Convergent Solid-phase Peptide Synthesis Performed in a CHCl3-phenol Mixed Solvent: Synthesis of Amyloid β-peptides as Examples with a Difficult Sequence

Convergent solid-phase peptide synthesis (CSPPS) involving the coupling of protected peptide segments on a solid support performed in a β-sheet disrupting solvent consisting of a mixture of CHCl₃ and phenol (v/v, 3/1), proceeded smoothly without danger of epimerization or of significant phenyl ester formation with the carboxyl component when diisopropylcarbodiimide (DIC) was used in the presence of 1-hydroxy-7-azabenzotriazole (HOAt) or 6-chloro-1-hydroxybenzotriazole (Cl-HOBt). In particular, this synthetic strategy using the CHCl₃ and phenol mixed solvent proved to be essential for coupling sparingly soluble segments even with difficult sequences. The present approach was successfully applied to the synthesis of amyloid β-peptide (Aβ) (1-40) and also its reversed Aβ (40-1) as an inactive control peptide.     

Synthesis of phosphopeptides by the Multipinast method: Evaluation of coupling methods for the incorporation of Fmoc- Tyr(PO3Bzl,H)-OH, Fmoc- Ser(PO3Bzl,H)-OH and Fmoc- Thr(PO3Bzl,H)-OH

astMultipin is a trademark of Chiron Technologies Pty. Ltd., Clayton, Victoria, Australia.The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiester-protected derivatives, Fmoc- Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO3H2)-Leu- Glu-Asp-Pro-Ala-NH2 (Xxx = Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9) DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc- Xxx(PO3Bzl,H)-OH derivatives. In the subsequent synthesis of the -helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys- Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO3H2)-Glu-Ala-Thr- His-Lys-NH2, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc- Tyr(PO3Bzl,H)-OH.     

Synthesis of phosphopeptides by the Multipinast method: Evaluation of coupling methods for the incorporation of Fmoc- Tyr(PO3Bzl,H)-OH,Fmoc- Ser(PO3Bzl,H)-OH and Fmoc- Thr(PO3Bzl,H)-OH

Summary The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiesterprotected derivatives, Fmoc-Tyr(PO₃Bzl,H)-OH, Fmoc-Ser(PO₃Bzl,H)-OH and Fmoc-Thr(PO₃Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO₃H₂)-Leu-Glu-Asp-Pro-Ala-NH₂ (Xxx=Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9)DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc-Xxx (PO₃Bzl,H)-OH derivatives. In the subsequent synthesis of the α-helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys-Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO₃H₂)-Glu-Ala-Thr-His-Lys-NH₂, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc-Tyr(PO₃Bzl,H)-OH. Multipin is a trademark of Chiron Technologies Pty. Ltd., Clayton, Victoria, Australia.     

Applications of BOP reagent in solid phase synthesis. Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1-29)-NH2

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.     

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8

Substitution of the -OSO3H group in the sulfated-tyrosine by the non-hydrolyzable-CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH2SO3Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the L- or the D-Phe(p-CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure L isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI approximately 1 nM) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The "stabilized-CCK8 peptide", easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.     

Real time monitoring of acylations during solid phase peptide synthesis: a method based on electrochemical detection

Monitoring of acylation reactions during solid phase peptide synthesis is important to ensure high coupling yields in all steps of the synthesis. We describe in this paper a simple and reliable method for monitoring the time course of the acylation steps as well as the washing and deprotection steps during computer-controlled solid phase peptide synthesis. The method is based on the continuous measurement of electrical conductivity in the reaction vessel. It is shown that there is a close correspondence between the degree of acylation (as determined from the amount of 9-fluorenylmethoxycarbonyl- (Fmoc) groups released during deprotection) and the conductivity profile obtained during coupling of the amino acids to the growing peptide chain. The measurements are fed back to the computer providing data for software control of the duration of the acylation, deprotection and washing steps. The method is demonstrated with pentafluorophenol esters, but is equally applicable to dihydroxybenzotriazole esters and symmetric anhydrides using the Fmoc-polyamide strategy in a continuous flow set-up with dimethylformamide (DMF) as the general solvent.     

Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI).

An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.     

Studies on epimerization-free methods for the preparation of aminosuccinyl peptides

Summary We have found that guanidine acetate catalyses the transformation of a -benzyl-aspartyl peptide (Boc-Asp-(OBzl)-Leu-Trp-OMe) to an aminosuccinyl peptide (Boc-Asu-Leu-Trp-OMe). The reaction was accompanied by partial epimerization. However, not even a small amount of epimerization could be detected when the aminosuccinyl peptide was synthesised from Boc-Asp-Leu-Trp-OMe with the addition of DIC, HOPfp and guanidine acetate (as a catalyst). This reaction seems to be suitable for the epimerization-free solid phase synthesis of aminosuccinyl peptides, e.g. Asu⁶-Lamprey-III-GnRH (Glp-His-Trp-Ser-His-Asu-Trp-Lys-Pro-Gly-NH₂).     

