1 Nature Protocols 2007 Vol: 2(12):3247-3256. DOI: 10.1038/nprot.2007.454

Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences

This protocol for solid-phase peptide synthesis (SPPS) is based on the widely used Fmoc/tBu strategy, activation of the carboxyl groups by aminium-derived coupling reagents and use of PEG-modified polystyrene resins. A standard protocol is described, which was successfully applied in our lab for the synthesis of the corticotropin-releasing factor (CRF), >400 CRF analogs and a countless number of other peptides. The 41-mer peptide CRF is obtained within ~80 working hours. To achieve the so-called difficult sequences, special techniques have to be applied in order to reduce aggregation of the growing peptide chain, which is the main cause of failure for peptide chemosynthesis. Exemplary application of depsipeptide and pseudoproline units is shown for synthesizing an extremely difficult sequence, the Asn(15) analog of the WW domain FBP28, which is impossible to obtain using the standard protocol.

Mentions
Figures
Figure 1: Nowadays, most of the desired peptide sequences can be obtained by chemosynthesis, using appropriate protocols. Figure 2: Special units used during assembly to prevent peptide chain aggregation.R: H/CH3 (Ser/Thr). (a) Hmb-amino acid. (b) Pseudoproline. (c) Depsidipeptide unit. Figure 3: Diketopiperazine (DKP) formation promoted by piperidine during Fmoc removal.For common peptide chains, for X = NH, DKP formation occurs mostly during deprotection of the amino acid following either a Pro or an N-alkylated residue (R = alkyl), and preferably when R" = H. By assembly of depsipeptides (X = O), DKP formation can always occur during deprotection of the second residue following the ester bond, in an extent which is strongly dependent on the sequence54. Figure 4: Depsipeptides are converted into the all-amide form through an O,N-acyl shift, which occurs quantitatively under mildly basic conditions over a short period.R: H/CH3 (Ser/Thr). Figure 5: Base-catalyzed aspartimide formation on an OtBu-protected aspartic acid residue and subsequent aminolysis by piperidine, yielding the corresponding piperidide; in dimethyl formamide (DMF) the -piperidide may preferably be formed.The mass of the aspartimide peptide corresponds to the mass of the target peptide (M*) -18, whereas the piperidide peptide shows a mass difference of +67. Figure 6: HPLC profile of the crude corticotropin-releasing factor (CRF) synthesized according to the standard protocol. Figure 7: HPLC profiles of the crude N(15)FBP28WW.The red arrows indicate the target product. (a) Product synthesized following a standard protocol, too complex to be purified. (b) Product obtained by synthesis of a depsipeptide analog, as described in Box 2. (c) Product synthesized using pseudoprolines, as described in Box 3. The improvement in product quality from A to B or C is impressive.
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References
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    • . . . The first successful coupling of two amino acids was performed via acyl chlorides by Emil Fischer in 1903, but at that time no suitable amino-protecting group was available for synthesizing longer peptides1 . . .
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    • . . . The introduction of the benzyloxycarbonyl-protecting group by Bergmann and Zervas2 and other inventions, such as the development of tetraethyl pyrophosphite as a coupling reagent by Anderson et al.3 and the successful protection of the mercapto group of Cys by the benzyl residue4, as well as the removal of S-benzyl and tosyl groups with sodium in liquid ammonia4 (for an overview, see ref. 5) allowed, for the first time, the synthesis of the neurohypophysial nonapeptide hormone oxytocin6, for which du Vigneaud was awarded the Nobel Prize in 1955 . . .
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    • . . . The introduction of the benzyloxycarbonyl-protecting group by Bergmann and Zervas2 and other inventions, such as the development of tetraethyl pyrophosphite as a coupling reagent by Anderson et al.3 and the successful protection of the mercapto group of Cys by the benzyl residue4, as well as the removal of S-benzyl and tosyl groups with sodium in liquid ammonia4 (for an overview, see ref. 5) allowed, for the first time, the synthesis of the neurohypophysial nonapeptide hormone oxytocin6, for which du Vigneaud was awarded the Nobel Prize in 1955 . . .
  5. Merrifield, R.B. History of protein synthesis. In Houben-Weyl. Methods of Organic Chemistry. Vol. E 22b: Synthesis of Peptides and Peptidomimetics (eds. Goodman, M., Felix, A., Moroder, L. & Toniolo, C.) 3-41 (Thieme, Stuttgart, New York, 2002) , .
    • . . . The introduction of the benzyloxycarbonyl-protecting group by Bergmann and Zervas2 and other inventions, such as the development of tetraethyl pyrophosphite as a coupling reagent by Anderson et al.3 and the successful protection of the mercapto group of Cys by the benzyl residue4, as well as the removal of S-benzyl and tosyl groups with sodium in liquid ammonia4 (for an overview, see ref. 5) allowed, for the first time, the synthesis of the neurohypophysial nonapeptide hormone oxytocin6, for which du Vigneaud was awarded the Nobel Prize in 1955 . . .
