A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Solid phase synthesis of an amphiphilic peptide modified for immobilisation at the C-terminus

The solid phase peptide synthesis (SPPS) of the amphiphilic peptide Ac-(Leu-Ala-Arg-Leu)(3)-linker, which is modified at the C-terminus with 1,8-diamino-3,6-dioxaoctane as linker moiety, has been investigated. Two different approaches that allow for the synthesis of C-terminally modified, side-chain protected peptides were examined. The solid phase peptide synthesis using aliphatic safety-catch resin followed by activation and aminolysis with the mono-Boc protected linker was compared with the synthesis on 1,8-diamino-3,6-dioxaoctane loaded 2-chlorotrityl resin.     

2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage

The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr.     

Heating and microwave assisted SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield

Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides.     

Peptides Derived from α-hydroxymethylserine: Aspects of solid-phase synthesis

Summary The solid-phase synthesis of peptides derived from the sterically hindered α-hydroxymethylserine (HmS) was investigated. The acid-sensitive,O,O-isopropylidene (Ipr) protection of HmS is compatible with the Fmoc chemistry, represented here by the Fmoc-HmS(Ipr)-OH and Fmoc-HmS(Ipr)-F derivatives. Three analogs of the opioid pentapeptide DADLE with a single or two consecutive HmS residue(s) were synthesized using Wang resin as the solid support. The HATU method has been shown to effectively accomplish ‘difficult’ couplings with the HmS(Ipr) residue. Wang resin is not suitable, for the synthesis of sequences with a C-terminal HmS because of the easy formation of the diketopiperazine resulting from the cyclization of the susceptible dipeptide sequence AA-HmS(Ipr) bound to the resin. A further drawback of the Wang resin methodology is the increased danger of the undersired N→O-acyl shift, when long-lasting acidic cleavage is applied. These side reactions are totally suppressed when the 2-chlorotrityl polystyrene is used as a solid support. The mild conditions (AcOH/TFE/DCM) applied for the peptide detachment from this resin do not affect the Ipr protection, affording highly pure fragments with HmS(Ipr) residues suitable for post-cleavage condensation, cyclization or controlled side-chain deprotection. This approach is documented by the efficient synthesis of linear and cyclic analogs of the opioid hexapeptide DTLET containing two residues of HmS or HmS(Ipr) in positions 2 and 6.     

Solid-phase synthesis of larger peptides by a new strategy of detachment from the resin

Attachment of the side-chain carboxyl of the protected aspartic or glutamic acid ester to the resin support has been established for the solid-phase synthesis of the asparagine or glutamine peptide. After further elongation of the α-amino deprotected resin-bound peptide ester with protected peptide fragments and the final detachment from the resin support by ammonolysis, the larger peptides containing, or preferably C-terminated with, asparagine or glutamine could be obtained. Thus, the C-peptide of human proinsulin was prepared by coupling to the resin-bound dipeptide derivative, Leu-Glu(OCH₂Ph®)·OtBu, with six fragments consecutively. It was obtained in an overall yield of 36% after detaching from the resin with alcoholic ammonia, followed by mild acidolysis, DEAE cellulose chromatography, and gel filtration. This procedure has now been applied to the synthesis of the C-terminal fragment of the insulin A chain ending in asparagine, and also to the synthesis of the threonine or serine peptide, where the anchorage to the resin was designed by the reaction of the sidechain hydroxyl with succinic anhydride in the presence of 4-dimethylaminopyridine to form the hemiester of succinic acid, which in turn was condensed to the aminomethyl resin by the DCC-HOBt procedure. Model experiments on the synthesis of the Z-Thr(CO-CH₂CH₂CONHCH₂Ph®)·OtBu and Bpoc-Lys(Boc)-Thr(COCH₂CH₂CONHCH₂Ph®)·OtBu, as well as their detachment from the resin by amminolysis or hydrazinolysis, have shown the potential for a milder process in the solid-phase synthesis of larger peptides.     

