23. A sample for quality measurements should be taken before
freezing the peptide. We run LC-MS and UPLC measurements
as a standard for all synthesized peptides after cleavage.
24. Boc-Asn is activated with HBTU and HOBt, Boc-Asn(Xan) is
activated with HBTU only.
25. For Gln couplings, DCM should be used for washing to avoid
Pca formation.
26. If high amounts of by-products appear, cool to À15
C by
using NaCl and ice.
27. The reaction can be monitored by converting the azide to a
thioester by adding DTT or MPAA and measuring it in LC-MS
and UPLC.
28. If an N-terminal Fmoc-synthesized hydrazine peptide is used
after activation via oxidation, add ligation buffer 2 to a final
concentration of 2 mM and remove cooling.
29. If the pH climbs very high above 7.0, the peptide thioester will
hydrolyze.
30. Depending on the protein stability it can also be incubated at
4
C or 37
C with increased or decreased reaction times,
respectively.
References
1. Merrifield RB (1963) Solid-phase peptide
synthesis. I. The synthesis of a tetrapeptide. J
Am Chem Soc 85:2149–2154
2. Palomo JM (2014) Solid-phase peptide synthesis: an overview focused on the preparation of
biologically relevant peptides. RSC Adv
4:32658–32672
3. Behrendt R, White P, Offer J (2016) Advances
in Fmoc solid-phase peptide synthesis. J Pept
Sci 22:4–27
4. Wo ¨hr T, Mutter M (1995) Pseudo-prolines in
peptide synthesis: direct insertion of serine and
threonine derived oxazolidines in dipeptides.
Tetrahedron Lett 36:3847–3848
5. Spare LK, Laude V, Harman DG, AldrichWright JR, Gordon CP (2018) An optimised
approach for continuous-flow solid-phase peptide synthesis utilising a rudimentary flow reactor. React Chem Eng 3:875–882
6. Varela Y, Vanegas Murcia M, Patarroyo M
(2018) Synthetic evaluation of standard and
microwave-assisted solid-phase peptide synthesis of a long chimeric peptide derived from four
plasmodium falciparum proteins. Molecules
23:2877
7. Dawson PE, Kent SB (2000) Synthesis of
native proteins by chemical ligation. Annu
Rev Biochem 69:923–960
8. Conibear AC, Watson EE, Payne RJ, Becker
CF (2018) Native chemical ligation in protein
synthesis and semi-synthesis. Chem Soc Rev
47:9046–9068
9. Johnson EC, Kent SB (2006) Insights into the
mechanism and catalysis of the native chemical
ligation reaction. J Am Chem Soc
128:6640–6646
10. Li J, Li Y, He Q, Li Y, Li H, Liu L (2014)
One-pot native chemical ligation of peptide
hydrazides enables total synthesis of modified
histones. Org Biomol Chem 12:5435–5441
11. Thompson RE, Liu X, Alonso-Garcı ´a N, Pereira PJB, Jolliffe KA, Payne RJ (2014) Trifluoroethanethiol: an additive for efficient one-pot
peptide ligation- desulfurization chemistry. J
Am Chem Soc 136:8161–8164
12. Huang YC, Chen CC, Gao S, Wang YH,
Xiao H, Wang F, Li YM (2016) Synthesis of
L-and D-ubiquitin by one-pot ligation and
metal-free desulfurization. Chem Eur J
22:7623–7628
13. Ghassemian A, Wang CIA, Yau MK, Reid RC,
Lewis RJ, Fairlie DP, Durek T (2013) Efficient
chemical synthesis of human complement protein C3a. Chem Commun 49:2356–2358
14. Torbeev VY, Kent SB (2007) Convergent
chemical synthesis and crystal structure of a
Synthetic PDZ Domains
215
freezing the peptide. We run LC-MS and UPLC measurements
as a standard for all synthesized peptides after cleavage.
24. Boc-Asn is activated with HBTU and HOBt, Boc-Asn(Xan) is
activated with HBTU only.
25. For Gln couplings, DCM should be used for washing to avoid
Pca formation.
26. If high amounts of by-products appear, cool to À15
C by
using NaCl and ice.
27. The reaction can be monitored by converting the azide to a
thioester by adding DTT or MPAA and measuring it in LC-MS
and UPLC.
28. If an N-terminal Fmoc-synthesized hydrazine peptide is used
after activation via oxidation, add ligation buffer 2 to a final
concentration of 2 mM and remove cooling.
29. If the pH climbs very high above 7.0, the peptide thioester will
hydrolyze.
30. Depending on the protein stability it can also be incubated at
4
C or 37
C with increased or decreased reaction times,
respectively.
References
1. Merrifield RB (1963) Solid-phase peptide
synthesis. I. The synthesis of a tetrapeptide. J
Am Chem Soc 85:2149–2154
2. Palomo JM (2014) Solid-phase peptide synthesis: an overview focused on the preparation of
biologically relevant peptides. RSC Adv
4:32658–32672
3. Behrendt R, White P, Offer J (2016) Advances
in Fmoc solid-phase peptide synthesis. J Pept
Sci 22:4–27
4. Wo ¨hr T, Mutter M (1995) Pseudo-prolines in
peptide synthesis: direct insertion of serine and
threonine derived oxazolidines in dipeptides.
Tetrahedron Lett 36:3847–3848
5. Spare LK, Laude V, Harman DG, AldrichWright JR, Gordon CP (2018) An optimised
approach for continuous-flow solid-phase peptide synthesis utilising a rudimentary flow reactor. React Chem Eng 3:875–882
6. Varela Y, Vanegas Murcia M, Patarroyo M
(2018) Synthetic evaluation of standard and
microwave-assisted solid-phase peptide synthesis of a long chimeric peptide derived from four
plasmodium falciparum proteins. Molecules
23:2877
7. Dawson PE, Kent SB (2000) Synthesis of
native proteins by chemical ligation. Annu
Rev Biochem 69:923–960
8. Conibear AC, Watson EE, Payne RJ, Becker
CF (2018) Native chemical ligation in protein
synthesis and semi-synthesis. Chem Soc Rev
47:9046–9068
9. Johnson EC, Kent SB (2006) Insights into the
mechanism and catalysis of the native chemical
ligation reaction. J Am Chem Soc
128:6640–6646
10. Li J, Li Y, He Q, Li Y, Li H, Liu L (2014)
One-pot native chemical ligation of peptide
hydrazides enables total synthesis of modified
histones. Org Biomol Chem 12:5435–5441
11. Thompson RE, Liu X, Alonso-Garcı ´a N, Pereira PJB, Jolliffe KA, Payne RJ (2014) Trifluoroethanethiol: an additive for efficient one-pot
peptide ligation- desulfurization chemistry. J
Am Chem Soc 136:8161–8164
12. Huang YC, Chen CC, Gao S, Wang YH,
Xiao H, Wang F, Li YM (2016) Synthesis of
L-and D-ubiquitin by one-pot ligation and
metal-free desulfurization. Chem Eur J
22:7623–7628
13. Ghassemian A, Wang CIA, Yau MK, Reid RC,
Lewis RJ, Fairlie DP, Durek T (2013) Efficient
chemical synthesis of human complement protein C3a. Chem Commun 49:2356–2358
14. Torbeev VY, Kent SB (2007) Convergent
chemical synthesis and crystal structure of a
Synthetic PDZ Domains
215