Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Protein-protein interactions (PPIs) play a pivotal role in most biological processes, from intracellular signal transduction to cell death¹. Hence, targeting PPIs is of fundamental importance to basic research and therapeutic applications. PPIs can be regulated by specific and stable antibodies, but antibodies are expensive and difficult to manufacture and have poor bioavailability. Alternatively, PPIs can be targeted by small molecules. Small molecules are easier to synthesize and inexpensive compared to antibodies; however, they are relatively less flexible and fit better to small cavities than to large protein-protein interfaces^2,3. Diverse studies have demonstrated that peptides, which are simpler and cheaper than antibodies and more flexible than small molecules, can bind protein interfaces and regulate PPIs^4,5. The global therapeutic peptide market was valued around fifteen billion dollars in 2013 and is growing 10.5% annually⁶. Furthermore, there are more than 50 marketed peptides, around 270 peptides in different phases of clinical testing, and about 400 peptides in advanced preclinical phases⁷. Although numerous peptides are being used as drugs, peptides still pose several challenges that limit their widespread application including poor bioavailability and stability, inefficiency in crossing cell membranes, and conformational flexibility^8,9. One alternative to surmount these drawbacks is to apply different modifications such as local (D-amino acid and N-alkylation) and global (cyclization) constraints^8,10-12. These modifications also occur naturally. For example, cyclosporin A, an immunosuppressant cyclic natural peptide, contains a single D-amino acid and undergoes N-alkylation modifications^13,14.

Modification of natural amino acids to induce local constraints, such as D- and N-alkylation, often affects the peptide's biological activity. However, cyclization, in which the sequence of interest can remain the same, is more likely to preserve biological activity. Cyclization is a highly attractive way to restrict peptide conformational space by reducing the equilibrium between different conformations. It usually increases biological activity and selectivity by restricting the peptide to the active conformation that mediates only one function. Cyclization also improves peptide stability by keeping the peptide in a conformation that is less recognized by degrading enzymes. Indeed, cyclic peptides were shown to have improved metabolic stability, bioavailability, and selectivity compared to their linear counterparts^15-17.

However, cyclization can be a double-edged sword since in some cases the restriction may prevent the peptides from achieving a bioactive conformation. To overcome this hurdle, a focused library in which all peptides have the same primary sequence and consequently constant pharmacophores can be synthesized. Peptides in the library differ in parameters that influence their structure, such as ring size and position, in order to subsequently screen for the most bioactive conformation^9,18.

Peptides can be synthesized both in solution and by a solid-phase peptide synthesis (SPPS) approach, which is now the more prevalent peptide synthesis approach and will be discussed further. SPPS is a process by which chemical transformations are performed on a solid support via a linker to prepare a wide range of synthetic compounds¹⁹. SPPS enables assembling peptides by consecutive coupling of amino acids in a stepwise manner from the C-terminus, which is attached to a solid support, to the N-terminus. The N-α-amino acid side-chains must be masked with protecting groups that are stable in the reaction conditions used during peptide elongation to ensure the addition of one amino acid per step. In the final step, the peptide is released from the resin and the side-chain protecting groups are concomitantly removed. While the peptide is being synthesized, all soluble reagents can be removed from the peptide-solid support matrix by filtration and washed away at the end of each coupling step. With such a system, a large excess of reagents at high concentration can drive coupling reactions to completion and all the synthesis steps can be performed in the same vessel without any transfer of material²⁰ .

Although SPPS has some limitations such as the production of incomplete reactions, side reactions, impure reagents, as well as difficulties monitoring the reaction²¹, the advantages of SPPS have made it the "gold standard" for peptide synthesis. These advantages include the option to incorporate non-natural amino acids, automation, easy purification, minimized physical losses, and the use of excess reagents, resulting in high yields. SPPS has been shown to be extremely useful in the synthesis of difficult sequences^21,22, fluorescent modifications²³, and peptide libraries^24,25. SPPS is also very useful for other poly-chain assemblies such as oligonucleotides^26,27, oligosaccharides^28,29, and peptide nucleic acids^30,31. Interestingly, in some cases, SPPS was shown to be advantageous for synthesizing small molecules that are traditionally made in solution^32,33. SPPS is used both in small scale for research and teaching^34,35 as well as large scale in industry^36-38.

Two synthesis strategies that are mainly used in SPPS methodology for the synthesis of peptides are butyloxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). The original strategy introduced for SPPS was Boc, which requires strong acid conditions to remove side-chain protecting groups and cleave the peptide from the resin. Fmoc-based peptide synthesis, however, utilizes moderate base conditions and is a milder alternative to the acid-labile Boc protocol³⁹. The Fmoc strategy utilizes orthogonal t-butyl (tBu) side-chain protection that is removed in the last step of the synthesis while cleaving the peptide from the resin under acid conditions.

The general principle for peptide synthesis on solid support is presented in Figure 1. The initial amino acid, masked by a temporary protecting group on the N-α-terminus, is loaded onto the resin from the C-terminus. A semi-permanent protecting group to mask the side chain is also used if necessary (Figure 1, Step 1). The synthesis of the target peptide is assembled from the C-terminus to the N-terminus by repetitive cycles of deprotection of the N-α-temporary protecting group (Figure 1, Step 2) and coupling of the next protected amino acid (Figure 1, Step 3). After the last amino acid is loaded (Figure 1, Step 4), the peptide is cleaved from the resin support and the semi-permanent protecting groups are removed (Figure 1, Step 5).

