Protected Peptide Segments Research Articles

Epimerization-Free Preparation of C-Terminal Cys Peptide Acid by Fmoc SPPS Using Pseudoproline-Type Protecting Group.

Preparation of C-terminal Cys-containing peptide acid by Fmoc solid-phase peptide synthesis (SPPS) is difficult due to base-mediated epimerization at Cys. In this paper, use of a C-terminal pseudoproline structure and Trt(2-Cl) resin achieved epimerization-free direct preparation of the C-terminal Cys-containing peptide acid by Fmoc SPPS. Additionally, the C-terminal Cys(ΨDmp,Hpro)-containing protected peptide segment was applied to an epimerization-free segment condensation reaction.

Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin.

We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-β-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.

Synthesis and application of Nα‐Fmoc‐Nπ‐4‐methoxybenzyloxymethylhistidine in solid phase peptide synthesis

The 4-methoxybenzyloxymethyl (MBom) group was introduced at the Nπ-position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)-assisted solid phase peptide synthesis (SPPS) with 9-fluorenylmethyoxycarbonyl (Fmoc) chemistry. The Nπ-MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side-chain-induced racemization during incorporation of the His residue even in the case of MW-assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His-containing peptide segments from acid-sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc-His(π-MBom)-OH with the aid of purification procedure by crystallization to effectively remove the undesired τ-isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large-scale production without any obstacles.

ChemInform Abstract: Polypeptide Synthesis by the Thioester Method

A novel method for polypeptide synthesis, in which partially protected peptide thioesters are used as building blocks, has been developed. Partially protected peptide thioesters are easily prepared by solid-phase methodology. The thioester moiety is converted to an active ester in the presence of a silver compound such as AgNO(3) or AgCl and an active ester component such as 1-hydroxybenzotriazole or 3,4-dihydro-3-hydro-4-oxo-1,2, 3-benzotriazine. Segment condensation can be accomplished using partially protected peptide segments. The consecutive condensation of the partially protected peptide segments is realized by the selective removal of the 9-flourenylmethoxycarbonyl group, for terminal amino protection, after segment condensation has been achieved. In this method, large peptide segments can easily be used. Thus, the products obtained by the thioester method can be separated from by-products by reverse phase high performance liquid chromatography, even when no purification process was performed during the prior segment condensation procedures. This indicates that proteins that have no specific features such as enzymatic or biological activities can be obtained after isolation, solely based on their chromatographic profiles. Thus, the thioester method will provide a new basis for protein studies including phosphorylated and glycosylated polypeptides.

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.

On-resin assembly of a linkerless lanthanide(III)-based luminescence label and its application to the total synthesis of site-specifically labeled mechanosensitive channels.

A synthesis strategy for the on-resin assembly of luminescent lanthanide chelates from commercially available compounds was developed. Advantages of the approach include the absence of spacers between the metal ion and the attachment site, and the compatibility with typical chemical protein synthesis protection schemes. Methoxycoumarin-labeled lysine and tris(tert-butyl)-DOTA were consecutively coupled with high efficiency to a free amino group in otherwise fully protected peptide segments using standard peptide synthesis methods. Addition of stoichiometric amounts of Tb(3+) to the modified, cleaved, and purified peptides yielded the desired lanthanide chelate. Incorporation of this label into a chemically synthesized, full-length mechanosensitive channel of large conductance (MscL) of E. coli and subsequent reconstitution into vesicles resulted in a functional mechanosensitive channel of comparable conductance to the wild-type channel. However, this channel required increased suction to gate. Excitation of the antenna molecule methoxycoumarin at 336 nm resulted in an emission spectrum typical for Tb(3+) and a luminescence lifetime of 0.67 ms. The location of the probe close to the backbone of this protein may provide precise information about conformational changes during channel opening from LRET studies.

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.

ChemInform Abstract: Cyclohexyl Ether as a New Hydroxy-Protecting Group for Serine and Threonine in Peptide Synthesis.

