Synthesis of glycopeptide dendrimer by a convergent method

Glycopeptide thioester comprising the sequence of extracellular matrix metalloproteinase inducer (emmprin) (34-58) was prepared and condensed with a dendrimer core having eight amino groups by the thioester method. The desired product, a glycopeptide dendrimer carrying an N-linked core pentasaccharide of about 30 kDa, was successfully isolated by preparative electrophoresis and characterized by mass analysis.     

Efficient microwave-assisted tandem N- to S-acyl transfer and thioester exchange for the preparation of a glycosylated peptide thioester

A peptide carrying a mercaptomethylated proline derivative at the C-terminus was prepared by solid-phase peptide synthesis (SPPS) and converted to the thioester of 3-mercaptopropionic acid (MPA) by aqueous MPA under microwave irradiation conditions. This post-SPPS thioesterification reaction was successfully applied to the synthesis of a glycopeptide thioester composed of 25 amino acid (AA) residues, which was then used for the preparation of a 61-AA glycopeptide by the thioester condensation method.     

Application of a novel thioesterification reaction to the synthesis of chemokine CCL27 by the modified thioester method

Aryl thioesters of peptide segments were prepared by the conventional 9-fluorenylmethoxycarbonyl (Fmoc) strategy using a novel N-alkyl cysteine (NAC)-assisted thioesterification reaction. The peptide carrying NAC at its C-terminus was prepared by the Fmoc strategy and converted to the aryl thioester by 4-mercaptophenylacetic acid (MPAA) treatment without significant side reactions. The peptide thioester was used for the efficient preparation of 95-amino acid (AA) chemokine CCL27 by an Ag(+)-free thioester method.     

Inhibition of mitosis by glycopeptide dendrimer conjugates of colchicine

Glycopeptide dendrimers have been prepared bearing four or eight identical glycoside moieties at their surface (beta-glucose, alpha-galactose, alpha-N-acetyl-galactose, or lactose), natural amino acids within the branches (Ser, Thr, His, Asp, Glu, Leu, Val, Phe), 2,3-diaminopropionic acid as the branching unit, and a cysteine residue at the core. These dendrimers have been used as drug-delivery devices for colchicine. Colchicine was attached to the dendrimers at the cysteine thiol group through a disulfide or thioether linkage. The biological activities of the glycopeptide dendrimer conjugates were evaluated in HeLa tumor cells and non-transformed mouse embryonic fibroblasts (MEFs). The concentrations of glycopeptide dendrimer drug conjugates required to achieve inhibition of cell proliferation by interference with the tubulin system were found to be higher (IC50 > 1 microM) compared to the required colchicine concentration. On the other hand, the glycopeptide dendrimer conjugates inhibited the proliferation of HeLa cells 20-100 times more effectively than the proliferation of MEFs. In comparison, non-glycosylated dendrimers and colchicine itself showed a selectivity of 10-fold or less for HeLa cells.     

N-Alkyl cysteine-assisted thioesterification of peptides

A new method for the preparation of peptide thioester by the post-solid phase peptide synthesis (SPPS) approach was developed. A series of N-alkyl cysteine derivatives were prepared and used as the C-terminus residue of the peptides prepared by the Fmoc SPPS. The synthetic peptides released from resin by TFA were readily converted to the peptide thioester in aqueous 3-mercaptopropionic acid (MPA) without significant side reactions.     

N-methyl-phenacyloxycarbamidomethyl (Pocam) group: a novel thiol protecting group for solid-phase peptide synthesis and peptide condensation reactions

In the so-called thioester method for the condensation of peptide segments, protecting groups for amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel thiol protecting group, N-methyl-phenacyloxycarbamidomethyl (Pocam). We used it for protection of cysteine side chains, and synthesized Pocam-containing peptides and peptide thioesters. These were condensed by the thioester method. After the condensation reaction, Pocam groups were cleaved by Zn/AcOH treatment. At the same time, the azido group, which was used for the protection of lysine side chains, was also converted to an amino group, demonstrating that this protecting group strategy simplified the deprotecting reaction after the peptide condensation reaction to only one step.     

N‐Linked Glycosyl Auxiliary‐Mediated Native Chemical Ligation on Aspartic Acid: Application towards N‐Glycopeptide Synthesis

A practical approach towards N‐glycopeptide synthesis using an auxiliary‐mediated dual native chemical ligation (NCL) has been developed. The first NCL connects an N‐linked glycosyl auxiliary to the thioester side chain of an N‐terminal aspartate oligopeptide. This intermediate undergoes a second NCL with a C‐terminal thioester oligopeptide. Mild cleavage provides the desired N‐glycopeptide.     

Efficient synthesis of MUC4 sialylglycopeptide through the new sialylation using 5-acetamido-neuraminamide donors

Sialylation reactions using a new sialyl donor, diethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-beta-D-glycero-D-galacto-2-nonulopyranosylonamide phosphite (Neu5Ac-1-amide-2-phosphite) derivatives, and the synthesis of the sialyl-T N-MUC4 glycopeptide are described. The sialylation was performed in CH2Cl2 solvent toward the 6-hydroxyl group of several monosugar acceptors and generated alpha-sialoside in good yield under low temperature and TMSOTf activation system. Amide derivatives of sialoside were easily converted into naturally occurring sialoside after hydrolysis of the amide group. Sialyl-alpha(2,6)-GalN3 was also prepared by this new sialylation protocol, and then this sialoside was further converted into a Fmoc-protected sialyl-TN serine derivative for solid-phase glycopeptides synthesis. The solid-phase glycopeptide synthesis using this sialyl-TN serine derivative in which the sugar hydroxyl group was free afforded the target sialyl-TN-MUC4 glycopeptide.     