  6. Du Vigneaud, V., Ressler, C., Swan, J.M., Roberts, C.W. & Katsoyannis, P.G. The synthesis of oxytocin. J. Am. Chem. Soc. 76, 3115-3121 , (1954) .
    • . . . The introduction of the benzyloxycarbonyl-protecting group by Bergmann and Zervas2 and other inventions, such as the development of tetraethyl pyrophosphite as a coupling reagent by Anderson et al.3 and the successful protection of the mercapto group of Cys by the benzyl residue4, as well as the removal of S-benzyl and tosyl groups with sodium in liquid ammonia4 (for an overview, see ref. 5) allowed, for the first time, the synthesis of the neurohypophysial nonapeptide hormone oxytocin6, for which du Vigneaud was awarded the Nobel Prize in 1955 . . .
  7. Merrifield, R.B. Solid phase peptide synthesis. 1. Synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149-2154 , (1963) .
    • . . . Merrifield, that is to assemble peptides onto a solid phase7 (Nobel Prize 1984), had an enormous impact on the further development of peptide synthesis . . .
    • . . . Those so-called 'difficult sequences'7, 42 might be more easily assembled by the Boc/Bzl than Fmoc/tBu strategy, because trifluoroacetic acid (TFA), which is used for removing the temporary Nα-protecting group in the first case, can destroy aggregates, in contrast to piperidine/DMF, which is mostly used for Fmoc-removal . . .
  8. Atherton, E., Clive, D.L. & Sheppard, R.C. Polyamide supports for polypeptide-synthesis. J. Am. Chem. Soc. 97, 6584-6585 , (1975) .
    • . . . Although Merrifield's solid support, cross-linked poly(styrene-divinylbenzene) is still in use, more polar resins gave better results8, and cross-linked poly(dimethylacrylamide) resins8 were developed, as well as combinations of soft polyamides with rigid, highly permeable matrices, constructed from kieselguhr or highly cross-linked polystyrene9 . . .
  9. Atherton, E., Brown, E. & Sheppard, R.J. Internal association in solid-phase peptide-synthesis-synthesis of cytochrome-C residues 66-104 on polyamide supports. J. Chem. Soc. Chem. Commun. 1151-1152 , (1981) .
    • . . . Although Merrifield's solid support, cross-linked poly(styrene-divinylbenzene) is still in use, more polar resins gave better results8, and cross-linked poly(dimethylacrylamide) resins8 were developed, as well as combinations of soft polyamides with rigid, highly permeable matrices, constructed from kieselguhr or highly cross-linked polystyrene9 . . .
  10. Bayer, E. Towards the chemical synthesis of proteins. Angew. Chem. Int. Ed. Engl. 30, 113-129 , (1991) .
    • . . . High mechanical stability, in combination with proper solvation behavior, was also successfully achieved by copolymerization of ethylene oxide and polystyrene10 or by grafting PEG chains onto polystyrene beads11 . . .
  11. Zalipsky, S., Chang, J.L., Albericio, F. & Barany, G. Preparation and applications of polyethylene glycol-polystyrene graft resin supports for solid-phase peptide-synthesis. React. Polym. 22, 243-258 , (1994) .
    • . . . High mechanical stability, in combination with proper solvation behavior, was also successfully achieved by copolymerization of ethylene oxide and polystyrene10 or by grafting PEG chains onto polystyrene beads11 . . .
  12. Albericio,, F. & Giralt, E. Handles and supports. In Houben-Weyl. Methods of Organic Chemistry. Vol. E 22a: Synthesis of Peptides and Peptidomimetics (eds. Goodman, M., Felix, A., Moroder, L. & Toniolo, C.) 685-709 (Thieme, Stuttgart, New York, 2002) , .
    • . . . Nowadays, commercially available resins are modified by appropriate handles, which enable anchoring of the protected C-terminal amino acid residue by the formation of ester or amide bonds, thus allowing the synthesis of peptide acids and peptide amides, respectively12 . . .
  13. Carpino, L.A. Oxidative reactions of hydrazines. 2. Isophthalimides. New protective groups on nitrogen. J. Am. Chem. Soc. 79, 98-101 , (1957) .
    • . . . For temporary protection of the N-terminal amino group, the Boc-group13 is ideally suited, because its urethane structure helps to minimize epimerization of activated amino acids, and deprotection can be achieved using various acidic agents under relatively mild conditions . . .
