Symmetrical anhydrides in Solid-Phase Peptide Synthesis
(Peptideweb protocol, www.peptideweb.com, “Neubauer D., Kamysz W., Gorski A.”)
Solid-phase peptide synthesis (SPPS) is one of the most common techniques used for obtaining peptides. The key principle of SPPS is that the peptide remains anchored to a solid support during chain elongation, with its release occurring at the final stage of the process. The peptide chain grows through consecutive steps of amino acid coupling and deprotection. Nowadays, Fmoc/tBu chemistry prevails in SPPS. There are different coupling methods, and the use of symmetrical anhydrides is one of them. It was found that this method is especially successful in the acylation of the N-terminus of highly hindered amino acids and secondary amines [1, 2]. Racemization in this coupling type strongly depends on various parameters, including protecting groups, additives (bases), temperature, and solvent [3, 4]. In some cases, it may be undetectable, but in others, it can pose a significant problem [5, 6]. These entities are very reactive but are of limited use, presumably because only half of the Fmoc-amino acid can be conjugated to the peptide chain. This can be explained by the reaction mechanism presented below:
Figure 1. Mechanism of coupling reaction with symmetrical anhydrides. R1, R4– amino acid side chains; R2, R3 vary according to carbodiimide used (DIC – N,N’-diisopropylcarbodiimide, DCC – N,N’-dicyclohexylcarbodiimide, EDC – 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)).
Considerations:
Some amino acids used in this type of coupling can lead to side-products. For instance, Fmoc-Gly-OH and Fmoc-Ala-OH may introduce diglycyl and dialanyl fragments into the peptide chain. Furthermore, Nτ-protected histidine and all arginine derivatives are not recommended for this coupling method due side reactions [7].
Fmoc-AA-OH exhibit varying solubility in DCM. Table 1 presents a summary of this issue.
Table 1. Solubility of Fmoc-AA-OH and their anhydrides ("+" soluble, "-" precipitate).
The anhydride can be dissolved by the further addition of a more polar solvent, e.g., DMF. In some cases, the anhydride may not completely dissolve; however, the addition of the mixture can still result in successful coupling, as part of the anhydride dissolves during the acetylation process.
A typical procedure for symmetrical anhydrides includes the use of the following reagents:
- Nα-(9-Fluorenylmethoxycarbonyl) - amino acid (Fmoc-AA-OH),
- DIC,
- resin,
- dichloromethane (DCM).
Procedure for coupling using symmetrical anhydrides:
1. Dissolve 4 to 10 equivalents of Fmoc-AA-OH in DCM. If the Fmoc-AA-OH does not completely dissolve, add a small amount of DMF, for example, 10% v/v. Note that some studies suggest that increasing solvent polarity can reduce coupling yields [1]. Therefore, the amount of DMF should be kept as low as possible.
2. Add DIC – from 2 to 5 equivalents – to maintain a 2:1 ratio between Fmoc-AA-OH and DIC.
3. Incubate the mixture at 8-10°C for 20 minutes.
4. Add the mixture to the resin to perform coupling of the anhydride with the amino group. Incubate at room temperature for 1 hour.
2. Add DIC – from 2 to 5 equivalents – to maintain a 2:1 ratio between Fmoc-AA-OH and DIC.
3. Incubate the mixture at 8-10°C for 20 minutes.
4. Add the mixture to the resin to perform coupling of the anhydride with the amino group. Incubate at room temperature for 1 hour.
References:
1. Fu Y, Hammer RP. Efficient acylation of the N-terminus of highly hindered C(alpha)(alpha)-disubstituted amino acids via amino acid symmetrical anhydrides. Org Lett. 2002 Jan 24;4(2):237-40.
2. El-Faham A, Albericio F. Peptide coupling reagents, more than a letter soup. Chem Rev. 2011 Nov 9;111(11):6557-602.
3. Benoiton NL, Lee YC, Chen FM. Racemization during aminolysis of mixed and symmetrical anhydrides of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide. Int J Pept Protein Res. 1988 May;31(5):443-6.