A new hydroxy-protecting group for Ser and Thr, cyclohexyl (Chx), has been developed, and its application to peptide synthesis is described. The Chx group is introduced to the hydroxy functions of Boc-Ser-OH and Boc-Thr-OH in two steps; Boc-Ser-OH and Boc-Thr-OH are treated with NaH and then allowed to react with 3-bromocyclohexene to afford N-Boc-O-(cyclohex-2-enyl)-Ser and N-Boc-O-(cyclohex-2-enyl)-Thr in satisfactory yields, which are hydrogenated in the presence of PtO2 to give Boc-Ser(Chx)-OH and Boc-Thr(Chx)-OH in good yields. The O-Chx group is stable to various acidic and basic conditions including TFA and 20% piperidine in DMF. It is not removed with catalytic hydrogenation over Pd–charcoal. The Chx group is, however, removed quantitatively with 1 mol dm−3 trifluoromethanesulfonic acid–thioanisole in TFA over a short period. These results indicate that the Chx group is suitable for the hydroxy-protection of Ser and Thr in peptide synthesis based on Boc-chemistry and can be used also in combination with either Nα-Fmoc or Nα-Z-protection. The apparent rate constant for removal of the Chx group with 50% TFA in CH2Cl2 (kChx) is found to be less than a twentieth of that of the Bzl group (kBzl), confirming the substantial stability of the Chx group under common Boc-deprotection conditions. Simulations of solid-phase peptide syntheses using kChx and kBzl indicate that the Chx group would be adequate for solid-phase synthesis of large peptides and proteins. Solid-phase synthesis of a peptide and conventional solution synthesis of a protected peptide segment using Chx protection are also demonstrated.

Polypeptide synthesis using an expressed peptide as a building block via the thioester method

An expressed peptide was proven to be useful as a building block in the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, [Lys(Boc) 15,25,31,48,57,68,80,95]-Max(14–101), was prepared via transamination of an N-terminal amino group from p21Max(13–101), which was obtained by expression using Escherichia coli. This peptide was utilized in the synthesis of [Ser(PO 3H 2) 2,11]-p21Max(1–101) via condensation with the peptide thioester, Boc-[Ser(PO 3H 2) 2,11]-p21Max(1–13)-SCH 2CH 2CO-Leu-OH.

Cyclohexyl ether as a new hydroxy-protecting group for serine and threonine in peptide synthesis

A new hydroxy-protecting group for Ser and Thr, cyclohexyl (Chx), has been developed, and its application to peptide synthesis is described. The Chx group is introduced to the hydroxy functions of Boc-Ser-OH and Boc-Thr-OH in two steps; Boc-Ser-OH and Boc-Thr-OH are treated with NaH and then allowed to react with 3-bromocyclohexene to afford N-Boc-O-(cyclohex-2-enyl)-Ser and N-Boc-O-(cyclohex-2-enyl)-Thr in satisfactory yields, which are hydrogenated in the presence of PtO2 to give Boc-Ser(Chx)-OH and Boc-Thr(Chx)-OH in good yields. The O-Chx group is stable to various acidic and basic conditions including TFA and 20% piperidine in DMF. It is not removed with catalytic hydrogenation over Pd–charcoal. The Chx group is, however, removed quantitatively with 1 mol dm−3 trifluoromethanesulfonic acid–thioanisole in TFA over a short period. These results indicate that the Chx group is suitable for the hydroxy-protection of Ser and Thr in peptide synthesis based on Boc-chemistry and can be used also in combination with either Nα-Fmoc or Nα-Z-protection. The apparent rate constant for removal of the Chx group with 50% TFA in CH2Cl2 (kChx) is found to be less than a twentieth of that of the Bzl group (kBzl), confirming the substantial stability of the Chx group under common Boc-deprotection conditions. Simulations of solid-phase peptide syntheses using kChx and kBzl indicate that the Chx group would be adequate for solid-phase synthesis of large peptides and proteins. Solid-phase synthesis of a peptide and conventional solution synthesis of a protected peptide segment using Chx protection are also demonstrated.

