Improving the aqueous solubility of HCV-E2 glycoprotein epitope mimics by cyclization using POLAR hinges

Abstract

In this research we describe the improvement of the water-solubility of cyclic epitope mimics based on the HCV E2 glycoprotein by incorporation of suitable polar hinges. The poor solubility of epitope mimics based on peptide sequences in the envelope (E2) protein hampered their synthesis and purification and made it very difficult to prepare the molecular constructs for evaluation of their bioactivity. Since changes in the amino acid composition are hardly possible in these epitope mimics in order to increase water-solubility, a polar cyclization hinge may offer a remedy leading to a significant increase of polarity and therefore water solubility. These polar hinges were applied in the synthesis of better water-soluble HCV-E2 epitopes. An azide functionality in the polar hinges allowed attachment of a tetraethylene glycol linker by Cu-catalyzed azide-alkyne cyclo-addition (CuAAC) for a convenient conjugation to ELISA plates in order to evaluate the bio-activity of the epitope mimics. The immunoassays showed that the use of more polar cyclization hinges still supported anti-HCV antibody recognition and did not negatively influence their binding. This significantly increased solubility induced by polar hinges should therefore allow for the molecular construction and ultimate evaluation of synthetic vaccine molecules.

Keywords: ELISA; cyclic peptides; cyclisation method; epitope mimics; hepatitis C virus; polar hinge; synthetic vaccine.

Figure 1

Chemical structures of cyclization hinges for preparation of bicyclic peptides 1,3,5‐tris (bromomethyl)benzene (TBMB) (A); 1,3,5‐triacryloyl‐1,3,5‐triazinane (TATA), N,N′,N″‐(benzene‐1,3,5‐triyl)‐tris(2‐bromoacetamide) (TBAB) and N,N′,N″‐benzene‐1,3,5‐triyltrisprop‐2‐enamide (TAAB) described by Chen et al. 9 and Rim et al. 12 (B); and 2,4,6‐tris (bromomethyl)‐s‐triazine (TBMT) and 1,1′,1″‐(1,3,5‐triazinane‐1,3,5‐triyl)tris(2‐bromoethanone) (TATB) described by Van de Langemheen et al. 16 (C)

Figure 2

Thiol‐maleimide conjugation of epitope mimics forming a plate‐surface with uniformly oriented peptides used for ELISA to evaluate epitope mimicry

Figure 3

1‐(azidomethyl)‐3,5‐bis (bromomethyl)‐s‐triazine (DBMT‐N₃) 1, azido triazinane‐tris(2‐bromoethanone) (TADB‐N₃) 2, and 1‐(azidomethyl)‐3,5‐bis (bromomethyl)benzene (DBMB‐N₃) 3 ^16,17

Scheme 1

Synthesis of the propargyl moiety containing PEG‐based thiol linker for cu‐catalyzed azide‐alkyne cyclo‐addition of cyclic peptides

Scheme 2

Cyclization of precursor peptides 7–10 with azido cyclization hinges DBMT‐N₃ 1, TADB‐N₃ 2, and DBMB‐N₃ 3. The reported yields are of purified products, after preparative reverse phase HPLC

Figure 4

Analytical HPLC traces overlay of peptides 7–9 cyclized on DBMT‐N₃ (1; purple), TADB‐N₃ (2; pink), and DBMB‐N₃ (3; black), cyclization hinges. Column: Dr. Maisch Reprosil gold 200 C18, 5 μm 250 x 4.6 mm; gradient: 0 to 100% buffer B in 30 minutes

Scheme 3

Cyclic epitope mimics with different hinges DBMT‐N₃ (11, 14, 17, 20), TADB‐N₃ (12, 15, 18, 21), and DBMB‐N₃ (13, 16, 19, 22) based on loops I (green), II (red), III (blue), and a scrambled negative control (black). The red crosses indicate the mimics, which were only obtained in trace amounts. The green tick marks indicate the mimics, which were obtained in satisfactory amounts

Figure 5

Mimics of epitopes I (green), II (red), III (blue), and scrambled negative control (black) partly based on the previously reported cyclization linker. Epitope mimics 35 and 37 were described previously17 and epitope mimics 34 and 36 were included here for completeness of the study

Figure 6

ELISA comparing immobilized epitope I mimics having (a): DBMT 23 and (B): TADB 24 hinges and immobilized scrambled negative controls (A): DBMT 31 and (B): TADB 32. Including immobilized epitope I mimic 34 and immobilized scrambled negative control 37 based on the previously published benzylic‐based linker 4 17

Figure 7

ELISA comparing immobilized epitope II mimics having (a): DBMT 26 and (B): TADB 27 hinges and immobilized scrambled negative controls (A): DBMT 31 and (B): TADB 32. Including immobilized epitope II mimic 35 and immobilized scrambled negative control 37 based on the previously published benzylic‐based linker 4 17

Figure 8

ELISA comparing immobilized epitope III mimics having TADB 30 hinge and immobilized scrambled negative controls 32. Including immobilized epitope III mimic 36 and immobilized scrambled negative control 37 based on the previously published benzylic‐based linker 4 17