  14. Stewart, J.M. Protection strategies. In Houben-Weyl. Methods of Organic Chemistry. Vol. E 22a: Synthesis of Peptides and Peptidomimetics (eds. Goodman, M., Felix, A., Moroder, L. & Toniolo, C.) 726-739 (Thieme, Stuttgart, New York, 2002 , .
    • . . . Although many peptides14, and even short proteins, have been successfully synthesized using the Boc/Bzl/HF technique, the potential hazards of HF and the requirement for HF-resistant equipment prompted the search for alternative . . .
    • . . . Under such conditions, the diffusion of reagents into the matrix is limited, coupling and deprotection reactions are often slow and incomplete, and the Kaiser test may give false negative results (reviewed in ref. 14) . . .
  15. Carpino, L.A. 9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. J. Am. Chem. Soc. 92, 5748 , (1970) .
    • . . . The introduction of the Fmoc-protecting group—developed by Carpino in 1970 (ref. 15)—into SPPS16 allowed the entire process of SPPS to be carried out using milder chemistry . . .
  16. Chang, C.D. & Meienhofer, J. Solid-phase peptide-synthesis using mild base cleavage of N-alphafluorenylmethyloxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostatin. Int. J. Pept. Protein Res. 11, 246-249 , (1978) .
    • . . . The introduction of the Fmoc-protecting group—developed by Carpino in 1970 (ref. 15)—into SPPS16 allowed the entire process of SPPS to be carried out using milder chemistry . . .
  17. Atherton, E. & Wellings, D.A. 9-Fluorenylmethoxycarbonyl/tert-butyl strategy. In Houben-Weyl. Methods of Organic Chemistry. Vol. E 22a: Synthesis of Peptides and Peptidomimetics (eds. Goodman, M., Felix, A., Moroder, L. & Toniolo, C.) 740-754 (Thieme, Stuttgart, New York, 2002) , .
    • . . . The orthogonal Fmoc/tBu chemistry was further improved by extension of the repertoire of novel side-chain protecting groups, such as Asn/Gln(Trt), Lys(Dde), Lys(Aloc), His(Trt), Arg(Pbf), Trp(Boc)17 . . .
  18. Sheehan, J.C. & Hess, G.P. A new method of forming peptide bonds. J. Am. Chem. Soc. 77, 1067-1068 , (1955) .
    • . . . Although more traditional coupling methods, such as diimide-based activation18, anhydride-mediated couplings19 and preactivated esters20 have been successfully applied, coupling reagents such as phosphonium21- or uronium/guanidinium (aminium)22-based structures are the most widely used today, especially in automated SPPS. . . .
  19. Wieland, T., Kern, W. & Sehring, R. Über anhydride von acylierten aminosäuren. Justus Liebigs Ann. Chem. 569, 117-121 , (1950) .
    • . . . Although more traditional coupling methods, such as diimide-based activation18, anhydride-mediated couplings19 and preactivated esters20 have been successfully applied, coupling reagents such as phosphonium21- or uronium/guanidinium (aminium)22-based structures are the most widely used today, especially in automated SPPS. . . .
  20. Schwyzer, R., Iselin, B. & Feurer, M. Über aktivierte ester. 1. Aktivierte ester der hippursäure und ihre umsetzungen mit benzylamin. Helv. Chim. Acta 38, 69-79 , (1955) .
    • . . . Although more traditional coupling methods, such as diimide-based activation18, anhydride-mediated couplings19 and preactivated esters20 have been successfully applied, coupling reagents such as phosphonium21- or uronium/guanidinium (aminium)22-based structures are the most widely used today, especially in automated SPPS. . . .
  21. Coste, J. Phosphonium salts. In Houben-Weyl. Methods of Organic Chemistry. Vol. E 22a: Synthesis of Peptides and Peptidomimetics (eds. Goodman, M., Felix, A., Moroder, L. & Toniolo, C.) 538-554 (Thieme, Stuttgart, New York, 2002) , .
    • . . . Although more traditional coupling methods, such as diimide-based activation18, anhydride-mediated couplings19 and preactivated esters20 have been successfully applied, coupling reagents such as phosphonium21- or uronium/guanidinium (aminium)22-based structures are the most widely used today, especially in automated SPPS. . . .
  22. Bienert, M., Henklein, P., Beyermann, M. & Carpino, L. A. Uronium/guanidinium salts. In Houben-Weyl. Methods of Organic Chemistry. Vol. E 22a: Synthesis of Peptides and Peptidomimetics (eds. Goodman, M., Felix, A., Moroder, L. & Toniolo, C.) 555-580 (Thieme, Stuttgart, New York, 2002) , .
    • . . . Although more traditional coupling methods, such as diimide-based activation18, anhydride-mediated couplings19 and preactivated esters20 have been successfully applied, coupling reagents such as phosphonium21- or uronium/guanidinium (aminium)22-based structures are the most widely used today, especially in automated SPPS. . . .
