In the Fuchs-Farthing method, an unprotected amino acid is treated with phosgene to give the amino acid isocyanate. In the next step, a nucleophilic attack of the carboxyl oxygen on the isocyanate closes the ring to the desired NCA. The major drawback of this synthesis is the use of gaseous, highly toxic phosgene, of which the exact amount can not be dosed. Endo improved the synthesis by using the solid triphosgene instead.[57] This is also highly toxic, but much easier to handle.
A very crucial step in the synthesis of NCAs is their isolation and purification after the reaction, since they are very reactive molecules. It is possible to do a very quick aqueous work-up with ice cold water and dilute NaHCO3-solution to remove HCl and to quench excess phosgene. After this work-up, the product is obtained by crystallization. The NCAs used in polymerizations have to be very pure in order to react properly to high molecular weight polymers with narrow polydispersity. The purification of the isolated NCAs is achieved by several recrystallization steps.
2.4.2 Synthesis Of Homo-D-(alt)-L-polypeptides
The synthesis of homo-peptides by ring opening polymerization of NCAs is well established and yields optical pure material in the desired length scale with small polydispersity index, but for more diverse polypeptides with defined amino acid sequences, no such sophisticated procedure is known so far.
The polypeptides investigated by Lotz, Heitz, and Spach (see 2.2.3) were synthesized by polycondensation reactions, yielding the resulting polypeptides within a very broad polydispersity.[58,59] The synthesis of these polymers is shown in Scheme 10.
Scheme 10: Synthetic approach to homo-D-(alt)-L-polypeptides by Heitz and Spach.
In order to obtain a polypeptide with strictly alternating stereochemistry, the D -(alt)-L information has to be incorporated into the monomer. A copolymerization of D- and L-configured NCAs would lead to statistical polymers with no determined sequence, i.e. stereocontrol. In a first attempt, Heitz and Spach were using the J-benzyl protected D-(alt)-L-glutamate dimer to synthesize the polypeptide. The N-terminus was protected with the N-o-nitrophenylsulphenyl group, which was cleaved with HCl in diethylether. The carboxylic acid was activated by transferring it into the 4-nitrophenyl ester (ONp) or the pentachlorophenyl ester (OPcp). This synthesis suffers from racemization and the formation of diketopiperazine. The use of OPcp gives the polypeptide in higher yields, but with a higher degree of racemization. To overcome the issue of diketopiperazine formation, they used the J-benzyl protected D-(alt)-L -glutamate tetramer as monomer. For the tetramer synthesis, the N-terminus was Boc protected, and the C-terminus was protected with a phenacyloxyphenyl ester. After successful tetramer synthesis, the Boc group was removed under acidic conditions and the phenacyloxyphenyl ester was transferred into the activating o-hydroxyphenyl ester with Zn/acetic acid. This tetrapeptide was now directly used in polymerization reactions to give high molecular weight polypeptides with no racemization observed.
2.5 Depsipeptides (Ester-isosteres)
The variations of the peptides mentioned so far were always maintaining the backbone integrity, only changing the residues in the sequence or their stereochemistry. Another option is the change of the chemical nature of the peptide chain by replacing amide bonds by amide analogues such as esters, ketomethylene, vinyl, amine or cyclopropene. The approach of replacing amide bonds in peptides by esters is also done by nature.[60] Therefore, D-amino acids in the chain are replaced by the corresponding D-hydroxy acids (Figure 15 a).
The resulting compounds are so called depsipeptides.
Figure 15: a) Peptide backbone variation by replacing an amino acid by the corresponding hydroxy carboxylic acid, b) hydrogen bonding patterns for peptides and depsipeptides.
Secondary amide linkages and esters have some key structural features in common. They are both planar, have electronic resonance structures and the alkyl substituent on the nitrogen or the oxygen atom prefers to be syn to the carbonyl oxygen. This replacement of amides by esters keeps the number of atoms in the backbone constant, so that the intramolecular distances remain comparable, but has extreme influences on the hydrogen bonding. The formal exchange of one nitrogen by one oxygen atom eliminates one hydrogen bond donor from the molecule, and hence can have drastic consequences for the hydrogen bonding pattern (Figure 15 b). In numerous works, the influence of amide-ester exchange on peptide structures has been investigated.[61-65] For helical structures, Katakai found by X-ray analysis that the ester group (even the carbonyl) was not involved into intramolecular H-bonding.[66-69] The depsipeptide solved the issue of a missing hydrogen bond by helix deformation.