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys⁷⁷–Cys¹⁰⁹ bond, followed by iodine oxidation to form the Cys⁶⁷–Cys⁹⁹ bond.     

Segmented condensation of peptides on a solid phase using subtilisin complexed with sodium dodecyl sulfate

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using the Met residue cleavage by BrCN.     

Preparation of tri(ethylene glycol) grafted core‐shell type polymer support for solid‐phase peptide synthesis

A core‐shell type polymer support for solid‐phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on‐bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core‐shell type polymer supports (TEG SURE) for efficient solid‐phase peptide synthesis and on‐bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on‐bead bioassays.     

Racemization free coupling of peptide segments. Synthesis of an insect neuropeptide.

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed alpha-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt/NMM always rendered highly optically impure products containing 10-20% of the D-epimer.     

Solid phase synthesis of apamin, the principal neurotoxin in bee venom. Isolation and characterization of acetamidomethyl apamin

The synthesis of apamin, the principal neurotoxin in bee venom, has been accomplished by the solid phase method on a benzhydrylamine resin, 2-Phenylisopropyloxycarbonyl amino acids were used throughout the synthesis except for the C-terminal histidine. Improved yields in the coupling steps in the N-terminal part of the molecule were obtained by coupling each amino acid both in dichloromethane and dimethylformamide. The use of acetamidomethyl as an S-protecting group for cysteine made it possible to isolate and purify the linear peptide. The deblocked and oxidized peptide was fractionated by ion-exchange chromatography (Bio-Rex 70) to obtain a highly purified apamin with full biological activity and with the same physical and chemical properties as the natural peptide. Circular dichroism (CD) spectra of the synthetic and natural apamin were identical.     

Chemical synthesis of human beta-endorphin(1-27) analogs by peptide segment coupling. Leucine and glycine residues bearing thiocarboxyl functions as junctions for peptide segment coupling.

[Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2, two analogs of human beta-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides. Msc-[Gly8]beta hEP(1-8)SH and Msc-[L-Leu8]beta hEP(1-8)SH, were synthesized by standard solid phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[alpha-(Boc-L-Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 in the segment coupling reaction were approximately 18%. Less than 1% of racemization of Leu-8 occurred during coupling of Msc-[L-Leu8]beta hEP(1-8)SH to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of beta-endorphin(1-27) and its analogs influenced the receptor binding activity.     

A new micro-test for the detection of incomplete coupling reactions in solid-phase peptide synthesis using 2,4,6-trinitrobenzenesulphonic acid.

A rapid micro-test using 2,4,6-trinitrobenzenesulphonic acid has been developed to detect incomplete coupling reactions in solid phase peptide synthesis. This new test will detect 3 nmol of free amino groups per milligram of resin.     

Solid phase peptide synthesis in water VI: evaluation of water-soluble coupling reagents for solid phase peptide synthesis in aqueous media

Solid phase peptide synthesis requires large amounts of organic solvents, the safe disposal of which is an important environmental issue. Peptide synthesis, if performed in water and using less or nontoxic reagents, circumvents the disposal problem. Our ultimate aim is to develop an "environment-friendly" solid phase peptide synthesis (SPPS) methodology. Previously, we showed that SPPS in water is feasible. To perform SPPS in water, the coupling reagent must be water-soluble and maintain its reactivity in water. For this report, we tested the efficacy of the water-soluble coupling reagents, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), towards SPPS in water. We successfully synthesized Leu-enkephalin amide on a solid support suspended in aqueous 50% EtOH using DMT-MM and 2-(4-sulfophenylsulfonyl)ethoxycarbonylamino acids.     

The solid phase coupling of peptide segments catalyzed by the subtilisin-sodium dodecyl sulfate complex

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using Met residue cleavage by BrCN. Deceased.     

Determination of the availability of carbodiimide-activated N-protected amino acids in solid phase peptide synthesis

A new method is described for the determination of the availability of carbodiimide-activated N-protected amino acids in solid phase peptide synthesis. The method involves the addition of a second nucleophile to a solid phase coupling reaction at different time intervals and measuring the amount of activated amino acid intercepted. Using the DCCI-mediated coupling reaction of Boc-Ala-OH and H-Gly-O-resin with H-Gly-O-tBu as the second nucleophile, it was determined that $\text{ca}$. 61% of the theoretical maximum amount of activated Boc-Ala-OH was available after 8 hr of reaction.     

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.