Novel amino-Li resin for water-based solid-phase peptide synthesis.

We report the first application of a novel amino-Li resin to water-based solid-phase peptide synthesis (SPPS) applying the Smoc-protecting group approach. We demonstrated that it is a suitable support for the sustainable water-based alternative to a classical SPPS approach. The resin possesses good swelling properties in aqueous milieu, provides significant coupling sites, and may be applicable to the synthesis of difficult sequences and aggregation-prone peptides.

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination.

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein-antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled ("conformational") antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA-aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.

Sustainable Peptide Synthesis Enabled by a Transient Protecting Group.

The growing interest in synthetic peptides has prompted the development of viable methods for their sustainable production. Currently, large amounts of toxic solvents are required for peptide assembly from protected building blocks, and switching to water as a reaction medium remains a major hurdle in peptide chemistry. We report an aqueous solid-phase peptide synthesis strategy that is based on a water-compatible 2,7-disulfo-9-fluorenylmethoxycarbonyl (Smoc) protecting group. This approach enables peptide assembly under aqueous conditions, real-time monitoring of building block coupling, and efficient postsynthetic purification. The procedure for the synthesis of all natural and several non-natural Smoc-protected amino acids is described, as well as the assembly of 22 peptide sequences and the fundamental issues of SPPS, including the protecting group strategy, coupling and cleavage efficiency, stability under aqueous conditions, and crucial side reactions.

Nanoscale Biodegradable Organic-Inorganic Hybrids for Efficient Cell Penetration and Drug Delivery.

We report a comprehensive study on novel, highly efficient, and biodegradable hybrid molecular transporters. To this end, we designed a series of cell-penetrating, cube-octameric silsesquioxanes (COSS), and investigated cellular uptake by confocal microscopy and flow cytometry. A COSS with dense spatial arrangement of guanidinium groups displayed fast uptake kinetics and cell permeation at nanomolar concentrations in living HeLa cells. Efficient uptake was also observed in bacteria, yeasts, and archaea. The COSS-based carrier was significantly more potent than cell-penetrating peptides (CPPs) and displayed low toxicity. It efficiently delivered a covalently attached cytotoxic drug, doxorubicin, to living tumor cells. As the uptake of fluorescently labeled carrier remained in the presence of serum, the system could be considered particularly attractive for the in vivo delivery of therapeutics.