二硫键的普兰林肽经RP-HPLC和MALDI-TOF-MS确证,粗品纯度在50.0%以上,粗品经半制备型反相高效液相色谱纯化,所得精肽的纯度大于95.0%,总收率为30.5%。结论:该方法简单,合成的产品成本低,纯度高,可为工业化生产提供借鉴。     

Synthesis of 13C6‐labeled Reyataz™ (BMS‐232632)

An efficient synthesis of ¹³C₆‐labeled Reyataz? (BMS‐232632) is described. ¹³C₆‐labeled Reyataz? was synthesized in eight steps from commercially available [ring‐¹³C₆]‐L ‐phenylalanine. The overall yield of the synthesis was 8% and the purity of the final product was greater than 98%. Copyright © 2005 John Wiley & Sons, Ltd.     

Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides

The use of disulfide bonds for peptide–resin linkage in solid-phase peptide synthesis was investigated using polyacrylic polymers (Expansin^TM) and automated Fmoc methodology. The disulfide moiety was bound to the support either by coupling a protected bifunctional handle or by an original stepwise procedure. Among the three different disulfide handles that were investigated, only the aminoethyldithio-2-isobutyric acid (AEDI) handle was stable enough to achieve peptide synthesis. A series of peptides of up to 10–20 amino acids were prepared in this manner, in good yield and purity. Rapid and quantitative peptide release was obtained by reduction with equimolecular amounts of dithiothreitol at pH 9 or tris(2-carboxymethyl) phosphine at pH 4.5. This allowed direct and rapid coupling of the released cysteamide peptides to an activated protein carrier and the use of free or resin-bound forms of the antigen in immunoassays.     

A facile and efficient synthesis of d3‐labelled Reyataz™

A facile and efficient synthesis of d₃‐labelled Reyataz? is described. The key step of synthesis involved the coupling of N‐(d₃‐methoxycarbonyl)‐L ‐tert‐leucine 2b with an advanced chiral non‐racemic building block 4 . The latter was synthesized in 4 steps from the readily accessible hydrazine derivative 8 . Copyright © 2005 John Wiley & Sons, Ltd.     

Microwave‐assisted synthesis and chiral HPLC separation of 18F‐labeled MaxiPost™. An agent for post‐stroke neuroprotection

The syntheses of (S)‐3‐(5‐chloro‐2‐methoxyphenyl)‐1,3‐dihydro‐3‐[¹⁸F]‐fluoro‐6‐(trifluoromethyl)‐1H‐indol‐2‐one was accomplished by the microwave assisted carrier‐added ¹⁸F fluorination of (R,S)‐3‐(5‐chloro‐2‐methoxyphenyl)‐1,3‐dihydro‐3‐chloro‐6‐(trifluoromethyl)‐1H‐indol‐2‐one, followed by chiral HPLC separation to afford the desired ¹⁸F‐labeled enantiomer in radiochemical yields of 5–15% (EOS) and synthesis and purification times of 60–67 min. Biodistribution studies in rodents were consistent with previously reported studies using racemic ¹⁸F‐radiolabeled material. Copyright © 2003 John Wiley & Sons, Ltd.     

A novel approach to the synthesis of EGF‐like domains: a method for the one‐pot regioselective formation of the three disulfide bonds of a human blood coagulation Factor VII EGF‐1 analogue

Abstract: The study of EGF‐like domains is of great general interest in protein science because of their participation in a multitude of protein–protein interactions. A common structural feature of EGF‐like modules is the presence of three disulfide bonds, the regioselective formation of which still remains a challenge to peptide chemists. We report here on a method for the one‐pot regioselective synthesis of an analogue of the EGF‐1 domain of human coagulation Factor VII (residues 45–83) comprising an Asn57β–Asp substitution. The cysteine protecting groups trityl, t‐butyl and acetamidomethyl were chosen for the three disulfide bond pairings. All three disulfide bridges were prepared directly from the crude starting product, obviating the need for the timely and costly purification of the intermediate folded products. The fully folded product was purified by preparative high‐pressure liquid chromatography prior to evaluation of its biological activity in an assay to detect inhibition of FVII/TF complex formation. In addition circular dichroism spectroscopy was employed to elucidate the main structural similarities between this peptide analogue and the native human Factor VII EGF‐1 domain.     