Synthesis of a Phosphorylated Polypeptide by a Thioester Method

Abstract A method for the synthesis of phosphorylated polypeptides was investigated through the synthesis of the phosphorylated cAMP response element binding protein 1 (19-106) amide, [Thr(PO3H2)69,71]-CRE BP1(19-106)-NH2, as a model compound. Three partially protected peptide segments: Boc-[Lys(Boc)23,46,48,54, Cys(Acm)27,32]-CRE BP1(19-56)-SCH2CH2CO-β-Ala-NH2, Fmoc-[Thr(PO3H2)69,71, Lys(Boc)77, Cys(Acm)79]-CRE BP1(57-83)-SCH2CH2CO-β-Ala-NH2, and [Lys(Boc)97,98,105,106]-CRE BP1(84-106)-NH2, were prepared by the Boc solid-phase method and were used as building blocks. The segment condensations were carried out sequentially by a thioester method in the presence of a silver compound, HOObt, and DIEA. The desired product was obtained in 19% yield, based on the C-terminal building block, after removal of all the protecting groups on the peptide. These results indicate that the thioester method is quite useful for the preparation of phosphorylated polypeptide even if it contains cysteine residues. One thing which remains to be improved is the efficiency of the method for the preparation of partially protected peptide thioesters, which contain phosphorylated amino acid residues.

Polypeptide synthesis by the thioester method.

A novel method for polypeptide synthesis, in which partially protected peptide thioesters are used as building blocks, has been developed. Partially protected peptide thioesters are easily prepared by solid-phase methodology. The thioester moiety is converted to an active ester in the presence of a silver compound such as AgNO(3) or AgCl and an active ester component such as 1-hydroxybenzotriazole or 3,4-dihydro-3-hydro-4-oxo-1,2, 3-benzotriazine. Segment condensation can be accomplished using partially protected peptide segments. The consecutive condensation of the partially protected peptide segments is realized by the selective removal of the 9-flourenylmethoxycarbonyl group, for terminal amino protection, after segment condensation has been achieved. In this method, large peptide segments can easily be used. Thus, the products obtained by the thioester method can be separated from by-products by reverse phase high performance liquid chromatography, even when no purification process was performed during the prior segment condensation procedures. This indicates that proteins that have no specific features such as enzymatic or biological activities can be obtained after isolation, solely based on their chromatographic profiles. Thus, the thioester method will provide a new basis for protein studies including phosphorylated and glycosylated polypeptides.

Preservation of the protective group under alkaline conditions by using CaCl 2. Applications in peptide synthesis

CaCl 2 is shown to dramatically increase the lifetime of the Fmoc protective group in alkaline iPrOH-H 2O but has little effect on hydrolytic processes. This enables efficient preparation of Fmoc-protected peptide segments by saponification of the corresponding C-terminal methyl or benzyl esters. Similarly, protected peptide segments prepared by Fmoc/tBu solid-phase synthesis can be selectively released from the support by CaCl 2-catalyzed alkaline hydrolysis of an N-acylurea-based linker.

Application of reversible amide-bond protection to suppress peptide segment epimerisation

Reversible alkylation of the C-terminal amide-bond of a protected peptide segment with 2-hydroxy-4-methoxybenzyl dramatically suppresses epimerisation during activation and coupling. However, due to the formation of a 4,5-dihydro-8-methoxy-1,4-benzoxazepin-2(3H)-one species upon activation the rate of coupling is low. A safety-catch amide-bond protecting group, 6-hydroxy-5-methyl-1,3-benzoxathiolyl, has been designed to suppress epimerisation and couple with excellent yield.

15] Convergent solid-phase peptide synthesis

Publisher Summary This chapter discusses solid-phase synthesis and coupling of protected peptides. There are two main strategies for the chemical synthesis of peptides: (1) chain elongation in linear synthesis is carried out by repetitive N α amino group deprotection and protected amino acid coupling steps, (2) convergent synthesis involves the synthesis and coupling of protected peptide segments. Both strategies can be carried out in solution or on a solid support, although solution and solid-phase methodologies can coexist in convergent synthesis. Protected peptide segments can be elaborated on solid supports and coupled in solution or vice versa. Convergent solid-phase peptide synthesis (CSPPS) involves (1) solidphase synthesis of protected peptides (these must retain the protecting groups of the N α -amino and the side-chain functions after cleavage from the resin), (2) purification and characterization of the protected peptides, and (3) their solid-phase coupling.

