Synthesis, solid-state structure, and surface properties of end-capped poly(L-lactide)

End-capped poly(L-lactide) (PLLA) samples with dodecyl or 2-(2-(2-methoxyethoxy)ethoxy)ethyl (MEEE) ester were synthesized by ring-opening polymerization of L-lactide in the presence of zinc dodecanoxide or zinc 2-(2-(2-methoxyethoxy)ethoxy)ethoxide as a catalyst, respectively. On the basis of NMR analysis, it was confirmed that the carboxylic acid chain ends of PLLA molecules were selectively substituted by dodecyl or MEEE ester groups. To evaluate the wettability on the surface of end-capped PLLA films, the advancing contact angle (thetaa) with water was measured. The amorphous PLLA films showed relatively similar thetaa values regardless of the chemical structure of the polymer chain end. In contrast, the thetaa values of semicrystalline films were varied over a wide range, dependent on the chemical structure of the chain end. In addition, the thetaa values of dodecyl ester end-capped PLLA film with low molecular weight increased with an increase in the crystallization temperature. Both the crystallinity and lamellar thickness of dodecyl ester end-capped PLLA films increased with the crystallization temperature. These results suggest that the segregation of the chain ends on the PLLA film surface was strongly affected by the crystallization conditions.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

Structure and activity of insulin, XV[1-5]. Further evidence for the importance of arginine residue B22 in the activity of insulin. Semisyntheses of despentapeptide-(B23 - 30)-insulins varied in B22 using desnonapeptide-(B22 - 30)-insulin and tetrapeptides

Insulin hexamethyl ester was digested by trypsin. The resulting desoctapeptide-(B23 - 30)-insulin pentamethyl ester was purified. This compound was digested by carboxypeptidase B to remove the arginine residue B22 at the end of the B chain. Then the N-terminal amino groups of the remaining desnonapeptide-(B22 - 30)-insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the glutamic acid residue B21 of this product was coupled to the following synthetic tetrapeptide esters: Arg-Gly-Phe-Phe-OMe, Lys(Boc)-Gly-Phe-Phe-OMe, Orn(Boc)-Gly-Phe-Phe-OMe, Cit-Gly-Phe-Phe-OMe, Ala-Gly-Phe-Phe-OMe and Gly-Gly-Phe-Phe-OMe. The syntheses of these peptide esters are described. After removal of all protecting groups, despentapeptide-insulin (B22-Arg) and analogues of this product with variation in position B22 could be obtained. They were purified by column chromatography. The biological activities of these components were determined by the mouse fall test. In the case of despentapeptide insulin (C-terminus Arg-Gly-Phe-Phe), the activity rose to the expected value of 34%. The insulin variants with amino acid residues other than arginine in position B22 had much lower activities: with lysine 13%, with ornithine 12%, with citrulline 9%, with alanine 8% and with glycine 6%. Desnonapeptide-insulin by itself posses an activity of 3%. These results demonstrate once more the essential nature of arginine residue B22 for insulin activity.     

Preparation of N-tBoc L-glutathione dimethyl and di-tert-butyl esters: versatile synthetic building blocks

The title l-glutathione derivatives, containing acid- and base-labile esters, respectively, were obtained in good overall yields. N-(t)Boc l-glutathione dimethyl ester was prepared via Fischer esterification of l-glutathione disulfide (GSSG) using HCl in dry methanol, protection of the amine with (t)Boc(2)O, and tributylphosphine cleavage of the disulfide in wet isopropanol. Alternatively, Fischer esterification and (t)Boc-protection of l-glutathione (GSH) also furnished N-(t)Boc glutathione dimethyl ester accompanied by a small amount of S-(t)Boc that was removed chromatographically. The di-tert-butyl ester was obtained by S-palmitoylation of GSH in TFA as solvent, N-(t)Boc-protection, esterification using (t)BuOH mediated by diisopropylcarbodiimide/copper(I) chloride, and saponification of the thioester. These l-glutathione derivatives are versatile synthetic building blocks for the preparation of S-glutathione adducts.     