    • . . . Although this protocol allowed smooth syntheses of a countless number of medium-sized peptides, it led to very poor raw products when applied to two classes of peptides: the first category comprises sequences that contain sterically hindered amino acid residues like Cα- and Nα-alkylated amino acids22, 40, 41, whereas the second category consists of sequences that show a strong tendency to aggregate under conditions of SPPS . . .
  23. Atherton, E. & Sheppard, R.C. Solid Phase Peptide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1999) , .
  24. Pennington, M.W. & Dunn, B.M. Peptide Synthesis Protocols (Humana Press, Totowa, New Jersey, 1994) , .
  25. Fields, G.B. Solid-Phase Peptide Synthesis (Academic Press, New York, 1997) , .
  26. Lloyd-Williams, P., Albericio, F. & Giralt, E. Chemical Approaches to the Synthesis of Peptides and Proteins (CRC Press, Boca Raton, Florida, 1997) , .
  27. Chan, W.C. & White, P.D. Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Oxford University Press, Oxford, UK, 2000) , .
  28. Sewald, N. & Jakubke,, H.-D. Peptides: Chemistry and Biology (Wiley-VCH, Weinheim, 2002) , .
  29. Goodman, M., Felix, A., Moroder, L. & Toniolo, C. (eds.) Houben-Weyl. Methods of Organic Chemistry. Vol. E 22a-e: Synthesis of Peptides and Peptidomimetics (Thieme, Stuttgart, New York, 2002) , .
  30. Amblard, M., Fehrentz, J.A., Martinez, J. & Subra, G. Methods and protocols of modern solid phase peptide synthesis. Mol. Biotechnol. 33, 239-254 , (2006) .
  31. Dawson, P.E. & Kent, S.B. Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 69, 923-960 , (2000) .
    • . . . As a result of this fruitful chemical research, nowadays the synthesis of many medium-sized 30–50-mer peptides can be smoothly accomplished by manual or automate-assisted SPPS and even longer protein-like peptides can be synthesized by coupling protected segments (for reviews see refs. 23–30) or more efficiently by chemical ligation31 of nonprotected purified sequences . . .
  32. Bray, B.L. Large-scale manufacture of peptide therapeutics by chemical synthesis. Nat. Rev. Drug Discovery 2, 587-593 , (2003) .
    • . . . SPPS has also been successfully applied to large-scale production of peptide pharmaceutics32 . . .
  33. Vale, W., Spiess, J., Rivier, C. & Rivier, J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213, 1394-1397 , (1981) .
    • . . . The standard protocol reported in the PROCEDURE was optimized for the synthesis of the 41-mer peptide human/rat corticotropin-releasing factor (CRF)33, SEEPP ISLDL TFHLL REFLE MARAE QLAQQ AHSNR KLMEI I-NH2, which is the principal neuroregulator of the basal and stress-induced secretion of ACTH, β-endorphin and other peptides from the anterior pituitary34 . . .
  34. Dauzenberg, F.M. & Hauger, R.L. The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol. Sci. 23, 71-77 , (2002) .
    • . . . The standard protocol reported in the PROCEDURE was optimized for the synthesis of the 41-mer peptide human/rat corticotropin-releasing factor (CRF)33, SEEPP ISLDL TFHLL REFLE MARAE QLAQQ AHSNR KLMEI I-NH2, which is the principal neuroregulator of the basal and stress-induced secretion of ACTH, β-endorphin and other peptides from the anterior pituitary34 . . .
  35. Beyermann, M., Fechner, K., Furkert, J., Krause, E. & Bienert, M. A single-point slight alteration set as a tool for structure-activity relationship studies of ovine corticotropin- releasing factor. J. Med. Chem. 39, 3324-3330 , (1996) .
    • . . . According to this protocol, several hundreds of CRF analogs have been prepared, in the context of ligand–receptor interaction studies35, 36, 37. (An alternative synthesis of CRF using the Boc/Bzl/HF strategy at elevated temperature is reported in ref. 38.) Although peptide synthesis is often performed using a peptide synthesizer, we describe here the manual procedure . . .
  36. Beyermann, M. et al. A role for a helical connector between two receptor binding sites of a long-chain peptide hormone. J. Biol. Chem. 275, 5702-5709 , (2000) .
    • . . . According to this protocol, several hundreds of CRF analogs have been prepared, in the context of ligand–receptor interaction studies35, 36, 37. (An alternative synthesis of CRF using the Boc/Bzl/HF strategy at elevated temperature is reported in ref. 38.) Although peptide synthesis is often performed using a peptide synthesizer, we describe here the manual procedure . . .
