Based on the formation of a stable bis-arylhydrazone bond between an aromatic hydrazine, i.e. HyNic (HydrazinoNicotinamide), and an aromatic aldehyde, i.e. 4FB (4-formylbenzamide, ( peptides can be directly conjugated to other biomolecules or surfaces. Figure 1 presents the chemistry of the conjugation of a HyNic-peptide (PepLink) to a 4FB-modified protein.The HyNic moiety is incorporated on peptides during their solid phase peptide synthesis.

Incorporation of the HyNic moiety on a peptide is straightforward, high yielding and can be added at either N-terminus , internally or C-terminus using 6-Boc-HyNic (1) (SoluLinK catalog # S-3003) or FMOC- Lys-(ε-6-BocHyNic)OH (2) (SoluLinK catalog # 3034).

Peptide Synthesis Service: Solulink offers HyNic-peptide synthesis service. The HyNic moiety can be incorporated on your peptide on the N-terminus, C-terminus or an internal position. For a quote submit your sequence to solulink@solulink.com .

Peptide Conjugation/Immobilization Service: Solulink offers services to conjugate or immobilze your peptide(s). Conjugation using Solulink’s HyNic/4FB bioconjugation couple is extremely efficient. We have experience conjugating HyNic-peptides to proteins and oligonucleotides and immobilizing the peptide to solid surfaces including magnetic beads.

Advantages

• Flexible: HyNic moiety can be incorporated on N-terminus, internally or C-terminus
• Simple: Conjugation and immobilization is accomplished by simply adding the HyNic-peptide to the 4-FB biomolecule or surface. The optimal pH 4.5-5.0 and if required can be catalyzed with aniline.
• Rapid and Efficient: Conjugation and immobilization is stoichiometrically efficient
• Quantifiable: Bis-arylhydrazone bond formed is chromophoric (354 nm, molar extinction coefficient 29000)
• Broad-based: Using a library of 4FB-linkers HyNic-peptides can be conjugated to proteins, oligonucleotides and carbohydrates with cleavable and non-cleavable 4FB-linkers.

 

Figure X: Scheme presenting the HyNic peptide monomers used to incorporate HyNic moieties on the N-terminus (top) and C-terminus (lower).

 

Peptide Conjugation/Immobilization Using HyNic/4FB Couple Examples

Protein/peptide conjugation

Figure 2: A 15-mer HyNic-peptide (5 equivalents) was added to 4FB-modified lactoglobulin (Lane 2; MW 18300) in 100 mM phosphate, 150 mM NaCl, pH 6.0 and incubated for 2 hours at room temperature. An aliquot of the crude conjugation reaction (Lane 3) was removed and examined using an SDS-PAGE gel (A). The HyNic-peptide efficiently conjugated to the 4FB-modified lactoglobulin with formation of the bis-aryl hydrazone bond structure shown in B. Overlaid spectra of 4FB-modified lactoglobulin and the lactoglobulin-peptide conjugate (C), illustrates the UV-traceable nature of the aromatic hydrazone linkage (λ 354, ε 29000).

Oligonucleotide/peptide conjugation

Figure 3: Two N-terminal HyNic-modified PepLink peptides 121 and 122 (lanes 2 and 3) were reacted with 5’-4FB-modified 18mer oligo (lane 1) to yield the peptide-oligo conjugate (lanes 4 and 5). Conjugate stability was tested at 94 OC in PBS for two hours. Gel results were visualized by UV back-shadow. As seen in lanes 4 and 5,, conjugate formation is efficient and the conjugate bond is stable to 94 OC , lanes 6 and7.

Peptide Immobilization

Figure 5: A 14mer N-terminus-HyNic-modified peptide was incubated @ 250 uM with Solulink’s NanoLink 4FB-magnetic beads in 100 mM MES buffer, pH 5.0 for 1 h. The course of the reaction could be monitored by a reduction in the peptide’s Å 280 solution phase absorbance as it became immobilized to the 4FB-beads.