N(3)-protection of thymidine with Boc for an easy synthetic access to sugar-alkylated nucleoside analogs

The use of Boc as a nucleobase-protecting group in the synthesis of sugar-modified thymidine analogs is reported. Boc was easily inserted at N(3) by a simple and high-yielding reaction and found to be stable to standard treatments for the removal of Ac and (t) BuMe(2) Si (TBDMS) groups, as well as to ZnBr(2) -mediated 4,4'-dimethoxytrityl (DMTr) deprotection. Boc Protection proved to be completely resistant to the strong basic conditions required to regioselectively achieve O-alkylation, therefore, providing synthetic access to a variety of sugar-alkylated nucleoside analogs. To demonstrate the feasibility of this approach, two 3'-O-alkylated thymidine analogs have been synthesized in high overall yields and fully characterized.     

Synthesis of oxytocin HYNIC derivatives as potential diagnostic agents for breast cancer

Two synthetic procedures for HYNIC oxytocin labeling were developed: one based on an orthogonal protection approach and the other with prelabeled (Boc)HYNIC-(Fmoc) amino acids. Both procedures were compared and applied to the preparation of several HYNIC-oxytocin derivatives where ligand position and amino acid (lysine and phenylalanine) were varied. Additionally, an oxytocin derivative labeled with HYNIC in the alpha-amino group of the Cys1 residue was also prepared. 99mTc-ethylendiaminediacetic acid (EDDA) labeling efficiencies were examined for all the derivatives, resulting in two candidates which showed affinity for the oxytocin receptor. Further biochemical experiments demonstrated that 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 could be used as a potential radiopharmaceutical for breast cancer diagnosis.     

Preparation of poly(L-lactide)-based microspheres having a cationic or anionic surface using biodegradable surfactants

Poly(L-lactide)-based microspheres having cationic or anionic surfaces were prepared using polydepsipeptide-block-poly(L-lactide)s as surfactants. Polydepsipeptide-block-poly(L-lactide)s having amino or carboxylic acid groups on their side chains were synthesized through anionic ring-opening polymerizations of L-lactide using the corresponding protected polydepsipeptides as macroinitiators and consequent deprotections. Since these amphiphilic copolymers consisting of hydrophobic segments and hydrophilic segments with amino or carboxylic acid groups could be converted to cationic or anionic block copolymers, they could act as surfactants preparing poly(L-lactide)-based microspheres by an oil-in-water emulsion method. The amount of ionic groups located on the surfaces of the obtained microspheres was found to increase with increasing the feed of charged polydepsipeptide-block-poly(L-lactide)s in the blend of poly(L-lactide) and block copolymers. The average diameters of the dried microspheres estimated by scanning electron microscopy were found to decrease with an increase in feed of block copolymers in polymer blends.     

A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope.

A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to approximately 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.     

Bioorganic synthesis of a recombinant HIV-1 fusion inhibitor,SC35EK,with an N-terminal pyroglutamate capping group

The bioorganic synthesis of an end-capped anti-HIV peptide from a recombinant protein was investigated. Cyanogen bromide-mediated cleavage of two Met-Gln sites across the target anti-HIV sequence generated an HIV-1 fusion inhibitor (SC35EK) analog bearing an N-terminal pyroglutamate (pGlu) residue and a C-terminal homoserine lactone (Hsl) residue. The end-capped peptide, pGlu-SC35EK-Hsl, had similar bioactivity and biophysical properties to the parent peptide, and an improved resistance to peptidase-mediated degradation was observed compared with the non-end-capped peptide obtained using standard recombinant technology.     

L-Phe end-capped poly(L-lactide) as macroinitiator for the synthesis of poly(L-lactide)-B-poly(L-lysine) block copolymer

A poly(L-lactide)-b-poly(Nepsilon-(Z)-L-lysine) (PLLA-b-PZLys) block copolymer was synthesized through the ring-opening polymerization of Nepsilon-(Z)-lysine-N-carboxyanhydride using L-Phe-terminated PLLA as a macroinitiator. The L-Phe-terminated PLLA was prepared through a novel three-step process. First, the hydroxyl-terminated PLLA was synthesized through the ring-opening polymerization of L-lactide initiated by n-butanol under the existence of tin(II) ethylhexanoate. Subsequently, the complete capping of the hydroxyl end group of PLLA with BOC-L-Phe was achieved by using a mixed anhydride of BOC-L-Phe under the catalysis of 4-(1-pyrrolidinyl) pyridine. Finally, the free amino end group was obtained by removal of the t-butoxycarbonyl group through trifluoroacetic acid treatment under anhydrous condition. All these treatments were conducted under mild conditions, thus avoiding the breakdown of the PLLA backbone. Poly(L-lactide)-b-poly(L-lysine) block copolymer was produced after deprotection treatment of PLLA-b-PZLys. The structure of the block copolymer was confirmed by 1H NMR, IR, and GPC. Adjustment of the ratio of the NCA monomer to the macroinitiator could control the chain length of the PLys block.     