Chemical synthesis and immunological properties of a cyclic eicosapeptide (Gly88,90) 82–101 of the beta subunit of human chorionic gonadotrophin

The eicosapeptide (Gly^88,90) 82–101 hCG-β was synthesized by the fragment condensation of the nonapeptide (Gly^88,90) 82–90 and the undecapeptide 91–101 followed by iodine oxidation to make the disulfide loop. Intramolecularity of the disulfide linking cysteines at 93 and 100 was confirmed. Anti-peptide antibodies were elicited in rabbits upon immunization with a conjugate of the peptide with tetanus toxoid. The repertoire of these antibodies was directed solely against the undecapeptide 91–101. These antibodies showed no recognition for hCG, hCG-β or ARCM hCG-β, suggesting that the conformational epitopes of hCG-β in the region 82–101 may not be accessible to antibodies.     

Incorporation of azaglutamine residues into peptides synthesised by the ultra-high load solid (gel)-phase technique

The solid (gel)-phase peptide synthesis of peptides, each containing an azaglutamine residue, has been examined. Procedures using various mono-, di- and tripeptide and carbazate fragments containing or relating to an azaglutamine (1) residue have been evaluated. N-Activation of the amino-terminus of a resin-bound peptide with bis-(2,4-dinitrophenyl)carbonate (2) yielded the terminal isocyanate species, which reacted with protected carbazates to give resin-bound protected peptides containing the aza-residue. By contrast, coupling of activated amino-acid derivates to the free amino-group of a resin-bound peptide with an aza-residue at the N-terminus was a slow and unsatisfactory process. It is concluded that the route yielding the best results involves the reaction of a protected amino-acyl carbazate to a resin-bound isocyanate-activated peptide.     

Studies on the carboxyl terminal peptides of human seminal plasma inhibin (HSPI)

Observation of contradictory results with the in vitro assays for inhibin-like activity of the carboxyl terminal 28 amino acid peptide 67–94 with a disulfide loop, of human seminal plasma inhibin (HSPI), prompted us to synthesize both the linear and the cyclic peptides and test their ability to suppress the circulating levels of follicle stimulating hormone (FSH) in vivo in adult male rats. The linear peptide [Cys(Acm)^73,87] 67–94 of HSPI was synthesized by solid-phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry and a continuous-flow technology. The peptide was cyclized by direct iodine oxidation of the S-diacetamidomethyl peptide in dilute solution. In the in vivo assay the linear peptide did not affect the levels of FSH, whereas the cyclic peptide suppressed the levels of FSH significantly. Thus, the carboxyl terminal region of HSPI does have inhibin-like activity and perhaps has the active core of the protein.     

Solid phase synthesis of a trypsin inhibitor isolated from the Cucurbitaceae Ecballium elaterium

The synthesis of a 28-residue peptide isolated from Ecballium elaterium of the Cucurbitaceae family which strongly inhibits trypsin activities (Ka = 8·10⁻¹¹ M), using BOP as the coupling reagent in a solid phase procedure is presented. This micro protein contains three disulfide bridges in its sequence and was obtained after oxidation of the six half-cystine residues either by air or with the use of carboethoxysulfenyl chloride. After purification by semi-preparative HPLC, the synthetic product was shown by trypsin inhibition tests to be identical with the trypsin inhibitor EETI II isolated from Ecballium elaterium.     