  37. Beyermann, M. et al. Achieving signalling selectivity of ligands for the corticotropin-releasing factor type 1 receptor by modifying the agonist's signalling domain. Br. J. Pharmacol. 151, 851-859 , (2007) .
    • . . . According to this protocol, several hundreds of CRF analogs have been prepared, in the context of ligand–receptor interaction studies35, 36, 37. (An alternative synthesis of CRF using the Boc/Bzl/HF strategy at elevated temperature is reported in ref. 38.) Although peptide synthesis is often performed using a peptide synthesizer, we describe here the manual procedure . . .
  38. Rivier, J.E. & Miranda, M.T.M. Solid-phase peptide synthesis at elevated temperature. In Houben-Weyl. Methods of Organic Chemistry. Vol. E 22a: Synthesis of Peptides and Peptidomimetics (eds. Goodman, M., Felix, A., Moroder, L. & Toniolo, C.) 806-813 (Thieme, Stuttgart, New York, 2002) , .
    • . . . According to this protocol, several hundreds of CRF analogs have been prepared, in the context of ligand–receptor interaction studies35, 36, 37. (An alternative synthesis of CRF using the Boc/Bzl/HF strategy at elevated temperature is reported in ref. 38.) Although peptide synthesis is often performed using a peptide synthesizer, we describe here the manual procedure . . .
  39. Kaiser, E., Colescot, R.L., Bossinge, C.D. & Cook, P.I. Color test for detection of free terminal amino groups in solid-phase synthesis of peptides. Anal. Biochem. 34, 595-598 , (1970) .
    • . . . In the manual synthesis, the number of (expensive) second couplings can be minimized by checking the completeness of the first coupling step at each cycle (see also Box 1) using Kaiser tests39. . . .
  40. Carpino, L.A., Beyermann, M., Wenschuh, H. & Bienert, M. Peptide synthesis via amino acid halides. Acc. Chem. Res. 29, 268-274 , (1996) .
    • . . . Although this protocol allowed smooth syntheses of a countless number of medium-sized peptides, it led to very poor raw products when applied to two classes of peptides: the first category comprises sequences that contain sterically hindered amino acid residues like Cα- and Nα-alkylated amino acids22, 40, 41, whereas the second category consists of sequences that show a strong tendency to aggregate under conditions of SPPS . . .
  41. Gilon, C., Dechantsreiter, M.A., Burkhart, F., Friedler, A. & Kessler, H. Synthesis of N-alkylated peptides. In Houben-Weyl. Methods of Organic Chemistry. Vol. E 22c: Synthesis of Peptides and Peptidomimetics (eds. Goodman, M., Felix, A., Moroder, L. & Toniolo, C.) 215-271 (Thieme, Stuttgart, New York, 2002) , .
    • . . . Although this protocol allowed smooth syntheses of a countless number of medium-sized peptides, it led to very poor raw products when applied to two classes of peptides: the first category comprises sequences that contain sterically hindered amino acid residues like Cα- and Nα-alkylated amino acids22, 40, 41, whereas the second category consists of sequences that show a strong tendency to aggregate under conditions of SPPS . . .
  42. Hyde, C., Johnson, T., Owen, D., Quibell, M. & Sheppard, R.C. Some "difficult sequences" made easy. A study of interchain association in solid-phase peptide synthesis. Int. J. Peptide Protein Res. 43, 431-440 , (1994) .
    • . . . Those so-called 'difficult sequences'7, 42 might be more easily assembled by the Boc/Bzl than Fmoc/tBu strategy, because trifluoroacetic acid (TFA), which is used for removing the temporary Nα-protecting group in the first case, can destroy aggregates, in contrast to piperidine/DMF, which is mostly used for Fmoc-removal . . .
  43. Fields, C. & Fields, G.B. Solvents for solid-phase peptide synthesis. In Peptide Synthesis Protocols (eds. Penningten, M.W. & Dunn, B.M.) 29-40 (Humana Press, Totowa, New Jersey, 1994) , .
    • . . . The synthesis of 'difficult sequences' may be improved by using polar solvents43 and intermediate acid washing steps44, but better results are obtained by applying reversible modifications to the peptide backbone . . .
  44. Beyermann, M. & Bienert, M. Synthesis of difficult peptide sequences: a comparison of Fmoc- and Boc-technique. Tetrahedron Lett. 33, 3745-3748 , (1992) .
    • . . . The synthesis of 'difficult sequences' may be improved by using polar solvents43 and intermediate acid washing steps44, but better results are obtained by applying reversible modifications to the peptide backbone . . .