4. Chen FM, Lee YC, Steinauer R, Benoiton NL, Mixed anhydrides in peptide synthesis. A study of urethane formation with a contribution on minimization of racemization. Can. J. of Chem., March 1987, 65(3):613-618.
5. Wang SS, Tam JP, Wang BS, Merrifield RB. Enhancement of peptide coupling reactions by 4-dimethylaminopyridine. Int J Pept Protein Res. 1981 Nov;18(5):459-67.
6. Fuse S, Otake Y, Nakamura H. Peptide Synthesis Utilizing Micro-flow Technology. Chem Asian J. 2018 Dec 18;13(24):3818-3832.
7. Grant G. Synthetic peptides: a user’s guide. Pages 124-125, W.H.Freeman & Co Ltd, 1992.
2. El-Faham A, Albericio F. Peptide coupling reagents, more than a letter soup. Chem Rev. 2011 Nov 9;111(11):6557-602.
3. Benoiton NL, Lee YC, Chen FM. Racemization during aminolysis of mixed and symmetrical anhydrides of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide. Int J Pept Protein Res. 1988 May;31(5):443-6.
4. Chen FM, Lee YC, Steinauer R, Benoiton NL, Mixed anhydrides in peptide synthesis. A study of urethane formation with a contribution on minimization of racemization. Can. J. of Chem., March 1987, 65(3):613-618.
5. Wang SS, Tam JP, Wang BS, Merrifield RB. Enhancement of peptide coupling reactions by 4-dimethylaminopyridine. Int J Pept Protein Res. 1981 Nov;18(5):459-67.
6. Fuse S, Otake Y, Nakamura H. Peptide Synthesis Utilizing Micro-flow Technology. Chem Asian J. 2018 Dec 18;13(24):3818-3832.
7. Grant G. Synthetic peptides: a user’s guide. Pages 124-125, W.H.Freeman & Co Ltd, 1992.
Exemplary protocols from different authors:
I) Procedure according to “Montero A, Albericio F, Royo M, Herradón B. Synthesis of a 24-membered cyclic peptide-biphenyl hybrid. Eur J Organ Chem. 2007;2007:1301–1308.”
1. DCC (3.0 equivalents) was added to a solution of Fmoc-AA-OH (6.0 equivalents) in DCM.
2. The reaction mixture was stirred at room temperature for at least 30 minutes.
3. The resulting DCU (N,N'-Dicyclohexylurea) was filtered off, and the filtrate was used in the subsequent step.
4. The resin, with its free amino functionality, was swollen in DCM. The prepared solution of the symmetric anhydride was then added, followed by triethylamine (3.0 equivalents), and the mixture was shaken for 4.5 hours.
II) Procedure for coupling Boc-protected amino acids, based on the method of "Van Regenmortel MHV, Muller JPBS, Plaué S. Chapter 2 solid-phase peptide synthesis. In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19, RH Burdon, PH Knippenberg (eds). Elsevier Science Publishers B.V. (Biomedical Division): Amsterdam, The Netherlands, 1988; 41–94”:
1. 4 equivalents of the Boc-protected amino acid are dissolved in a minimal volume of DCM (with a minimal amount of DMF, if needed).
2. 2 equivalents of DIC are then added, and the mixture is stirred at room temperature for 10-20 minutes.
3. The DCM is subsequently removed by evaporation at 40°C. The resulting symmetrical anhydride is then dissolved in a minimal volume of DMF, and this solution is added to the resin for 15-20 min.
III) Procedure according to “Alsina J, Kates SA, Barany G, Albericio F. Backbone amide linker strategies for the solid-phase synthesis of C-terminal modified peptides. Methods Mol Biol. 2005;298:195-208.”:
1. The Fmoc-amino acid (10 equiv) and DCC (8.8 mg, 5 equiv) were dissolved in DCM (1 mL) and allowed to react at 25 °C for 10 min.
