User Guide

Step 1: Choosing Peptide Chain Length

In the first step, the user can choose any integral value as a peptide chain length up to 5. In one go, the user can only get one chain length.

Step 2: Selecting Amino Acids

It is possible to select or deselect any of the 20 canonical amino acids represented by uppercase FASTA identifiers, or their 19 enantiomers represented by lowercase FASTA identifiers. Glycine, being achiral, has no enantiomer.

Canonical Amino Acids

Step 3: Providing Custom Amino Acids

Now, the user can provide the SMILES of any custom amino acid in non-zwitterion form (unionized carboxylic and amino groups). The non-canonical amino acid is denoted by 'X' in identifiers. If the text box is left empty, it automatically ignores 'X'. Any form of D- or L-amino acid can be used.

Note: If the side chain of the non-canonical amino acid carries an ionizable functional group, its SMILES string should reflect this ionization state. This avoids an extra step of ionization later.

Step 4: Applying Filters

In this section, a set of four filters is present:

• Filter I: Peptides having at least one noncanonical amino acid X. This filter is useful when studying the effect of a noncanonical amino acid on a certain property.
• Filter II: Peptides having no more than 50% hydrophobic residues. Hydrophobic amino acids (A, F, I, L, M, V, W, Y, and their enantiomers) profoundly affect solubility. This filter removes peptides with more than 50% hydrophobic residues.
• Filter III: Peptides with no more than two cystines. This filter helps avoid undesired disulfide bond formation during synthesis.
• Filter IV: Peptides with two prolines not next to each other. This filter prevents issues like deletions or cis/trans isomerization during synthesis.

Step 5: Cyclization Types

In this step, the type of cyclization is selected. There are seven cyclization options (N-C, S-S, K-D, K-E, K-C, D-N, and E-N), with an eighth option of "None" for linear peptides. If selected, both D- and L-amino acids could involve in cyclizations. Duplicates generated due to cyclization are removed to provide a unique set of molecules.

Here, N = N-terminal, C = C-terminal, S = sulfur of cysteine, K = lysine side-chain, D = aspartic acid side-chain, E = glutamic acid side-chain.

Cyclization Types

Step 6: Capping Groups

The user can choose a capping group for the N or C terminal. For the N-terminal, draw the capping group in ChemDraw or RCSB Chemical-Sketch and connect a wildcard * to the atom that needs to be connected to the -NH₂ group via a single bond. Examples include:

• N-methyl: C[*]
• N-ethyl: CC[*]
• N-acetyl: CC([*])=O
• N-benzyl: [*]CC1=CC=CC=C1

The C-terminal capping group is replaced by -OH, connected to the carbonyl carbon of the C-terminal carboxylic acid.

• Methyl ketone: C[*]
• Methyl ester: CO[*]
• Ethyl ester: CCO[*]
• Benzyl ester: [*]OCC1=CC=CC=C1

Note: If cyclization is selected, required functional groups will be used, overriding any intended capping.

Step 7: Ionization State

Ionizable side chains of amino acids are ionized in the generated SMILES strings. N and C terminals, if not involved in cyclization or capped, are also ionized.

Step 8: Identifiers and File Names

Canonical amino acids are represented by uppercase FASTA codes, their enantiomers by lowercase. "X" is used for noncanonical amino acids. Cyclization is represented by codes preceded by an underscore. File names include the type of peptide (linear or cyclic), chain length (n), number of peptides in the file (N), date, and time, e.g., Linear/cyclization_n_N_yyyymmdd_hhmmss.txt.

Step 9: Visual Guides

Number of Linear and Cyclic Peptides