Figure 1. General scheme of solid phase peptide synthesis. The N-α-protected amino acid is anchored using the carboxyl group via a linker to the resin (Step 1). The desired peptide is assembled in a linear fashion from the C-terminus to the N-terminus by repetitive cycles of deprotection of the temporary protecting group (TPG) from the N-α (Step 2) and amino acid coupling (Step 3). After accomplishing the synthesis (Step 4), the semi-permanent protecting groups (SPG) are deprotected during peptide cleavage (Step 5). Please click here to view a larger version of this figure.

After assembly of the complete peptide chain, cyclization can be achieved by several alternatives: (A) head-to-tail cyclization — this is a convenient way but limited since it provides only one option for cyclization (Figure 2A), (B) cyclization using the amino acids from the sequence of interest that contain bioactive functional groups — however, the use of these amino acids may influence the biological activity (Figure 2B), and (C) cyclization by adding amino acids (or other building blocks) without disturbing the bioactive sequence. Introducing these molecules is widespread as it allows production of focused libraries without modifying the sequence of interest (Figure 2C).

Figure 2. Alternative peptide cyclization strategies. (A) head to tail cyclization, through a peptide bond between the C-terminus and N-terminus; (B) cyclization between functional groups such as a disulfide bond between cysteine residues (1), or an amide bond between the side chains of lysine to aspartic/glutamic acid (2), or side chain to N- or C-terminus (3-4); (C) cyclization by adding extra amino acids or amino acid derivatives or small molecules, for example before (R0) and after (R7) the bioactive sequence. Please click here to view a larger version of this figure.

Microwave-assisted synthesis uses microwave irradiation to heat reactions, thus accelerating organic chemical transformations^40,41. Microwave chemistry is based on the ability of the reagent/solvent to absorb the microwave energy and convert it to heat⁴². Before the technology became widespread, major drawbacks had to be overcome, including the controllability and reproducibility of synthesis protocols and lack of available systems for adequate temperature and pressure controls^43,44. The first report of microwave-assisted peptide synthesis was done using a kitchen microwave to synthesize several short peptides (7-10 amino acids) with significant improvement of the coupling efficiency and purity ⁴⁵. Moreover, microwave energy was shown to decrease chain aggregation, reduce side reactions, limit racemization, and improve coupling rates, which are all critical for difficult and long sequences^46-53.

Currently the use of microwave irradiation for the synthesis of peptides or related compounds on a solid support is extensive, including (A) synthesis in water instead of organic solvent⁵⁴; (B) synthesis of peptides with common post-translational modifications, such as glycopeptides^55-58 or phosphopeptides^59-61, whose synthesis is typically difficult due to the low coupling efficiency of sterically hindered amino acid derivatives; (C) synthesis of peptides with modification in the backbone, such as azapeptides, which can be formed by the replacement of the C(α) of an amino acid residue with a nitrogen atom⁶², or peptoids, whose side chain is connected to the amide nitrogen rather than the Cα atom^63,64; (D) synthesis of cyclic peptides^65-71; and (E) synthesis of combinatorial libraries^51,72. In numerous cases, the authors reported higher efficiency and reduced synthesis time using microwave irradiation as compared to the conventional protocol.

Using a rational design^73-75, we developed anti-parasitic peptides that were derived from the scaffold Leishmania's receptor for activated C-kinase (LACK). LACK plays an important role in the early phase of Leishmania infection⁷⁶. Parasites expressing lower levels of LACK fail to parasitize even immune-compromised mice⁷⁷ as LACK is involved in essential parasite signaling processes and protein synthesis⁷⁸. Therefore, LACK is a key scaffold protein⁷⁹ and a valuable drug target. Focusing on sequences in LACK that are conserved in the parasites, but not in the host mammalian homolog RACK, we identified an 8 amino acid peptide (RNGQCQRK) that decreased Leishmania sp. viability in culture.

Here, we describe a protocol for the synthesis of backbone cyclic peptides derived from the LACK protein sequence described above. The peptides were synthesized on a solid support using microwave heating by SPPS methodology with Fmoc/tBu protocol. Peptides were conjugated to a TAT_47-57 (YGRKKRRQRRR) carrier peptide through an amide bond as part of the SPPS. TAT-based transport of a variety of cargoes into cells has been used for over 15 years and delivery of the cargo into subcellular organelles has been confirmed⁸⁰. Four different linkers, succinic and glutaric anhydride as well as adipic and pimelic acid, were used to perform the cyclization to generate carboxylic acid linkers of two to five carbons. Cyclization was done using microwave energy, and the final cleavage and side-chain deprotection steps were done manually without microwave energy. The use of an automated microwave synthesizer improved the product purity, increased the product yield, and reduced the duration of the synthesis. This general protocol can be applied to other studies that utilize peptides to understand important molecular mechanism in vitro and in vivo and further develop potential drugs for human diseases.