Synthesis of a cyclic analogue of the C-loop region of epidermal growth factor,containing 1-aminocyclopropane-1-carboxylic acid

We report the synthesis of a cyclic analogue of epidermal growth factor sequence 33–42 with substitution of 1-aminocyclopropane-1-carboxylic acid for glycine at position 39 (N-acetyl-Cys-Val-Ile-Gly-Tyr-Ser-ACPCA-Asp-Arg-Cys-NH₂). The analogue was synthesised by solid-phase methods, using t-Boc chemistry and acid-labile side-chain protecting groups. The use of the 4-methoxybenzyl protecting group for C- and N-terminal cysteine residues resulted in the spontaneous formation of the desired intramolecular disulfide bond after HF deprotection.     

A novel strategy for the synthesis of the cysteine-rich protective antigen of the malaria merozoite surface protein (MSP-1)

The most promising antigen for a protective malaria vaccine is a cysteine-rich domain at the carboxyl terminus of the merozoite surface protein (MSP-1). Passive transfer of anti-MSP-1 antibody or immunization of MSP-1 against infection challenge confers protection in primate and rodent models. The antigen belongs to the three-disulfide epidermal growth factor (EGF) family based on the alignment of the six cysteines. In the K1 strain there are, however, only four cysteines corresponding to the four carboxyl cysteines of EGF. Furthermore, disulfide pairing would produce a non-EGF pattern. Because this cysteine-rich antigen is conformation-dependent, and reduction of the disulfide bonds abolishes antigenicity, we used a synthetic analog to investigate the probable disulfide pairing of this antigen. This paper describes the synthesis, folding and disulfide pairings of two 50-residue cysteine-rich peptides. One contains two disulfides (VK-50) derived from the native sequence of MSP-1 of the Thailand K1 strain (aa 1629–1679). The other contains an EGF-like, three-disulfide [Cys-9,14]VK-50 peptide. Both peptides were synthesized by a solid-phase method using Fmoc-chemistry. The crude peptide of VK-50 was folded, and the disulfide was oxidized by the DMSO method to obtain a structure with an expected disulfide pairing of 3–4, and 5–6. The specific pairing pattern of 1–3, 2–4 and 5–6 in [Cys 9,14]VK-50 corresponding to EGF in [Cys 9,14]VK-50 was obtained using a ‘knowledge-based’ (KB) strategy for their formation. Purified VK-50 and [Cys-9,14]VK-50 had the correct molecular weight, as shown by Cf-252 fission ionization mass-spectrometry. The disulfide pairings were confirmed by enzymatic digestion. These two peptides can be conjugated to the multiple peptide antigen core for immunization. The immunological results will allow us to conclude the correct disulfide pairing and the conformational importance of this antigen.     

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.     

Solid-phase synthesis of protected peptides using new cobalt(III) ammine linkers†

Cobalt(III) ammine complexes of the type cis-[CoL₄(4-AMB)O-AA-Boc](CF₃SO₃)₂, where L₄= bisethylenediamine (en)₂ or tetraammine (NH₃)₄, and 4-AMB = 4-(aminomethyl)benzoic acid, have been synthesized and used as linkers to polystyrene resins for solid-phase synthesis of protected peptides. Boc/t-Bu-protected [Leu⁵]enkephalin was assembled on the two different Co(III) resins, and then cleaved from the resins by reduction of the Co(III) center in 93–96%; yield. HPLC-purified protected [Leu⁵]enkephalin was obtained in 67–69% overall yield and characterized by amino acid analysis and ¹H NMR. Stepwise synthesis on the Co(en)₂-resin was also used in the assembly of Boc-Asp(OcHex)-Arg(Mts)-Gly-Asp(OcHex)-Ala-Pro-Lys(2Cl-Z)-Gly-OH, a sequence from collagen α1 Type 1. The protected peptide was cleaved from the Co(III) resin in 74% yield, and the HPLC-purified nonapeptide was characterized by amino acid analysis, ¹H NMR and liquid secondary-ion mass spectrometry (LSIMS). New routes are described for the synthesis of isomerically pure Co(III) anchor complexes. The Co(III) resins were found to be compatible with both the tert-butyloxycarbonyl (Boc) and the 9-fluorenylmethoxycarbonyl (Fmoc) N^α-protecting group strategies used in solid-phase peptide synthesis.     