Orthogonal protecting groups for N(alpha)-amino and C-terminal carboxyl functions in solid-phase peptide synthesis

For the controlled synthesis of even the simplest dipeptide, the N(alpha)-amino group of one of the amino acids and the C-terminal carboxyl group of the other should both be blocked with suitable protecting groups. Formation of the desired amide bond can now occur upon activation of the free carboxyl group. After coupling, peptide synthesis can be continued by removal of either of the two protecting groups and coupling with the free C-terminus or N(alpha)-amino group of another protected amino acid. When three functional amino acids are present in the sequence, the side chain of these residues also has to be protected. It is important that there is a high degree of compatibility between the different types of protecting groups such that one type may be removed selectively in the presence of the others. At the end of the synthesis, the protecting groups must be removed to give the desired peptide. Thus, it is clear that the protection scheme adopted is of the utmost importance and makes the difference between success and failure in a given synthesis. Since R. B. Merrifield introduced the solid-phase strategy for the synthesis of peptides, this prerequisite has been readily accepted. This strategy is usually carried out using two main protection schemes: the tert-butoxycarbonyl/benzyl and the 9-flourenylmethoxycarbonyl/tert-butyl methods. However, for the solid-phase preparation of complex or fragile peptides, as well as for the construction of libraries of peptides or small molecules using a combinatorial approach, a range of other protecting groups is also needed. This review summarizes other protecting groups for both the N(alpha)-amino and C-terminal carboxyl functions.     

Preparation of protected peptide amides using the Fmoc chemical protocol. Comparison of resins for solid phase synthesis.

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 (1) was synthesized on both the acid-labile 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanol, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

Chemical synthesis of NS-1 region of hepatitis C-viral polyprotein fragments: a comparison of PS-BDODMA resin and merrifield resin

Three peptide fragments selected from the NS-1 region of hepatitis C-viral polyprotein (Leu-Ile-Asn-Thr-Asn-Ala-Ser-Trp-His-Ala-Asn-Arg-Thr-Ala-Leu-Ser Asn-Asp Ser-Lys Leu Asn Thr-Gly Ala NH(2), Leu-lle Asn Thr Asn Ala Ser-Trp-His-Ala-Asn-Arg-Thr Ala NH(2) and Leu-Asn-Cys(Acm)-Asn-Asp-Ser-Leu-Asn-Thr-Ala-NH(2)) have been synthesized on PS-BDODMA resin. The synthetic capability of the resin PS-BDODMA resin was compared with Merrifield resin. The peptides were synthesized by the stepwise fluoren-9-yl methoxycarbonyl (Fmoc) solid-phase method. The synthesized peptides were purified by HPLC and the identity of the peptides was established by mass spectrum and amino acid analysis. The synthesis of these peptides illustrates the application of the PS-BDODMA resin for the synthesis of long chain peptides in high yield and homogeneity compared to the Merrifield resin.     

Chemistry of alpha-hydroxymethylserine: problems and solutions

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize "difficult" sequences containing a C-terminal HmS residue, MeA-HmS or consecutive HmS. Preparative methods for orthogonal N- and/or C-protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid-phase peptide chemistry protocols allows synthetic flexibility toward HmS-containing peptides. In the synthesis of the sterically hindered dipeptides with the C-terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid-phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C-terminal HmS(Ipr) or HmS residues, thus precluding their C-->N elongation. The successful protocols involve: (i) the 2+1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2-chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N-->O-acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin.     

两种树脂对固相法合成丙型肝炎疫苗多肽的影响

本实验采用固相法合成丙型肝炎疫苗多肽,比较Wang树脂和二氯三苯甲基树脂（以下简称二氯树脂）作为载体时的连接率以及目标肽的产率和纯度。通过质谱鉴定分析所得目标多肽的分子量,用茚三酮法对两种树脂与氨基酸的连接率进行比较,用反相高效液相色谱（RP-HPLC）进行粗肽的分析和纯化。结果显示以二氯树脂作载体,第一个氨基酸的连接率,以及目标肽的纯度和产率都要明显高于Wang树脂,因此二氯树脂作载体合成丙型肝炎疫苗多肽更适合大规模工业化生产的要求。     

Facile SPS of peptides having C-terminal Asn and Gln

Attachment of Fmoc-asparagine or glutamine to p-alkoxybenzyl alcohol type resins has always been difficult and not very effective. A very simple and effective method for the preparation of peptides terminating in asparagine or glutamine is described. The method involves quantitative attachment of Fmoc-Asp-OtBu or Fmoc-Glu-OtBu via their side-chain carboxyl group to a resin functionalized with our TMBPA linker for peptide amides. Peptide synthesis is performed using our standard Fmoc chemistry, and treatment with acid, e.g. TFA/DCM/scavenger mixtures, releases the Asn or Gln peptides.     

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.     

Solid support-dependent alkylation of tryptophan residues in SPPS using a 2-methoxybenzyl alcohol-based linker

Summary Several side reactions can be encountered in the synthesis of Trp-containing peptides, due to molecular species originating from side chain-protecting groups or from the linker during acidolytic cleavage of the peptide from the resin. The linker can be the source of both alkylation in solution of the indole moiety of the tryptophan side chain and permanent readdition of the cleaved peptide to the resin. We report that both these reactions occur at a high level during the synthesis of Trp-containing peptides on a PEG-PS resin containing a 2-methoxybenzyl alcohol-based linker, in spite of the presence of suitable scavengers in the TFA-based cleavage mixture. Both side reactions are efficiently prevented by the use of a protected analogue of tryptophan, namely N^im-Boc-Trp, previously reported for the synthesis of peptides containing tryptophan and arginine residues.     