2. The resultant insoluble N,N‘-dicyclohexylurea was removed by filtration, the filtrate was concentrated in vacuo.
3. Symmetric anhydride was dissolved in DCM and added to the resin for 1h.
IV) Procedure according to “Chang CD, Felix AM, Jimenez MH, Meienhofer J. Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids. Int J Pept Protein Res. 1980 May;15(5):485-94.”:
1. Symmetrical anhydrides were synthesized by combining Fmoc-amino acid (3.8 mmol, 4.37 equiv.) in DCM:DMF (1:1, 10 mL) with DCC (392 mg, 1.9 mmol, 2.19 equiv.) in DCM (3 mL) at 0°C.
2. After a 30 minutes of reaction, the resulting DCU precipitate was removed by filtration and washed with DCM, bringing the total volume to 25 mL. The combined filtrate and washings were then used for the coupling reaction. Each coupling was performed in duplicate using freshly prepared symmetrical anhydride.
3. The coupling reaction proceeded for 30 minutes. After 15 minutes 10% DIPEA in DCM (1 mL per gram of resin) was added.
1. DCC (3.0 equivalents) was added to a solution of Fmoc-AA-OH (6.0 equivalents) in DCM.
2. The reaction mixture was stirred at room temperature for at least 30 minutes.
3. The resulting DCU (N,N'-Dicyclohexylurea) was filtered off, and the filtrate was used in the subsequent step.
4. The resin, with its free amino functionality, was swollen in DCM. The prepared solution of the symmetric anhydride was then added, followed by triethylamine (3.0 equivalents), and the mixture was shaken for 4.5 hours.
II) Procedure for coupling Boc-protected amino acids, based on the method of "Van Regenmortel MHV, Muller JPBS, Plaué S. Chapter 2 solid-phase peptide synthesis. In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19, RH Burdon, PH Knippenberg (eds). Elsevier Science Publishers B.V. (Biomedical Division): Amsterdam, The Netherlands, 1988; 41–94”:
1. 4 equivalents of the Boc-protected amino acid are dissolved in a minimal volume of DCM (with a minimal amount of DMF, if needed).
2. 2 equivalents of DIC are then added, and the mixture is stirred at room temperature for 10-20 minutes.
3. The DCM is subsequently removed by evaporation at 40°C. The resulting symmetrical anhydride is then dissolved in a minimal volume of DMF, and this solution is added to the resin for 15-20 min.
III) Procedure according to “Alsina J, Kates SA, Barany G, Albericio F. Backbone amide linker strategies for the solid-phase synthesis of C-terminal modified peptides. Methods Mol Biol. 2005;298:195-208.”:
1. The Fmoc-amino acid (10 equiv) and DCC (8.8 mg, 5 equiv) were dissolved in DCM (1 mL) and allowed to react at 25 °C for 10 min.
2. The resultant insoluble N,N‘-dicyclohexylurea was removed by filtration, the filtrate was concentrated in vacuo.
3. Symmetric anhydride was dissolved in DCM and added to the resin for 1h.
IV) Procedure according to “Chang CD, Felix AM, Jimenez MH, Meienhofer J. Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids. Int J Pept Protein Res. 1980 May;15(5):485-94.”:
1. Symmetrical anhydrides were synthesized by combining Fmoc-amino acid (3.8 mmol, 4.37 equiv.) in DCM:DMF (1:1, 10 mL) with DCC (392 mg, 1.9 mmol, 2.19 equiv.) in DCM (3 mL) at 0°C.
2. After a 30 minutes of reaction, the resulting DCU precipitate was removed by filtration and washed with DCM, bringing the total volume to 25 mL. The combined filtrate and washings were then used for the coupling reaction. Each coupling was performed in duplicate using freshly prepared symmetrical anhydride.
3. The coupling reaction proceeded for 30 minutes. After 15 minutes 10% DIPEA in DCM (1 mL per gram of resin) was added.
