Chemical cell-surface receptor engineering using affinity-guided, multivalent organocatalysts.

Catalysts hold promise as tools for chemical protein modification. However, the application of catalysts or catalyst-mediated reactions to proteins has only recently begun to be addressed, mainly in in vitro systems. By radically improving the affinity-guided DMAP (4-dimethylaminopyridine) (AGD) catalysts that we previously reported (Koshi, Y.; Nakata, E.; Miyagawa, M.; Tsukiji, S.; Ogawa, T.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 245.), here we have developed a new organocatalyst-based approach that allows specific chemical acylation of a receptor protein on the surface of live cells. The catalysts consist of a set of 'multivalent' DMAP groups (the acyl transfer catalyst) fused to a ligand specific to the target protein. It was clearly demonstrated by in vitro experiments that the catalyst multivalency enables remarkable enhancement of protein acylation efficiency in the labeling of three different proteins: congerin II, a Src homology 2 (SH2) domain, and FKBP12. Using a multivalent AGD catalyst and optimized acyl donors containing a chosen probe, we successfully achieved selective chemical labeling of bradykinin B(2) receptor (B(2)R), a G-protein coupled receptor, on the live cell-surface. Furthermore, the present tool allowed us to construct a membrane protein (B(2)R)-based fluorescent biosensor, the fluorescence of which is enhanced (tuned on) in response to the antagonist ligand binding. The biosensor should be applicable to rapid and quantitative screening and assay of potent drug candidates in the cellular context. The design concept of the affinity-guided, multivalent catalysts should facilitate further development of diverse catalyst-based protein modification tools, providing new opportunities for organic chemistry in biological research.

Controlled release of protein drugs from newly developed amphiphilic polymer-based microparticles composed of nanoparticles.

A novel formulation of biodegradable microparticles was developed for the sustained release of peptide and protein drugs. The microparticles were formed by the aggregation of protein nanoparticles through water-in-oil (W/O) emulsion-lyophilization and subsequent solid-in-oil-in-water (S/O/W) emulsion-solvent evaporation. Amphiphilic copolymers were used as an emulsifier in the W/O emulsion and matrix of the microparticles. Among the various copolymers investigated, poly(lactide-co-glycolide)-grafted dextran (Dex-g-PLGA) was chosen as the best candidate on the basis of the encapsulation efficiency and in vitro release profile, the near zero-order release without a significant initial burst, of human growth hormone (hGH). The release rate of hGH was controllable by changing the composition of Dex-g-PLGA. The in vivo release studies using normal mice revealed that the plasma concentration of hGH was maintained for 1week without a significant initial burst. The enhancement of biological activity of hGH by sustained release was confirmed by measuring the IGF-1 concentration and body weight of hypophysectomized mice. These results suggest the high potential of the newly developed microparticles for the sustained release of biopharmaceuticals.

Affinity-labeling-based introduction of a reactive handle for natural protein modification.

A new chemical method to site-specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity-labeling-based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime-exchange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target-specific multiple modification in a protein mixture. The availability of various (photo)affinity-labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site-selective incorporation of many functional molecules into proteins.

Target-specific chemical acylation of lectins by ligand-tethered DMAP catalysts.

Because sugar-binding proteins, so-called lectins, play important roles in many biological phenomena, the lectin-selective labeling should be useful for investigating biological processes involving lectins as well as providing molecular tools for analysis of saccharides and these derivatives. We describe herein a new strategy for lectin-selective labeling based on an acyl transfer reaction directed by ligand-tethered DMAP (4-dimethylaminopyridine). DMAP is an effective acyl transfer catalyst, which can activate an acyl ester for its transfer to a nucleophilic residue. To direct the acyl transfer reaction to a lectin of interest, we attached the DMAP to a saccharide ligand specific for the target lectin. It was clearly demonstrated by biochemical analyses that the target-selective labeling of Congerin II, an animal lectin having selective affinity for Lactose/LacNAc (N-acetyllactosamine), was achieved in the presence of Lac-tethered DMAPs and acyl donors containing probes such as fluorescent molecules or biotin. Conventional peptide mapping experiments using HPLC and tandem mass-mass analysis revealed that the acyl transfer reaction site-specifically occurred at Tyr 51 of Cong II. This strategy was successfully extended to other lectins by changing the ligand part of the ligand-tethered DMAP. We also demonstrated that this labeling method is applicable not only to purified lectin in test tubes, but also to crude mixtures such as E. coli lysates or homogenized animal tissue samples expressing Congerin.