Synthesis of a molt-inhibiting hormone of the American crayfish, Procambarus clarkii, and determination of the location of its disulfide linkages

A molt-inhibiting hormone (Prc-MIH) of the American crayfish, Procambarus clarkii, a member of the type II CHH family, was chemically synthesized and the location of its three disulfide linkages was determined. Prc-MIH consists of 75 amino acid residues and was synthesized by a thioester method. Two peptide segments, Boc-[Cys(Acm)(7,24,27), Lys(Boc)(19)]-Prc-MIH(1-39)-SCH(2)CH(2)CO-Nle-NH(2) and H-[Cys(Acm)(40,44,53), Lys(Boc)(42,51,67)]-Prc-MIH(40-75)-NH(2), were prepared using peptides obtained via the Boc solid-phase method. Condensation of the building blocks in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, and N, N-diisopropylethylamine, followed by removal of the protecting groups, gave the reduced form of Prc-MIH(1-75)-NH(2). This product was converted to the native form of Prc-MIH (synthetic Prc-MIH) in a buffer which contained cysteine and cystine. The synthetic Prc-MIH showed the same behavior by RP-HPLC and biological activity assays as the natural Prc-MIH. The disulfide bond between Cys7 and Cys44 was determined by isolation of a fragment from an enzymatic digest of the synthetic Prc-MIH by RP-HPLC, followed by mass analysis. The disulfide bonds between Cys24 and Cys40 and between Cys27 and Cys53 were determined by comparing the elution position of an enzymatic digest of the synthetic Prc-MIH with authentic chemically synthesized samples, which contained three types of possible disulfide linkages.     

Biocompatible and biodegradable ultrafine fibrillar scaffold materials for tissue engineering by facile grafting of L-lactide onto chitosan

A chitosan derivative was prepared with good yields using a "one pot" approach by grafting L-lactide oligomers via ring opening polymerization. Side chains are primarily attached to hydroxyl groups located on carbons 3 and 6 of the glucosamine ring, while the amine group remains nonfunctionalized. By increasing the L-lactide to chitosan ratio, side chain length is controlled. This allows the manipulation of the biodegradation rate and hydrophilicity of the tissue engineering scaffold material. This general synthetic route renders functionalized chitosan soluble in a broad range of organic solvents, facilitating formation of ultrafine fibers via electrospinning. Cytotoxicity tests using fibroblasts (L929 cell line) performed on electrospun L-lactide modified chitosan fibers showed that the specimen with the highest molar ratio of L-lactide (1:24) investigated in this study is the most promising material for tissue engineering purposes, while less stable formulations might still find application in drug delivery vehicles.     

Solid phase synthesis of porcine LH-releasing hormone with hydrogen chloride in formic acid as the deblocking reagent.

The decapeptide corresponding to the amino acid sequence of porcine luteinizing hormone-releasing hormone (LH-RH) which involves 1 mol of tryptophan was synthesized via solid phase synthesis with two different deblocking procedures which used hydrogen chloride in formic acid and hydrogen chloride in acetic acid containing 1% 2-mercaptoethanol. After some fundamental studies on the former reagent with respect to deblocking efficiency toward the Boc group, 0.5 M hydrogen chloride (a 10-fold molar excess with respect to the N-terminal Boc group) in formic acid was used in the present synthesis. The two synthetic products exhibited the same chemical and biological properties as an authentic LH-RH. Hydrogen chloride in formic acid has proved effective without a scavenger although loss of peptide from the resin occurred to a somewhat greater extent than that with hydrogen chloride in acetic acid. A derivative of the synthetic LH-RH formylated at the indole nitrogen had a greatly diminished biological activity, indicating that the intact indole side chain is essential for the activity.     