1,4-diazepine-2,5-dione ring formation during solid phase synthesis of peptides containing aspartic acid beta-benzyl ester.

The Fmoc-based SPPS of H-Xaa-Asp(OBzl)-Yaa-Gly-NH(2) sequences results in side reactions yielding not only aspartimide peptides and piperidide derivatives, but also 1,4-diazepine-2,5-dione-peptides. Evidence is presented to show that the 1,4-diazepine-2,5-dione derivative is formed from the aspartimide peptide. The rate of this ring transformation depends primarily on the tendency to aspartimide and piperidide formation, which is influenced by the nature of the amino acid following the aspartic acid beta-benzyl ester (Xaa). However the bulkiness of the amino acid side chain preceeding the aspartic acid beta-benzyl ester (Yaa) is also important. Under certain conditions the 1,4-diazepine-2,5-dione peptide derivative may even be formed dominantly, which is a highly undesirable side reaction in peptide synthesis, but which provides a new way for the synthesis of diazepine peptide derivatives with targeted biological or pharmacological activity.     

Comparative Study of Chemical Approaches to the Solid-Phase Synthesis of a Tumor-Seeking α-MSH Analogue

The synthesis of a cyclic melanocortin analogue (H-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂), where the Boc-protected derivative of a metal-chelating pyrazolyl ligand (pz) was inserted as N-terminal residue, was addressed by several different Fmoc/tBu and Boc/Bzl solid-phase strategies. On-resin cyclization was achieved immediately following incorporation of Asp, by condensation of the Asp side chain carboxyl with the Lys side chain primary amine after selective and simultaneous removal of side chain protecting groups. The success of the synthesis was highly dependent on the chemical strategy employed, with Boc/Bzl chemistry giving the best results. On the light of our findings, Fmoc/tBu strategies are not advantageous for the solid-phase synthesis of this particular type of lactam-bridged peptides. Last, but not least, the target peptide was recently found to have promising tumor-seeking properties (J Biol Inorg Chem 13:449–459, 2008).     

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.     

Site-specific biotinylation. A novel approach and its application to endothelin-1 analogs and PTH-analog.

We have developed an expeditious method for the incorporation of the biotinylaminocaproyl moiety on the epsilon-amino group of a lysine residue within a peptide chain in a site-specific manner. Using t-Boc chemistry for the solid phase synthesis approach and a base labile, acid stable protecting group (Fmoc-) for the epsilon-amino group of the target lysine, we prepared fully protected resin bound peptides which are site-specifically biotinylated. Following HF cleavage, the uniquely biotinylated peptides were obtained in a high degree of purity. Using this approach, a number of biotinylaminocaproyllysyl derivatives of a monocyclic Endothelin-1 analog were prepared. Synthesis of selected bicyclic analogs of high affinity monocycles led to the preparation of the bicyclic [Nle7]ET-1 analog containing epsilon-biotinylaminocaproyllysine at position-9. This peptide, with Kd = 0.08 nM, has 1000-fold higher affinity for the ETA receptor than the commercially available N alpha-biotinylated Endothelin-1. The general utility of this biotinylation methodology was demonstrated by the synthesis of a site-specifically biotinylated PTH analog which contained several side chain functionalized amino acid residues in its sequence. The synthetic method reported here is convergent in that it allows the facile variation of the length of the spacer and also offers the potential to introduce in a site specific manner other groups such as affinity labels and fluorescent tags.     

The production of anti-hexapeptide antibodies which recognize the S7, L6 and L13 ribosomal proteins of Escherichia coli.

Here we report the synthesis of the N-terminal hexapeptide H-Pro-Arg-Arg-Arg-Val-Ile-OH of the E. coli ribosomal protein S7. the C-terminal hexapeptide H-Lys-Glu-Ala-Lys-Lys-Lys-OH of L6 and the C-terminal hexapeptide H-Pro-Gln-Val-Leu-Asp-Ile-OH of L13. All peptides were prepared by SPPS following the Fmoc-strategy, using DIC/HOBt and/or HBTU as coupling reagents and 2-chlorotrityl chloride resin as the solid support. The carrier linked synthetic peptides were injected into rabbits and elicited an anti-peptide response. These anti-hexapeptide antibodies were found to recognize the corresponding peptides and proteins.