  45. Narita, M., Fukunaga, T., Wakabayashi, A., Ishikawa, K. & Nakano, H. Syntheses and properties of tertiary peptide bond-containing polypeptides. 1. Syntheses and properties of oligo(L-leucine)S containing proline of glycyl-N-(2,4-dimethoxybenzyl)-L-leucine residues. Int. J. Peptide Protein Res. 23, 306-314 , (1984) .
    • . . . The observation that sequences containing Nα-alkyl-amino acids and Pro are often synthesized without difficulties45 led to the development of reversibly Nα-alkylated amino acids46, to be incorporated instead of the corresponding nonalkylated amino acids into the peptide chain (preferentially (Hmb)Gly-derivatives, commercially available; Fig. 2a) and reconverted into the native residue by TFA treatment . . .
  46. Johnson, T., Quibell, M. & Sheppard, R.C. N,O-bis Fmoc derivatives of N-(2-hydroxy-4-methoxybenzyl)-amino acids: useful intermediates in peptide synthesis. J. Pept. Sci. 1, 11-25 , (1995) .
    • . . . The observation that sequences containing Nα-alkyl-amino acids and Pro are often synthesized without difficulties45 led to the development of reversibly Nα-alkylated amino acids46, to be incorporated instead of the corresponding nonalkylated amino acids into the peptide chain (preferentially (Hmb)Gly-derivatives, commercially available; Fig. 2a) and reconverted into the native residue by TFA treatment . . .
  47. Quibell, M., Turnell, W.G. & Johnson, T. Preparation and purification of beta-amyloid (1-43) via soluble, amide backbone protected intermediates. J. Org. Chem. 59, 1745-1750 , (1994) .
    • . . . Using this strategy, syntheses of various difficult sequences, such as β-amyloid-derived peptides47, have been improved . . .
    • . . . The wild type was successfully synthesized using (Hmb)Gly at position 16, which prevents aspartimide formation and helps also to reduce the aggregation tendency of the growing peptide chain47, 65 . . .
  48. Wöhr, T. & Mutter, M. Pseudo-prolines in peptide synthesis: direct insertion of serine and threonine-derived oxazolidines in dipeptides. Tetrahedron Lett. 36, 3847-3848 , (1995) .
    • . . . The structure of Pro is mimed in the so-called 'pseudoprolines'48, 49, residues of Ser or Thr in which the β-hydroxyl function is reversibly bound through an alkyl bridge to the α-amino group (Fig. 2b) . . .
  49. Wöhr, T. et al. Pseudo-prolines as a solubilizing, structure-disrupting protection technique in peptide synthesis. J. Am. Chem. Soc. 118, 9218-9227 , (1996) .
    • . . . The structure of Pro is mimed in the so-called 'pseudoprolines'48, 49, residues of Ser or Thr in which the β-hydroxyl function is reversibly bound through an alkyl bridge to the α-amino group (Fig. 2b) . . .
  50. Toniolo, C., Bonora, G.M., Mutter, M. & Pillai, V.N.R. Linear oligopeptides. 78. The effect of the insertion of a proline residue on the solution conformation of host peptides. Macromol. Chem. Phys. 182, 2007-2014 , (1981) .
    • . . . Pseudoprolines, introduced into a peptide by coupling-preformed dipeptide derivatives (commercially available), destabilize peptide folding in β-sheets50 and efficiently reduce the formation of aggregates . . .
  51. Carpino, L.A. et al. Synthesis of "difficult" peptide sequences: application of a depsipeptide technique to the Jung-Redemann 10- and 26-mers and the amyloid peptide A(1-42). Tetrahedron Lett. 45, 7519-7523 , (2004) .
    • . . . Alternatively, difficult peptides may be obtained by the synthesis of depsipeptide (also named O-peptide, or O-acyl isopeptide) analogs51, 52, 53 . . .
    • . . . Compared with the target peptide, the corresponding depsipeptide isomer is more soluble in aqueous media, due to the presence of an additional ionizable moiety provided by the depsipeptide unit, and therefore can be more easily purified, as reported for the Alzheimer Aβ (1–42) peptide51, 52, 53 . . .
  52. Mutter, M. et al. Switch peptides in statu nascendi: induction of conformational transitions relevant to degenerative diseases. Angew. Chem. Int. Ed. Engl. 43, 4172-4178 , (2004) .
    • . . . Alternatively, difficult peptides may be obtained by the synthesis of depsipeptide (also named O-peptide, or O-acyl isopeptide) analogs51, 52, 53 . . .
  53. Sohma, Y., Sasaki, M., Hayashi, Y., Kimura, T. & Kiso, Y. Design and synthesis of a novel water-soluble A(1-42) isopeptide: an efficient strategy for the preparation of Alzheimer's disease-related peptide, A(1-42), via O-N intramolecular acyl migration reaction. Tetrahedron Lett. 45, 5965-5968 , (2004) .