Structural and conformational properties of (Z)-beta-(1-naphthyl)- dehydroalanine residue

To understand how chemical structure of beta-substituted alpha, beta-dehydroalanine (particularly size and pi conjugation of beta substituent) affects conformational property, x-ray crystallographic analysis was performed on Boc-Ala-Delta(Z) Nap-Val-OMe [Boc: t-butoxycarbonyl; Delta(Z) Nap: (Z)-beta-(1-naphthyl)dehydroalanine; OMe: methoxy] having the naphthyl group as a bulky beta substituent. Single crystals were grown by slow evaporation from an ethanol solution in the triclinic space group P1 with a = 9.528 (3) A, b = 12.410(4) A, c = 5.975(2) A, alpha = 96.77(3) degrees, beta = 102. 81(2) degrees, gamma = 88.74(3) degrees, V = 684.1(4) A3, and Z = 1. Phase determination was carried out by a direct method (SHELEXS), and the final structure was refined to R = 8.1% and R(w) = 9.0% for 1964 observed reflections. The bond lengths and bond angles of the Delta(Z)Nap residue, characterized by a sp(2) hybridized C(alpha) atom, did not differ from those of other dehydroresidues such as Delta(Z) Phe, Delta(Z) Leu, and DeltaVal essentially. The peptide backbone took a type II beta-turn conformation involving an intramolecular hydrogen bond between CO(Boc) and NH(Val), similar to di- or tripeptides containing a Delta(Z) Phe or Delta(Z) Leu residue in the second positions. Here the naphthyl group was found to be nonplanar [chi(2) = 55(1) degrees ] relative to the C(alpha)==C(beta)==C(gamma) plane. The nonplanarity was supported by conformational energy calculation. The molecular packing was stabilized by two kinds of intermolecular hydrogen bonds and van der Waals interactions. Naphthyl groups were arranged in a partially overlapped face-to-face orientation with a center-to-center distance of 5.97 A. For additional information, peptide Boc-(Ala-Delta(Z) Nap-Leu)(2)-OMe was synthesized and its solution conformation was investigated by (1)H-NMR spectroscopy. The hexapeptide showed the tendency to form a 3(10)-helical conformation in solution essentially. Conformational properties of Delta(Z) Nap residue, characterized by a type II beta-turn and 3(10)-helix, were supported by a conformational energy contour map of the Delta(Z)Nap residue.     

Synthesis of a membrane protein with two transmembrane regions.

A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent.     

Synthesis and biological evaluation of analogues of the marine cyclic depsipeptide obyanamide

On the basis of the total synthesis of obyanamide, 20 analogues of this marine cyclic depsipeptide have been synthesized by (i) preparation of the tripeptide fragments in the western hemisphere using Z/OtBu protocol; (ii) preparation of the dipeptide fragments in the eastern hemisphere using Boc/OMe protocol; and (iii) fragments coupling, removal of protecting groups (Boc and OtBu, in one pot), and macrocyclizaion in the last step. The cytotoxic test showed that three synthetic compounds exhibited moderate activities against HL‐60, KB, LOVO, and A549 cell lines. According to the results, the β‐amino acid residue was found to play a critical role in the biological activities. Additionally, the ester bond along with the Ala(Thz) moiety was also essential for biological activities. However, it seems too early to draw a conclusion that the N‐methylation of Val/Phe can lead to higher or lower cytotoxic activities. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Amphiphilic poly(L-lactide)-b-dendritic poly(L-lysine)s synthesized with a metal-free catalyst and new dendron initiators: chemical preparation and characterization

