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Three new triterpenoid glycosides, 2

We described here the synthesis of oxytocin by an improved Fmoc solid-phase peptide synthesis (SPPS) method with a Rink-Amide resin as the solid support, HBTU as the coupling reagent, Fmoc-protected amino acids as the building blocks, and piperazine for Fmoc removal as a substitute for the standard reagent piperidine. Unlike previously reported syntheses, the removal of the S-Acm protecting group of Cys and cyclization forming the disulfide bond were carried out by using iodine on the resin with the fully protected peptide chains. Finally, a crude oxytocin with a purity of 92% was obtained by simultaneous cleavage of the peptide chains from the resin and removal of all side-chain protecting groups with trifluoroacetic acid containing the scavengers (yield 85%). The crude peptide was purified by using preparative RP-HPLC to obtain oxytocin (high purity 99.3%) with a bioactivity of 588 IU/mg, the highest reported so far in the literature. This investigation provides a contribution in efforts for the large-scale synthesis of oxytocin in high purity under mild conditions with iodine for on-resin disulfide bond formation and a substitute for the standard Fmoc-deprotecting reagent piperidine, a controlled substance.

We describe the use of native chemical ligation (NCL) reaction to covalently cross-link soluble polymers into hydrogels. Macromonomers consisting of a four-armed poly(ethylene glycol) (PEG) core end-functionalized with either thioester or N-terminal cysteine peptide were designed and synthesized. Upon mixing aqueous solutions of the thioester and N-terminal cysteine macromonomers, rigid hydrogels formed within minutes. The gelation time was affected by choice of buffer, pH, polymer concentration, reaction temperature, and chemical composition of the N-terminal cysteine conjugate. The kinetics of gel formation and the viscoelastic behavior of selected hydrogels were further studied by oscillatory rheology, which demonstrated a minimum gel formation time of approximately two minutes and the formation of an elastic cross-linked hydrogel via the NCL reaction. A useful feature of this hydrogel strategy is the regeneration of thiol functional groups as a result of the NCL reaction, thereby allowing functionalization of the polymer hydrogel with biomolecules. This was demonstrated by conjugation of a maleimide-GRGDSPG-NH(2) peptide to an NCL hydrogel, permitting the attachment of human mesenchymal stem cells (hMSCs) on the hydrogel. Due to the mild reaction conditions, chemoselectivity, and potential for biological functionalization, our approach may prove useful as a general method for hydrogel formation, including hydrogels intended for biomedical applications.

We developed a new strategy for the functionalization of hyaluronic acid by chemical modification of its C-6 hydroxyl groups through an ether bond to obtain a cysteine-hyaluronic acid conjugate. This conjugate is suitable to prepare injectable and in situ formed hydrogels cross-linked by native chemical ligation and Michael addition under mild conditions.

A new biomimetic strategy for modification of biomaterial surfaces with poly(ethylene glycol) (PEG) was developed. The strategy exploits the adhesive characteristics of 3,4-dihydroxyphenylalanine (DOPA), an important component of mussel adhesive proteins, to anchor PEG onto surfaces, rendering the surfaces resistant to cell attachment. Linear monomethoxy-terminated PEGs were conjugated either to a single DOPA residue (mPEG-DOPA) or to the N-terminus of Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (mPEG-MAPD), a decapeptide analogue of a protein found in Mytilus edulis adhesive plaques. Gold and titanium surfaces were modified by adsorption of mPEG-DOPA and mPEG-MAPD from solution, after which surface analysis by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectroscopy confirmed the presence of immobilized PEG on the surface. The ability of modified surfaces to resist cell attachment was examined by culturing 3T3 fibroblasts on the surfaces for up to 14 days. Quantitative image analysis revealed that cell adhesion to mPEG-DOPA and mPEG-MAPD modified surfaces decreased by as much as 98% compared to control surfaces. Modified Ti surfaces exhibited low cell adhesion for up to 2 weeks in culture, indicating that the nonfouling properties of mPEG-DOPA and mPEG-MAPD treated surfaces persist for extended periods of time. This strategy paradoxically exploits the strong fouling characteristics of MAP analogues for antifouling purposes and may be broadly applied to medical implants and diagnostics, as well as numerous nonmedical applications in which the minimization of surface fouling is desired.

Cyclic ethyl orthoformate (Ceof) was utilized as a protecting group to protect the catechol hydroxyl groups of 3,4-dihydroxyphenylalanine (DOPA). This protecting group is stable to strong bases and nucleophiles, and can be removed efficiently by 1 M trimethylsilyl bromide in trifluoroacetic acid in the presence of scavengers at 0°C for 60 min. Fmoc-DOPA(Ceof)-OH was synthesized in high yield and applied along with other Fmoc-amino acids to the solid-phase peptide synthesis of a DOPA-containing decapeptide from a mussel adhesive protein.

A new tetrasaccharide flavonol glycoside was isolated from the aerial parts of Epimedium acuminatum, along with three known flavonoids. The structure of the new compound, named acuminatoside [1], was established to be anhydroicaritin-3-O-alpha-L-rhamnopyranosyl(1----2)-alpha-L-rhamno pyranoside-7- O-beta-D-glucopyranosyl-(1----2)-beta-D-glucopyranoside by means of spectroscopic techniques (uv, eims, fdms, fabms, 1H nmr, 1H-1H COSY, 2D-J, 13C nmr, APT, and 1H-13C HETCOR) and chemical methods (acid hydrolysis, enzymatic hydrolysis, and tlc-densitometry). The known compounds were identified as icariin, epimedoside A, and kaempferitrin.