    • . . . Alternatively, difficult peptides may be obtained by the synthesis of depsipeptide (also named O-peptide, or O-acyl isopeptide) analogs51, 52, 53 . . .
  54. Coin, I. et al. Depsipeptide methodology for solid-phase peptide synthesis: circumventing side reactions and development of an automated technique via depsidipeptide units. J. Org. Chem. 71, 6171-6177 , (2006) .
    • . . . Depsipeptide units are assembled via O-acylation directly onto the resin-bound peptide, or more conveniently incorporated by coupling preformed depsidipeptide blocks54, 55, some of which are commercially available . . .
    • . . . During depsipeptide assembly, care must be taken during Fmoc removal from the second amino acid residue following the ester bond at the N-terminal side, where diketopiperazine (DKP) formation58 can occur54 (Fig. 3) . . .
    • . . . By assembly of depsipeptides (X = O), DKP formation can always occur during deprotection of the second residue following the ester bond, in an extent which is strongly dependent on the sequence54. . . .
    • . . . In Box 2 we describe the application of the depsipeptide, and in Box 3 of the pseudoproline method to the assembly of an extremely difficult sequence, the Asn(15)-amide analog of the WW domain FBP28, a small, 37-residue peptide, GATAV SEWTE YKTAD GKTYY YNNRT LESTW EKPQE LK, recently used as a model system in studies about β-sheet stability and folding61, 62, and which is impossible to synthesize using standard protocols54, 62 . . .
    • . . . The depsipeptide strategy was compared with the pseudoproline method and was shown to be equally well suited for SPPS54. . . .
  55. Sohma, Y. et al. 'O-Acyl isopeptide method' for the efficient synthesis of difficult sequence-containing peptides: use of 'O-acyl isodipeptide unit'. Tetrahedron Lett. 47, 3013-3017 , (2006) .
    • . . . Depsipeptide units are assembled via O-acylation directly onto the resin-bound peptide, or more conveniently incorporated by coupling preformed depsidipeptide blocks54, 55, some of which are commercially available . . .
  56. Coin, I., Schmieder, P., Bienert, M. & Beyermann, M. The depsipeptide technique applied to peptide segment condensation: scope and limitations. J. Pept. Sci. DOI: doi: 10.1002/psc.928 , (2007) .
    • . . . In this case, coupling is best performed via carbodiimide in nonpolar solvents56, 57 . . .
  57. Taniguchi, A. et al. 'O-Acyl isopeptide method' for peptide synthesis: solvent effects in the synthesis of A1-42 isopeptide using 'O-acyl isodipeptide unit'. J. Pept. Sci. DOI: doi: 10.1002/psc.905 , (2007) .
    • . . . In this case, coupling is best performed via carbodiimide in nonpolar solvents56, 57 . . .
  58. Pedroso, E., Grandas, A., de las Heras, X., Eritja, R. & Girald, E. Diketopiperazine formation in solid-phase peptide-synthesis using P-alkoxybenzyl ester resins and Fmoc-amino acids. Tetrahedron Lett. 27, 743-746 , (1986) .
    • . . . During depsipeptide assembly, care must be taken during Fmoc removal from the second amino acid residue following the ester bond at the N-terminal side, where diketopiperazine (DKP) formation58 can occur54 (Fig. 3) . . .
  59. Carpino, L.A. et al. New family of base- and nucleophile-sensitive amino-protecting groups. A Michael-acceptor-based deblocking process. Practical utilization of the 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc)group. J. Am. Chem. Soc. 119, 9915-9916 , (1997) .
    • . . . The use of Bsmoc59 for Nα-protection at this position can prevent DKP formation, because this group is removed faster and under less basic conditions than Fmoc . . .
  60. Fujino, M., Wakimasu, M., Shinagawa, S., Kitada, C. & Yajima, H. Synthesis of the nonacosapeptide corresponding to mammalian glucagons. Chem. Pharm. Bull. 26, 539-548 , (1978) .
    • . . . Final conversion of the depsi into the amide form (Fig. 4) is smoothly achieved after peptide purification through an O,N-acyl shift60 under weakly alkaline conditions. . . .
  61. Marcias, M.J., Gervais, V., Civera, C. & Oschkinat, H. Structural analysis of WW domains and design of a WW prototype. Nat. Struct. Biol. 7, 375-379 , (2000) .
    • . . . In Box 2 we describe the application of the depsipeptide, and in Box 3 of the pseudoproline method to the assembly of an extremely difficult sequence, the Asn(15)-amide analog of the WW domain FBP28, a small, 37-residue peptide, GATAV SEWTE YKTAD GKTYY YNNRT LESTW EKPQE LK, recently used as a model system in studies about β-sheet stability and folding61, 62, and which is impossible to synthesize using standard protocols54, 62 . . .
