Scaffolds and Foldamers with a Click!
Jonel Saludes and Joan Go

Biologically compatible scaffolds for drug delivery and structural support has been widely used in biomedical and immunological applications. They function as inert conjugates for the delivery of small molecule ligands and to covalently crosslink macromolecules. We synthesize novel scaffolds containing azide, alkyne and other functionalizable groups using 1) polyethylene glycol (PEG) and 2) sialic acid derivatives as backbones.

PEG linkers

Heterofunctional azide and alkyne PEG-linkers have been synthesized and site specifically conjugated to single chain fragments (scFv) via a reactive thiol functionality. Copper (I) catalyzed 1,3-dipolar cycloaddition reaction or “click” chemistry was utilized to covalently link two scFv (Scheme 1). Multivalent scFv constructs (Figure 1) showed improved pharmacokinetics, lower off-rates and increased affinity to cancer cells compared to monovalent scFv (Figure 2).

Scheme 1. Construction of divalent-scFv utilizing click chemistry and PEG linkers


Figure 1. Mulitivalent scFv constucted from scFv which are derived from VH and VL regions of the intact monoclonal antibody (MAb)	Figure 2. Divalent scFv (bottom panel) tested on sections of human breast and human prostate cancer cells showed 2-4 times increased binding compared to scFv (top panel) by immunohistochemistry (IHC).

For enhanced conjugation and multivalent scFv ligation, current studies conducted in the laboratory involve the synthesis of various PEG linkers with modified: 1) PEG linker length, 2) alkyne core, 3) thiol reactive functional group, and 4) novel sugar backbones.

Sialic Acid-Derived Scaffold

Polymeric O-glycosides of sialic acid (N-acetylneuraminic acid; Neu5Ac), a sugar delta-amino acid, are helical. Unnatural amide-linked oligomers constructed by solid phase peptide synthesis (Figure 3) form stable secondary (2°) structures in water as evidenced by Circular Dichroism (CD) (Figure 4). These molecules act as rigid scaffolds with defined 2° structures. Further analogues of these oligomers were prepared and more are currently under investigation in our laboratory to extend the possible oligomeric constructs derived from Neu5Ac. Our new generation of scaffolds can be functionalized via “Click” chemistry to introduce ligands for multivalent ligand display.


Figure 3. Homooligomers from amide-linked Neu5Ac derivatives	Figure 4. CD spectra in H2O for α,β-unsaturated amide-linked oligosaccharides. Trimers to octamers have stable 2° structures.

Computational studies done on these constructs revealed that the s-trans isomer is much more stable than the cis with phi angle at 180°. The most stable conformation of the α,β-unsaturated octamer is a left-handed helix with eight residues per turn (Figure 5).

Figure 5. Computational studies on α,β-unsaturated octamer showing the most stable conformation.

We have demonstrated that biotinylated scaffolds have high plating efficiency on the ELISA plate coated with Neutravidin and non-functionalized scaffolds do not bind to gp120 (Figure 6). This provides us the ability to functionalize with different recognition elements for multivalent ligand display without interference from the scaffold itself. We are currently constructing 1-->5-amide linked functionalizable homo- and heterooligomers using the α,β-unsaturated derivative of Neu5Ac and Glu and characterize their secondary structure by NMR and CD.
		Figure 6. Graph for binding affinities towards gp120 indicating poor binding affinities of scaffolds

Acknowledgments
This work was supported by NSF CHE-0518010 and NIH HODG207 grants. Funding for the NMR spectrometers used on this project were provided by NIH Grants RR11973 and GM075093

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