This study presents investigations on new approaches to novel biodegradable amphiphilic poly(L-lactide)-b-dendritic poly(L-lysine)s bearing well-defined structures. First, two new Boc-protected poly(L-lysine) dendron initiators G(2)OH 4 (generation = 2) and G(3)OH 6 (generation = 3) with hydroxyl end functional groups were efficiently derived from corresponding precursors 3 and 5 via methyl ester substitution with ethanolamine. Subsequently, two series of new diblock copolymers of poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s (S1-S2, S3-S4) were prepared in chloroform through ring-opening copolymerization of poly(L-lactide)s with a metal-free catalyst of organic 4-(dimethylamino) pyridine (DMAP) in the presence of a corresponding new poly(L-lysine) dendron initiator. Further, molecular structures of the prepared new dendron initiators as well as those of poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s bearing different dendron blocks and PLLA lengths were examined by means of nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), mass spectrometry (ESI-MS, MALDI-FTMS), and thermal gravimetric analysis (TGA). The results demonstrated successful formation of the synthetic precursors, functional dendron initiators, and new diblock copolymers. In addition, the very narrow molecular weight distributions (PDI = 1.10-1.14) of these poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s further indicated their well-defined molecular structures. After the efficient Boc-deprotection for the dendron amino groups with TFA/CH(2)Cl(2), new diblock poly(L-lactide)-b-dendritic poly(L-lysine)s bearing lipophilic PLLA and hydrophilic dendritic PLL were finally prepared. It was noteworthy that the MALDI-FTMS result showed that no appreciable intermolecular chain transesterification happened during the ROP of L-lactide catalyzed by the DMAP. Moreover, self-assembly of these new biodegradable amphiphilic copolymers in diverse solvents were also preliminarily studied.     

Determination of substrate specificity of fucosyltransferase from rat brain using synthetic acceptors

The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic acceptors. Of 16 acceptors tested, only those containing the Gal beta 1-4GlcNAc beta 1-R fragment were subjected to enzymic fucosylation. The isomer with a 1-3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GlcNAc were not fucosylated whereas Fuc alpha 1-2Gal beta 1-4GlcNAc displayed the same substrate properties as Gal beta 1-4GlcNAc. FT from cerebellum and forebrain was shown to have the specificity similar to that of mammalian FT IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from forebrain, the specificity profiles being similar.     

Structural effects of terminal groups on nonenzymatic and enzymatic degradations of end-capped poly(L-lactide)

Poly(L-lactide) (PLLA) with various alkyl ester chain end groups were synthesized by ring-opening polymerization of L-lactide in the presence of zinc alkoxide as a catalyst. The structural effect of chain end groups on the rate of enzymatic and nonenzymatic degradations for amorphous films of PLLA were investigated at 37 degrees C in a Tris-HCl buffer solution (pH 8.6) with proteinase K and at 60 degrees C in a phosphate buffer solution (pH 7.4), respectively. The rate of enzymatic degradation for PLLA films was dependent on the carbon numbers of alkyl ester chain end groups, and the rates of PLLA samples with dodecyl (C12), tridecyl (C13), and tetracocyl (C14) ester end groups were much lower than those of the other samples. The surface morphologies of PLLA films after enzymatic degradation were characterized by scanning electron microscopy. After the enzymatic degradation, non-end-capped PLLA, PLLA with methyl (C1) and hexyl (C6) ester chain ends, were degraded homogeneously by proteinase K and the film surface was very smooth. In contrast, the PLLA with alkyl ester chain ends of carbon numbers over 12 were degraded heterogeneously by the enzyme, and the sponge-like network structure was formed on the film surface. These results indicated that the long alkyl ester groups at the chain ends of PLLA molecules aggregated in the amorphous films and the erosion rate was depressed due to the coverage of the aggregated terminal groups on the film surface. For the nonenzymatic degradation, the molecular weight of non-end-capped PLLA was remarkably decreased with progress of degradation. In contrast, the molecular weight of the end-capped PLLA gradually reduced at the initial stage of degradation and then the rate of degradation was accelerated. The decreases of molecular weight of PLLA by autocatalyzed degradation were retarded by the capping of carboxyl chain ends.     

N(3)‐Protection of Thymidine with Boc for an Easy Synthetic Access to Sugar‐Alkylated Nucleoside Analogs

The use of Boc as a nucleobase‐protecting group in the synthesis of sugar‐modified thymidine analogs is reported. Boc was easily inserted at N(3) by a simple and high‐yielding reaction and found to be stable to standard treatments for the removal of Ac and ^tBuMe₂Si (TBDMS) groups, as well as to ZnBr₂‐mediated 4,4′‐dimethoxytrityl (DMTr) deprotection. Boc Protection proved to be completely resistant to the strong basic conditions required to regioselectively achieve O‐alkylation, therefore, providing synthetic access to a variety of sugar‐alkylated nucleoside analogs. To demonstrate the feasibility of this approach, two 3′‐O‐alkylated thymidine analogs have been synthesized in high overall yields and fully characterized.