Design of Cyclic Peptides - Peptide and Protein Design for Biopharmaceutical Applications - page 138

specific structure, and even differing sequences that lead to the same
cyclic peptide. For more details, the reader is referred to the recent
literature [42].
One of the most efficient methods of cyclizing a linear peptide strand
from head to tail is to use a combination of diphenylphosphoryl azide
(DPPA) and NaHCO 3 in dimethylformamide (DMF). DPPA is a mild
cyclization reagent that helps to suppress racemization. On one hand,
NaHCO 3 is a weak base which helps to reduce racemization of the
amino acid building blocks; on the other, it is insoluble in DMF.
Therefore the reaction occurs only at the surface of the bicarbonate
(solid base method) and the low concentration of deprotonated ammo-
nium groups prevents the formation of cyclic dimers and higher multi-
mers of the peptide strand. Another useful cyclization reagent
especially suited for secondary aminesis2-(7-aza-1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), as
the C-terminus is more strongly activated and racemization rates are
still low.
Increasing yields are found when the conformation of the linear
precursor allows the N- and C-terminal groups of the peptide chain
to come close to each other. This is the case in sequences containing
both D -and L -residues, proline, or other N-alkylated amino acids, since
preformation of turns is induced, which reduces the distance between
the termini. For example, linear pentapeptides exclusively built of
L -aminoacidsaredifficulttocyc ize.However,whenanN-or
C-terminal D -amino acid is present, cyclization occurs smoothly [42].
Also, N-alkylated amino acids can favour cyclization, e.g. a linear
tripeptide containing solely L -amino acids is hardly convertible into a
cyclic tripeptide since it usually forms cyclic dimers, the hexapeptides.
On the other hand, there is no problem in the cyclization of triproline
or trisarcosine.
Apart from cyclizing linear peptide strands in solution, modern
orthogonal protecting group strategies allow ring closure on solid
support with standard coupling reagents. For example, the 4-(N-[1-
(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino)benzyl
(Dmab) protection group for acid functionalities is cleaved under mild
conditions and can be combined with the Fmoc strategy to synthesize
cyclic, resin-bound peptides. However, one should be aware that the
concentrationofthegrowingpeptidechainontheresinisrelatively
high. Hence, usually higher amounts of unwanted cyclic dimers or even
higher oligomers are formed compared to cyclization in solution where
low concentrations are present (principle of dilution).