This deformation was depending on the number of incorporated esters and their location in the backbone. Andersen and Koeppe were investigating the influence of amide-ester exchange in Gramicidin A on ion conductance and found a drastic change in channel formation of the depsi-Gramicidin A.[70] They replaced the Val-Gly-amide bond of the first two residues in the sequence by a Val-O-Gly ester to give the depsipeptide. The ester bond in this location of the sequence influences intramolecular and intermolecular hydrogen bonding, of which the
latter one is essential for the head-to-head channel formation of Gramicidin. The investigations showed that the channel events were very short and could not be analyzed quantitatively. Experiments with the depsi-Gramicidin/Gramicidin-heterodimer showed a measurable ion conductance providing that the E-helical conformation was possible for depsi-Gramicidin. So in the case of Gramicidin, an amide-ester exchange between the first two amino acids led to a remarkable destabilization of the E-helical conformation and also decreased the head-to-head dimerization ability to afford the channel structure.
2.6 Triazole Isosteres
The use of triazoles as amide mimics in peptides and proteins has become very popular within the last years. This has two main reasons: First, the triazole unit meets the structural and electronical requirements for amide replacement quite well and second, it is synthetically easy accessible. The replacement of an amide by a triazole is structurally more drastic than the replacement by an ester, since it usually elongates the peptide backbone by one atom (Figure 16).
Figure 16: Elongation of the peptide chain by amide-triazole exchange and retrosynthetic approach (hydrogen bonding pattern is indicated with dotted lines).
The rise of triazole as amide mimics was also driven by its easy and high yielding synthesis and the compatibility of the reaction conditions with peptides and proteins. Triazoles can be obtained by the copper-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes.[71] This reaction is atom-efficient, regioselective and proceeds in high yields at room temperature in water. The regioselectivity of the reaction yields only 1,4- and no 1,5-triazoles. The
reaction is copper-catalyzed, involving copper center(s) in the catalytic cycle.
Yet, the exact mechanism of this reaction has not been elucidated.[72] The easy accessibility of the azide- and alkyne-derivatives of amino acids makes the so called “Click”-reaction a versatile tool for triazole incorporation into peptides and proteins.[73] As depicted in Figure 16, azido amino acids and amino acid propargylamides can react to the desired triazoles.
Ghadiri was investigating the synthesis and structure of cyclic peptides with D-L -alternating stereochemistry and their self-assembly to tubular structures.[74,75]
In his work, he also incorporated triazole units into the backbone by synthesizing heterocycles consisting of four amino acids and two triazole units.[76, 77] In the solid state, these heterocycles self-assembled to solvent-filled nanotubes, which were held together by an extended network of intermolecular amide backbone hydrogen bonds.
Arora replaced every peptide bond by 1,4-triazoles and by this synthesized nonpeptidic foldamers from amino acids (Figure 17 a).[78] The triazole dimer unit could adopt two anti and two syn conformations, which are defined based on the relative direction of the dipoles in adjacent rings (Figure 17 b).
Figure 17: a) Structure of synthesized nonpeptidic foldamers, b) different orientations of the triazoles (qualitative representation of dipole moments with red arrows).
Molecular mechanics and ab initio calculations predicted the anti conformation to be more stable due to the large triazole dipole (~5 D). Solution NMR analysis suggested that the tetramers shown in Figure 17a adopt a zigzag conformation, which closely mimics the E-strand structure.
The results of these works and the ease of the synthesis show that triazole units can be an interesting mimic for amide bonds and thereby lead to backbone motifs with interesting (structural) properties.
2.7 Peptide Dendrimers
All peptide modifications mentioned so far were focused on side chains and their stereochemistry and on backbone modifications by amide replacement.
Branching of peptides differs from these variations since it does not maintain a linear backbone in the molecule, but introduces branching points where the main chain is splitting. This leads to a drastic change in secondary structure formation since the molecules become spherical and cannot adopt the classical peptide secondary structures such as helix, sheet or loop anymore. Branching of peptides requires a functional group in the side chain of the amino acid, that can be addressed. Consequent branching of a peptide leads to a dendritic growth of the molecule, generation for generation.
Peptide dendrimers are totally chiral, rendering them interesting for biomedical applications and recognition processes. Denkewalter reported the first synthesis of a chiral polylysine dendritic macromolecule in 1983, however little attention has been paid to these molecules and their full characterization has never been reported.[79] Mitchell published the synthesis of a fully chiral glutamate dendrimer.[80,81] He used the bifunctional glutamic acid as branching unit and could synthesize the glutamate dendrimer up to the third generation (Scheme 11 and Scheme 12).