Synthesis of phospho-urodilatin by combination of global phosphorylation with the segment coupling approach.

The chemical synthesis of biologically active phosphorylated urodilatin (CDD/ANP-95-126) was achieved by using a strategy of coupling protected peptide segments in solution. Three protected peptide segments corresponding to urodilatin (1-14) with side chain-unprotected Ser10, (15-24) and (25-32) were prepared manually using Fmoc chemistry on an aminopropyl polystyrene resin with the super acid-labile HMPB linker. For the coupling of segments, the carboxy group of the C-terminal segment (25-32) was converted into the tert-butyl ester by treatment with TBTA. The protected peptide segments were coupled in the presence of EDC/HOOBt or TBTU/HOBt to yield fully protected urodilatin with a free hydroxy function at Ser10. Introduction of the phosphate was performed with Et2NP(OtBu)2 and tetrazole followed by oxidation of the phosphite. Alternatively, a prephosphorylated protected segment (1-14) was used in the segment condensation. Our investigations indicate that both pathways, phosphorylation of protected urodilatin in solution and use of a prephosphorylated building block, are suitable methods to obtain a large phosphopeptide of high purity without formation of H-phosphonates or other by-products.

Solid-phase assembly of backbone amide-protected peptide segments: an efficient and reliable strategy for the synthesis of small proteins

In the preceding paper we showed that epimerization of the C-terminal residue of a fully protected peptide segment was minimized when activation (and subsequent coupling) was performed using 1-hydroxybenzotriazole-catalysed reaction with diisopropylcarbodiimide in dichloromethane (DCM). Good solubility of the segment in DCM was ensured by the incorporation of appropriately placed N-(2-acetoxy-4-methoxybenzyl)(AcHmb) backbone amide protection into the fully protected peptide. This low-epimerization protocol, combined with our previously described straightforward purification of AcHmb-substituted fully protected segments, provides an efficient, reliable and flexible strategy for the solid-phase assembly of small proteins. We describe here the preparation of HIV-1Brutat[1–72, Cys(Acm)22,25,27,30,31,34,37] protein through the sequential solid-phase assembly of five backbone-protected segments (11–17 residues). Individual addition of each segment was very efficient, achieving > 95% acylation using a two-fold excess with reaction for 6 h. The target 72-mer was readily purified and isolated in 38.4% overall yield.

Convergent solid‐phase peptide synthesis 12. * Chromatographic techniques for the purification of protected peptide segments

The purification of a range of protected peptide segments has been carried out using modified reversed-phase chromatographic techniques in which DMF was added to the water and acetonitrile mixtures used as eluents. The purity of the recovered peptides was excellent and recoveries were high in all cases, even for longer hydrophobic segments. In several cases purifications were carried out on the hundreds of milligrams scale. For protected peptide segments containing Met, protection as the sulfoxide avoids its unwanted alkylation and oxidation, and the increased overall polarity can be useful in the purification of protected peptides incorporating this residue.

Facile synthesis of protected C-terminal peptide segments by Fmoc/But solid-phase procedures on N-Fmoc-9-amino-xanthen-3-yloxymethyl polystyrene resin

Side-chain protected peptidyl amides, peptidyl tert-butyl esters, and the novel peptidyl 4-[N-(oxyacetyl)-β-alaninamide]-benzyl ester (HMP-β-Ala-NH2 ester) have been synthesised in excellent yields on N-Fmoc-9-amino-xanthen-3-yloxymethyl polystyrene resin and selectively cleaved from the solid support using 1% trifluoroacctic acid in dichloromethane within 7–8 min.

Synthesis and applications of a new base-labile fluorene derived linker for solid-phase peptide synthesis

The handle N-[(9-hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS) is reported for the preparation of protected peptide segments in combination with a Boc/Bzl protection scheme. Treatment of peptide-resins with morpholine in DMF renders protected peptides in high yields and purities.