Difficult couplings in stepwise solid phase peptide synthesis: predictable or just a guess?

Stepwise solid phase peptide synthesis, Fmoc-approach, of 88 peptides varying in length from 4 to 24 amino acid residues was performed using a uniform procedure for coupling, monitoring and deprotection steps. The data of 696 couplings have been incorporated into a computer programme in order to study whether the degree of coupling can be predicted. Parameters studied are the nature of the amino acid to be coupled, the amino acid to be acylated and the length of the growing peptide chain. Using this information, prediction of "good" or "difficult" sequences, that is, peptides that can be synthesized without appreciable repeated couplings or the opposite, seems possible.     

Enantioselective synthesis of (2R, 3S)- and (2S, 3R)-4,4,4-trifluoro-N-Fmoc-O-tert-butyl-threonine and their racemization-free incorporation into oligopeptides via solid-phase synthesis

An efficient method for the enantioselective synthesis of (2R, 3S)- and (2S, 3R)-4,4,4-trifluoro-N-Fmoc-O-tert-butyl-threonine on multigram scales was developed. Absolute configurations of the two stereoisomers were ascertained by X-ray crystallography. Racemization-free coupling conditions for the incorporation of tfT into oligopeptides were then explored. For solution-phase synthesis, tfT racemization was not an issue under conventional coupling conditions. For solid-phase synthesis, the following conditions were identified to achieve racemization-free synthesis: if tfT (3.0 equiv) was not the first amino acid to be linked to the resin (1.0 equiv), the condition is 2.7 equiv DIC/3.0 equiv HOBt as the coupling reagent at 0 degrees C for 20 h; if tfT (3.0 equiv) was the first amino acid to be linked to the resin (1.0 equiv), then 1.0 equiv of CuCl(2) needs to be added to the coupling reagent.     

Fmoc-based chemical synthesis and selective binding to supercoiled DNA of the p53 C-terminal segment and its phosphorylated and acetylated derivatives.

The C-terminal domain of p53 comprises a linker, the tetramerization domain and the regulatory domain, and contains at least seven sites of potential post-translational modification. An improved strategy was developed for the synthesis of large peptides that contain phosphorylated amino acids and p53(303-393), a 91-amino acid peptide, and three post-translationally modified derivatives were synthesized through the sequential condensation of three partially protected segments. Peptide thiolesters were prepared using the sulfonamide-based 'safety-catch' resin approach and employing Fmoc-based solid-phase peptide synthesis. At the N-terminus of the middle building block, a photolabile protecting group, 3,4-dimethoxy-6-nitrobenzyloxycarbonyl, was incorporated to differentiate the N-terminal amino group from the side-chain amino groups. Two sequential couplings were accomplished following this protection strategy. The synthetic products, p53(303-393) and its phosphorylated or acetylated derivatives, exhibited the ability to bind specifically to supercoiled DNA, which is one of the characteristics of this domain.     

'100 years of peptide synthesis': ligation methods for peptide and protein synthesis with applications to beta-peptide assemblies.

A brief survey of the history of peptide chemistry from Theodore Curtius to Emil Fischer to Bruce Merrifield is first presented. The discovery and development of peptide ligation, i.e. of actual chemical synthesis of proteins are described. In the main chapter, 'Synthesis of Proteins by Chemical Ligation' a detailed discussion of the principles, reactivities and mechanisms involved in the various coupling strategies now applied (ligation, chemical ligation, native chemical ligation) is given. These include coupling sites with cysteine and methionine (as well as the seleno analogs), histidine, glycine and pseudo-prolines, 'unrestricted' amino-acid residues (using the Staudinger reaction), as well as solid-phase segment coupling by thioligation of unprotected peptides. In another section, 'Synthesis of beta-peptides by Thioligation', couplings involving beta2- and beta3-peptides are described (with experimental details).     

Inclusion volume solid-phase synthesis

Solid-phase synthesis of peptides was carried out using only the volume of the solvent included in the swollen solid-phase resin beads [inclusion volume synthesis]. This approach enables (i) the use of higher concentrations of activated amino acids, resulting in increased coupling rates, (ii) drastically decreased consumption of solvents, and (iii) the construction of multiple peptide synthesizers having virtually no reaction vessels.     

Studies in solid-phase peptide synthesis: a personal perspective

By the early 1970s it had became apparent that the solid-phase synthesis of ribonuclease A could not be generalized. Consequently, virtually every aspect of solid-phase peptide synthesis (SPPS) was reexamined and improved during the decade of the 1970s. The sensitive detection and elimination of possible side reactions (amino acid insertion, N(alpha)-trifluoroacetylation, N(alphaepsilon)-alkylation) were examined. The quantitation of coupling efficiency in SPPS as a function of chain length was studied. A new and improved support for SPPS, the "PAM-resin," was prepared and evaluated. These and many other studies from the Merrifield laboratory and elsewhere increased the general acceptance of SPPS leading to the 1984 Nobel Prize in Chemistry for Bruce Merrifield.     