Design of a hybrid biosensor for enhanced phosphopeptide recognition based on a phosphoprotein binding domain coupled with a fluorescent chemosensor.

Protein-based fluorescent biosensors with sufficient sensing specificity are useful analytical tools for detection of biologically important substances in complicated biological systems. Here, we present the design of a hybrid biosensor, specific for a bis-phosphorylated peptide, based on a natural phosphoprotein binding domain coupled with an artificial fluorescent chemosensor. The hybrid biosensor consists of a phosphoprotein binding domain, the WW domain, into which has been introduced a fluorescent stilbazole having Zn(II)-dipicolylamine (Dpa) as a phosphate binding motif. It showed strong binding affinity and high sensing selectivity toward a specific bis-phosphorylated peptide in the presence of various phosphate species such as the monophosphorylated peptide, ATP, and others. Detailed fluorescence titration experiments clearly indicate that the binding-induced fluorescence enhancement and the sensing selectivity were achieved by the cooperative action of both binding sites of the hybrid biosensor, i.e., the WW domain and the Zn(II)-Dpa chemosensor unit. Thus, it is clear that the tethered Zn(II)-Dpa-stilbazole unit operated not only as a fluorescence signal transducer, but also as a sub-binding site in the hybrid biosensor. Taking advantage of its selective sensing property, the hybrid biosensor was successfully applied to real-time and label-free fluorescence monitoring of a protein kinase-catalyzed phosphorylation.

A fluorescent lectin array using supramolecular hydrogel for simple detection and pattern profiling for various glycoconjugates.

Because sugar and its derivatives play important roles in various biological phenomena, the rapid and high-throughput analysis of various glycoconjugates is keenly desirable. We describe herein the construction of a novel fluorescent lectin array for saccharide detection using a supramolecular hydrogel matrix. In this array, the fluorescent lectins were noncovalently fixed under semi-wet conditions to suppress the protein denaturation. It is demonstrated by fluorescence titration and fluorescence lifetime experiments that the immobilized lectins act as a molecular recognition scaffold in the hydrogel matrix, similar to that in aqueous solution. That is, a bimolecular fluorescence quenching and recovery (BFQR) method can successfully operate under both conditions. This enables one to fluorescently read-out a series of saccharides on the basis of the recognition selectivity and affinity of the immobilized lectins without tedious washing processes and without labeling the target saccharides. Simple and high-throughput sensing and profiling were carried out using the present lectin array for diverse glycoconjugates, which not only included a simple glucose, but also oligosaccharides, and glycoproteins, and, furthermore, the pattern recognition and profiling of several types of cell lysates were also accomplished.

Double-modification of lectin using two distinct chemistries for fluorescent ratiometric sensing and imaging saccharides in test tube or in cell.

The site-selective incorporation of two different fluorophores into a naturally occurring protein (lectin, a sugar-binding protein) has been successfully carried out using two distinct orthogonal chemical methods. By post-photoaffinity labeling modification, Con A, a glucose- and mannose-selective lectin, was modified with fluorescein in the proximity of the sugar binding site (Tyr100 site), and the controlled acylation reaction provided the site-selective attachment of coumarin at Lys114. In this doubly modified Con A, the fluorescein emission changed upon the binding to the corresponding sugars, such as the glucose or mannose derivatives, whereas the coumarin emission was constant. Thus, the doubly modified Con A fluorescently sensed the glucose- and mannose-rich saccharides in a ratiometric manner while retaining the natural binding selectivity and affinity, regardless of the double modification. On the benefit of the ratiometric fluorescent analysis using two distinct probes, the sugar trimming process of a glycoprotein can be precisely monitored by the engineered Con A. Furthermore, the doubly modified Con A can be used not only for the convenient fluorescent imaging of saccharides localized on a cell surface, such as the MCF-7, a breast cancer cell having rich high-mannose branch, but also for the ratiometric fluorescent sensing of the glucose concentration inside HepG2 cells. These results demonstrated that the semisynthetic lectin modified doubly by two distinct chemistries is superior to the singly modified one in function, and thus, it may be potentially useful in cell, as well as in test tube.