  62. Tremmel, S. et al. C-13-labeled tyrosine residues as local IR probes for monitoring conformational changes in peptides and proteins. Angew. Chem. Int. Ed. Engl. 44, 4631-4635 , (2005) .
    • . . . In Box 2 we describe the application of the depsipeptide, and in Box 3 of the pseudoproline method to the assembly of an extremely difficult sequence, the Asn(15)-amide analog of the WW domain FBP28, a small, 37-residue peptide, GATAV SEWTE YKTAD GKTYY YNNRT LESTW EKPQE LK, recently used as a model system in studies about β-sheet stability and folding61, 62, and which is impossible to synthesize using standard protocols54, 62 . . .
  63. Nicolás, E., Pedroso, E. & Girald, E. Formation of aspartimide peptides in Asp-Gly sequences. Tetrahedron Lett. 30, 497-500 , (1989) .
    • . . . We synthesized Asn(15)-analogs, because a considerable piperidine-catalyzed aspartimide63, 64 formation (Fig. 5) was observed at position Asp(15)-Gly(16) for the assembly of the wild type . . .
  64. Dölling, R. et al. Piperidine-mediated side product formation for Asp(OtBu)-containing peptides. J. Chem. Soc. Chem. Commun. 853-854 , (1994) .
    • . . . We synthesized Asn(15)-analogs, because a considerable piperidine-catalyzed aspartimide63, 64 formation (Fig. 5) was observed at position Asp(15)-Gly(16) for the assembly of the wild type . . .
  65. Offer, J., Quibell, M. & Johnson, T. On-resin solid-phase synthesis of asparagine N-linked glycopeptides: use of N-(2-acetoxy-4-methoxybenzyl)(AcHmb) aspartyl amide-bond protection to prevent unwanted aspartimide formation. J. Chem. Soc. Perkin Trans. 1, 175-182 , (1996) .
    • . . . The wild type was successfully synthesized using (Hmb)Gly at position 16, which prevents aspartimide formation and helps also to reduce the aggregation tendency of the growing peptide chain47, 65 . . .
  66. Wade, J.D., Bedford, J., Sheppard, R.C. & Tregear, G.W. DBU as an N-alpha-deprotecting reagent for the fluorenylmethoxycarbonyl group in continuous flow solid-phase peptide synthesis. Pept. Res. 4, 194-199 , (1991) .
  67. Kates, S.A., Solé, N.A., Beyermann, M., Barany, G. & Albericio, F. Optimized preparation of deca(L-alanyl)-L-valinamide by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis on polyethylene glycol-polystyrene (PEG-PS) graft supports, with 1,8-diazobicyclo[5.4.0]-undec-7-ene (DBU) deprotection. Pept. Res. 9, 106-113 , (1996) .
  68. Thaler, A., Seebach, D. & Cardinaux, F. Improvement of degree of resin swelling and of efficiency of coupling in solid-phase synthesis. Helv. Chim. Acta 74, 628-643 , (1991) .
  69. Pugh, K.C., York, E.J. & Stewart, J.M. Effects of resin swelling and substitution on solid phase synthesis. Int. J. Pept. Protein Res. 40, 208-213 , (1992) .
  70. Pennington, M.W. & Byrnes, M.E. Procedures to improve difficult couplings. In Peptide Synthesis Protocols (eds. Pennington, M.W. & Dunn, B.M.) 1-16 (Humana Press, Totowa, New Jersey, 1994) , .
  71. Wade, J.D., Mathieu, M.N., Macris, M. & Tregear, G.W. Base-induced side reactions in Fmoc-solid phase peptide synthesis: minimization by use of piperazine as N-alpha-deprotection reagent. Lett. Pept. Sci. 7, 107-112 , (2000) .
  72. Alsina, J., Giralt, E. & Albericio, F. Use of N-tritylamino acids and PyAOP for the supression of diketopiperazine formation in Fmoc/(t)Bu solid-phase peptide synthesis using alkoxybenzyl ester anchoring linkages. Tetrahedron Lett. 37, 4195-4198 , (1996) .
  73. Schnölzer, M., Alewood, P., Jones, A., Alewood, D. & Kent, S.B.H. In situ neutralization in Boc-chemistry solid-phase peptide synthesis-rapid, high-yield assembly of difficult sequences. Int. J. Peptide Protein Res. 40, 180-193 , (1992) .
  74. Krause, E. et al. Studies on thioether modifications: S-oxidation, S-oxide reduction and regeneration of methionine peptides from their S-benzyl-sulfonium derivatives. In Peptides: Chemistry and Biology (eds. Smith, J.A. & Rivier, J.E.) 478-479 (ESCOM, Leiden, the Netherlands, 1992) , .
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