DEPBT as an efficient coupling reagent for amide bond formation with remarkable resistance to racemization

3-(Diethoxyphosphoryloxy)-1, 2, 3-benzotriazin-4(3H)-one (DEPBT) is an effective coupling reagent for synthesis of linear and cyclic peptides by both solution and solid-phase peptide synthesis. DEPBT mediates amide bond formation with remarkable resistance to racemization. When DEPBT is used as a coupling reagent, it is not necessary to protect the hydroxy group of the amino component (such as tyrosine, serine, and threonine) and the imidazole group in the case of histidine. The high efficiency of DEPBT and its utility have been demonstrated in the syntheses of complex natural products such as ramoplanin A2, ramoplanose aglycon, ustiloxin, and teicoplanin aglycon.     

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.     

Problem of aspartimide formation in Fmoc-based solid-phase peptide synthesis using Dmab group to protect side chain of aspartic acid.

The sequence-dependent, acid- or base-catalysed aspartimide formation is one of the most serious side reactions in solid-phase synthesis of peptides containing aspartic acid. In the present work, we investigated the susceptibility of 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino)benzyl (Dmab), an aspartic acid beta-carboxy side-chain protecting group, for aspartimide formation. As a model, 15-amino acid-residue galanin fragment analogue containing the Asp-Ala motif was used during Fmoc-based solid-phase synthesis. Our study showed a strong tendency of Dmab-protected peptide to form aspartimide with unusual high efficiency. Furthermore, to investigate the susceptibility of Asp-Ala motif for aspartimide formation during the synthesis using Asp(ODmab), a 5-amino acid-residue galanin fragment LGPDA, different types of resin linkers, variety of Fmoc-deprotection conditions and coupling methods were applied.     

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.     

Investigation of the automated solid-phase synthesis of a 38mer peptide with difficult sequence pattern under different synthesis strategies

Difficult peptides are a constant challenge in solid-phase peptide synthesis. In particular, hydroxyl amino acids such as serine can cause severe breakdowns in coupling yields even several amino acids after the insertion of the critical amino acid. This paper investigates several methods of improving synthesis yields of difficult peptides including the use of different resins, activators and the incorporation of a structure-breaking pseudoproline dipeptide building block both alone and in combination with each other.     

Combinatorial approaches to harmacophoric heterocycles: A solid-phase synthesis of 3,1-benzoxazine-4-ones

An efficient solid-phase synthesis of 3,1-benzoxazine-4-ones is described. Immobilized amino acid based functionalized urea derivatives 2 undergo a high yielding heterocyclization under mild conditions in presence of coupling reagents (DIC, TsCl/Py, or Ac2O) to afford 3,1-benzoxazine-4-ones 6. The method offers broad scope for structural and chemical diversity, and is amenable for combinatorial synthesis of 3,1-benzoxazine-4-ones libraries with potential for discovery of novel serine protease inhibitors. Copyright 1998 John Wiley & Sons, Inc.     

An efficient, convenient solid-phase synthesis of amino acid-modified peptide nucleic acid monomers and oligomers

An efficient and highly versatile method for the synthesis of amino acid-modified peptide nucleic acid (PNA) monomers is described. By using solid-phase Fmoc techniques, such monomers can be assembled readily in a stepwise manner and obtained in high yield with minimal purification. Protected neutral hydrophilic, acidic, and basic amino acids were coupled to 2-chlorotrityl chloride resin. Following Fmoc removal, innovative conditions for the key step, reductive alkylation with N-Fmoc-aminoacetaldehyde, were developed to circumvent problems encountered with previously reported methods. Activation and coupling of pyrimidine and purine nucleobases to the resulting secondary amines afforded amino acid-modified PNA monomers. The mild reaction conditions utilized were compatible with sensitive and labile functional groups, such as tert-butyl ethers and tert-butyl esters. PNA monomers were obtained in 36-42% overall yield and very high purity, after cleavage and purification. Using standard solid-phase Fmoc chemistry, two of these monomers were incorporated with high coupling efficiency into a variety of modified PNA oligomers, including four tetradecamers designed to target bcl-2 mRNA. Such modified oligomers have the potential to enhance water solubility and cell portability, while maintaining hybridization affinity and promoting favorable biodistribution properties.     