Synthesis of 3H and 14C labeled (S)‐3‐(5‐chloro‐2‐methoxyphenyl)‐1,3‐dihydro‐3‐fluoro‐6‐(trifluoromethyl)‐2H‐indol‐2‐one,maxipost™. An agent for post‐stroke neuroprotection

The syntheses of tritium labeled (S)‐3‐(5‐chloro‐2‐[OC³H₃]methoxyphenyl‐1,3‐dihydro‐3‐fluoro‐6‐(trifluoromethyl)‐1H‐indol‐2‐one, and carbon‐14 (S)‐3‐(5‐chloro‐2‐methoxyphenyl)‐1,3‐dihydro‐3‐fluoro‐6‐(trifluoromethyl)‐2H‐[2,3‐¹⁴C₂] indol‐2‐one are reported. The ³H‐labeled compound was prepared in a two‐step synthesis from C³H₃I. The final product was purified via chiral HPLC to yield the desired enantiomer in a 4% radiochemical yield and a specific activity of 60 Ci/mmol. The ¹⁴C‐labeled compound was prepared in a four‐step synthesis from diethyl [carboxylate‐¹⁴C₁,₂] oxalate. The final product was purified via chiral HPLC to yield the desired enantiomer in a 20% radiochemical yield and a specific activity of 28.4 μCi/mg. Copyright © 2002 John Wiley & Sons, Ltd.     

Syntheses of [2H3, 15N], [14C]Nexavar™ and its labeled metabolites

Nexavar?, Sorafenib tosylate (BAY 43‐9006 tosylate) is a potent small molecule Raf kinase inhibitor for the treatment of hyperproliferative disorders such as cancer. Both radiolabeled and stable isotope labeled compounds were required for drug absorption, distribution, metabolism and excretion (ADME) and quantitative mass spectrometry bio‐analytical studies. Nexavar? labeled with carbon‐14 in the carboxamide group was prepared in two steps in an overall radiochemical yield of 42% starting from 4‐chloro‐N‐methyl‐2‐pyridine‐[¹⁴C]carboxamide. The [²H₃,¹⁵N] version of Nexavar? was prepared in 75% yield based on 4‐chloro‐N‐[²H₃]methyl‐2‐pyridine‐[¹⁵N]carboxamide. The pyridine N‐oxide metabolite labeled with carbon‐14 as well as with deuterium and nitrogen‐15 and was synthesized by oxidation in yields of 59% and 87%, respectively. Starting from [²H₂, ¹³C]formaldehyde the N‐hydroxymethyl metabolite was labeled with carbon‐13 and deuterium in one step in a 45% overall yield. Copyright © 2006 John Wiley & Sons, Ltd.     

Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-α

The putative receptor-binding region of human transforming growth factor-α (TGF α) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)₂ on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70%, yield. In contrast, the conventional scheme of using I₂ to form the intermolecular disulfide between the Cys(Acm) of the A-chain and C-fragment with the preformed intramolecular disulfide bond in solution phase did not result in any product. The purified two-chain analogs were found to be unstable and rearranged to the homo-dimers. This reaction was greatly accelerated in I₂, which explained the difficulty associated with the conventional scheme. When assayed against A431 and NRK clone 49F cells, both the A-chain and the C-fragment did not exhibit any biological activity independently, but the two-chain analogs showed low receptor-binding activity with an IC₅₀ at 0.3 mM level. Unexpectedly, dimeric C-fragment, which resulted from the rearrangement reaction, also showed receptor-binding activity. Our results demonstrate that the two-chain analogs exhibit low but distinct biological activity and provide evidence that the putative TGFα receptor binding region may be discontinuous. In addition, we also provide an efficient approach to further explore the two-chain receptor-binding analogs of TGFα.