Three thiazole and oxazole containing amino acids were synthesized in good yields by condensation-cyclization. The active functional groups used, a C-terminal imino ester or a C-terminal aldehyde, reacted with both the amino groups and side chains of either serine or cysteine within 5 minutes at rt to form oxazolines or diasteriomeric mixtures of thiazolidines, respectively. The intermediate heterocyclic rings were then dehydrogenated to form the more stable, fully aromatic, rings. Ready availability of N-protected thiazole and oxazole-containing building blocks facilitates the solid-phase synthesis of natural products such as microcin B17 and other peptide-derived natural products that contain 2,4-linked thiazole and oxazole rings.

Building blocks A, B, and C allowed us to create a large number of novel compounds in combinatorial libraries which were inspired by natural products such as microcin B17 and bleomycin. As a side benefit, the availability of these building blocks will facilitate the chemical synthesis of natural products which contain these motifs. Microcin B17 is the first known example of a peptide-based inhibitor of topoisomerase II [4] and library compound 39 is a fragment of microcin B17. A set of compounds prepared using combinations of these three building blocks and naturally-occurring amino acids contained peptidomimetics that inhibited the growth of Vibrio anguillarum, a fish pathogen, but they were less active than was a synthetic fragment of microcin B17 (Fig. 2 39). All of the active compounds had the oxazolylthiazole building block at their N-terminus. Of the peptidomimetics synthesized, only two compounds (Fig. 2., L2-6 and L2-9) showed substantial antibacterial activity against Vibrio anguillarum. The N-acetyl C-G-A-C-(3-aminopropamide) compound (L2-3 not shown) was inactive. Since L2-3 differs from L2-6 only by substitution of a thiazole for an oxazole ring near the C-terminus, the net antibacterial activity is apparently determined by more than one building block.

The ability to present cell adhesion molecule (CAM) ligands in controlled amounts on a culture surface would greatly facilitate the control of cell growth and differentiation. Supported lipid monolayer/bilayer systems have previously been developed that allow for presentation of CAM ligands for cell interaction; however, these systems have employed peptide loadings much higher than those used in poly(ethylene glycol) (PEG)-based immobilization systems. We report the development of synthetic methods that can be used for the efficient and versatile creation of many linear and cyclic lipid-linked peptide moieties. Using RGD-based peptides for the alpha5beta1 integrin as a model system, we have demonstrated that these lipopeptides support efficient cell binding and spreading at CAM ligand loadings as low as 0.1 mol %, which is well below that previously reported for supported lipid systems. Engineered lipopeptide-based surfaces offer unique presentation options not possible with other immobilization systems, and the high activity at low loadings we have shown here may be extremely useful in presenting multiple CAM ligands for studying cell growth, differentiation, and signaling.

Short peptide substrates with high specificity toward transglutaminase (TGase) enzyme were designed, characterized, and coupled to a biocompatible polymer, allowing for rapid enzymatic cross-linking of peptide-polymer conjugates into hydrogels. Eight acyl acceptor Lys-peptide substrates and three acyl donor Gln-peptide substrates were rationally designed and synthesized. The kinetic constants of these peptides toward tissue transglutaminase were measured by enzyme assay using RP-HPLC analysis with the aid of LC-ESI/MS. Several acyl donor and acyl acceptor peptides with high specificities toward TGase were identified, including a few containing the unusual amino acid l-3,4-dihydroxylphenylalanine (DOPA), which is found in the adhesive proteins secreted by marine and freshwater mussels. Acyl donor and acyl acceptor peptides with high substrate specificities were separately coupled to branched poly(ethylene glycol) (PEG) polymer molecules. Equimolar solutions of these polymer-peptide conjugates rapidly formed hydrogels in less than 2 min in the presence of transglutaminase under physiological conditions. The use of biocompatible building blocks, their rapid solidification from a liquid precursor under physiologic conditions, and the ability to incorporate adhesive amino acid residues using biologically benign enzymatic cross-linking are advantageous properties for the use of such materials for tissue repair, drug delivery, and tissue engineering applications.

Ex vivo expansion of hematopoietic stem cells (HSCs) would greatly facilitate cell and gene therapies. However, HSC division in culture is associated with differentiation. This contrasts with sustained HSC expansion in vivo, and has led to the hypothesis that a stem cell niche supports self-renewal. It is likely that multiple aspects of the niche will have to be mimicked to substantially enhance HSC self-renewal. We are developing a defined culture surface for the presentation of cytokines and cell adhesion molecule (CAM) ligands that are thought to be in the HSC niche. Peptide mimics of CAM ligands and cytokines conjugated to dipalmitoyl glycerol via a polyethylene glycol tether are incorporated into dipalmitoylphosphatidylcholine (DPPC) vesicles and deposited onto a hydrophobic surface to create a lipid monolayer. We have previously shown that this system effectively presents adhesive peptide ligands (Jensen et al., JACS 126:15223, 2004). The strategy for immobilizing lipopeptides has been extended to the presentation of a peptide mimetic for the hematopoietic growth factor thrombopoietin (TPO). The lipopeptide mimetic of TPO is based on the branched dimer mimic (TPOm) developed by Cwirla et al. (Science 276:1696, 1997). We have synthesized two versions of TPOm lipopeptide, the first linked to a lipid at both of the amine termini (TPOm-2L) and the second is linked by a single lipid at the carboxy terminus (TPOm-1L). This immobilization strategy does not interfere with the bioactivity of the TPOm as evidenced by cell adhesion and signaling assays. Adhesion was measured with a normal force assay at 30g using the TPO-responsive M07e cell line. We observed a dose-dependent increase in adhesion, with