Developments in peptide and amide synthesis

The solid-phase methodology is key for an effective synthesis of peptides, from a milligram scale for research to a multi-kilo scale for drug production. Indeed, small peptides containing up to 20-30 amino acids are most readily synthesized by a solid-phase strategy. Larger peptides (up to 60 amino acids) should be synthesized by a convergent approach (i.e. synthesis of protected constituent peptides in solid-phase and combination of these units in solution). Larger peptides and proteins are prepared by chemical ligation, where unprotected segments have been prepared in solid-phase.     

DNA display III. Solid-phase organic synthesis on unprotected DNA

DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl–based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide–DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation.     

Automated solid-phase peptide synthesis: use of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate for coupling of tert-butyloxycarbonyl amino acids.

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) has been adapted for use as a coupling reagent for tert-butyloxycarbonyl (Boc) amino acids in automated solid-phase peptide synthesis. When compared to the existing preformed symmetrical anhydride procedure employing dicyclohexyl-carbodiimide (DCC), the use of TBTU in the presence of 1-hydroxybenzotriazole (HOBt) provides a more efficient coupling procedure for Boc-amino acid derivatives. Overall cycle times using TBTU/HOBt coupling reagents (30 min) compare favorably to those of the DCC-mediated procedure (approx 65 min). Dimethylformamide can be used as the sole solvent for both activation and coupling reactions. Implementation of TBTU/HOBt coupling conditions does not require replumbing of any lines of the Applied Biosystems Model 430A instrument and necessitates changes to only three reagent bottle positions. The variable coupling efficiencies of Boc-asparagine following activation with TBTU/HOBt (as low as 89%) can be overcome by protection of the amide function of Boc-asparagine with the 9-xanthyl group. Examples of the synthesis and characterization of a number of peptides ranging in length from 13 to 29 residues are given.     

Synthesis and conformational characterization of the epidermal growth factor-like domain of blood coagulation factor IX carrying xylosyl-glucose

Solid-phase synthesis of glycopeptide generally requires the protection of both peptide side chains and hydroxyl groups of the carbohydrate portion. However, if the mild coupling conditions are used, the protection of the carbohydrate portion can be omitted. In this paper, we demonstrated it by the synthesis of Fmoc-serine carrying unmasked xylosyl glucose followed by the solid-phase synthesis of epidermal growth factor (EGF)-like domain of factor IX (45-87) using the unit. The product was well characterized by enzymatic digestion, amino acid analysis and mass spectrometry. The secondary structure of the product as well as glucosylated and non-glycosylated EGF-like domain was characterized by circular dichroism (CD) spectroscopy.     

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.     

Utilization of Proteases from Aspergillus Species for Peptide Synthesis via the Kinetically Controlled Approach

We examined Aspergillus melleus protease (Amano protease P) and A. oryzae protease (Amano protease A) as catalysts for peptide bond formation via the kinetically controlled approach. As the coupling efficiency was only moderate, even with a good amino acid substrate as the carboxyl component, in acetonitrile as a solvent (with or without a small amount of added water) that we had mainly employed previously in α-chymotrypsin catalyzed couplings, other solvent systems were sought. In 1,1,1,3,3,3-hexafluoro-2-propanol-DMF (1:1) without added water, these Aspergillus proteases were found to remain active for a long period of time and to be utilizable for peptide synthesis when the carbamoylmethyl ester was employed as the acyl donor, though the coupling efficiencies were dependent rather largely on the combination of the amino acid residues at the coupling site. The superiority of the carbamoylmethyl ester to conventional esters, for example the methyl ester, was once again established. Furthermore, some segment condensations were also achieved by the same procedure.     

Solid-phase synthesis of functionalized bis-peptides

We demonstrate the first solid-phase synthesis of highly functionalized bis-peptides. Bis-peptides are ladder oligomers composed of stereochemically pure, cyclic bis-amino acids joined by substituted diketopiperazine linkages. They have a shape-programmable backbone that is controlled by controlling the stereochemistry and sequence of the monomers within each oligomer. Functionalized bis-peptides are assembled using a new amide bond forming reaction (acyl-transfer coupling) that we have previously developed and a novel activation strategy that allows the sequential formation of penta- and hexa-substituted diketopiperazines from extremely hindered N-alkyl-alpha,alpha-disubstituted amino acids. We present mechanistic evidence that acyl-transfer coupling is competitive with direct acylation in the formation of hindered amide bonds. We also detail the synthesis of four functionalized bis-peptides, and that by combining bis-peptides with amino acids through diketopiperazine linkages, bis-peptides can mimic the display of residues i, i+4, i+7 of an alpha-helical peptide.