Facile peptide thioester synthesis via solution-phase tosylamide preparation

Preparation of peptide thioester is essential for native chemical ligation and block condensation. Our novel methodology involves conversion of the carboxylic acid of a peptide into a thioester using p-toluenesulfonyl isocyanate, followed by alkylation, then thiol substitution. Our methodology can also be used for the preparation of glycopeptide thioesters. Furthermore, it is possible to carry out the reaction as a sequential peptide chemical ligation.     

An efficient peptide ligation using azido-protected peptides via the thioester method

Azido-protected Fmoc-Lys-OH (Fmoc-Lys(N₃)-OH) was synthesized from Fmoc-Lys-OH by the copper(II)-catalyzed diazo transfer method, and introduced to a peptide by the ordinary Fmoc-based solid-phase peptide synthesis. This azido peptide could be condensed with a peptide thioester by the Ag⁺-free thioester method without any significant side reactions. The azido group was easily reduced to an amino group by Zn powder after peptide condensation.     

Synthesis,characterization, and thermal properties of dendrimer‐star,block‐comb copolymers by ring‐opening polymerization and atom transfer radical polymerization

Novel and well‐defined dendrimer‐star, block‐comb polymers were successfully achieved by the combination of living ring‐opening polymerization and atom transfer radical polymerization on the basis of a dendrimer polyester. Star‐shaped dendrimer poly(?‐caprolactone)s were synthesized by the bulk polymerization of ?‐caprolactone with a dendrimer initiator and tin 2‐ethylhexanoate as a catalyst. The molecular weights of the dendrimer poly(?‐caprolactone)s increased linearly with an increase in the monomer. The dendrimer poly(?‐caprolactone)s were converted into macroinitiators via esterification with 2‐bromopropionyl bromide. The star‐block copolymer dendrimer poly(?‐caprolactone)‐block‐poly(2‐hydroxyethyl methacrylate) was obtained by the atom transfer radical polymerization of 2‐hydroxyethyl methacrylate. The molecular weights of these copolymers were adjusted by the variation of the monomer conversion. Then, dendrimer‐star, block‐comb copolymers were prepared with poly(L ‐lactide) blocks grafted from poly(2‐hydroxyethyl methacrylate) blocks by the ring‐opening polymerization of L ‐lactide. The unique and well‐defined structure of these copolymers presented thermal properties that were different from those of linear poly(?‐caprolactone). © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 6575–6586, 2006     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

Pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction

In one of the peptide condensation methods termed thioester method, an amino protecting group is required in the lysine side chain. In this study, to investigate the efficiency of the pyruvoyl group as an amino protecting group, we synthesized N^α-fluorenylmethoxycarbonyl (Fmoc)-N^ε-pyruvoyl-lysine and introduced it into peptides and glycopeptides by the ordinary Fmoc-based solid phase peptide synthesis. The pyruvoyl peptide could be condensed with a peptide thioester by the thioester method, and this protecting group was easily removed by o-phenylenediamine treatment without significant side reactions.     

Peptide bond formation mediated by 4,5-dimethoxy-2-mercaptobenzylamine after periodate oxidation of the N-terminal serine residue

[reaction in text] A thiol linker-attached peptide was prepared from a nonprotected peptide via an N(alpha)()-alpha-oxoacyl peptide. Selective oxidation of the N-terminal serine with sodium periodate gave the N(alpha)-glyoxyloyl peptide, reductive amination of which with 4,5-dimethoxy-2-(triphenylmethylthio)benzylamine gave an N(alpha)-4,5-dimethoxy-2-mercaptobenzyl glycyl peptide after removal of the trityl group. The N(alpha)-4,5-dimethoxy-2-mercaptobenzyl peptide can be condensed with a peptide thioester, and the linker is removable. This strategy provides a useful method for the synthesis of peptides using recombinant proteins.     

Semisynthesis of Biologically Active Glycoforms of the Human Cytokine Interleukin 6

Human interleukin 6 (IL‐6) is a potent cytokine with immunomodulatory properties. As the influence of N‐glycosylation on the in vivo activities of IL‐6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL‐6 was established by sequential native chemical ligation. The four cysteines of IL‐6 are convenient for ligations and require only the short synthetic glycopeptide 43–48. The Cys‐peptide 49–183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1–42 was accessible by the simultaneous cleavage of two inteins, leading to the 1–42 thioester with the native N‐terminus. Ligation and refolding studies showed that the inherently labile Asp? Pro bond 139–140 was detrimental for the sequential C‐ to N‐terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full‐length IL‐6 glycoproteins, which showed full bioactivity after efficient refolding and purification.     

Chemical synthesis of 23 kDa glycoprotein by repetitive segment condensation: a synthesis of MUC2 basal motif carrying multiple O-GalNAc moieties

Peptide thioester corresponding to a MUC2 tandem repeat unit, which retains seven GalNAc moieties, was prepared by the Fmoc method followed by the low TfOH treatment to remove benzyl groups at the carbohydrate portions. The glycosylated peptide thioester was then consecutively joined by the activation of a thioester group by silver ions to obtain a MUC2 tandem repeat model composed of 141 amino acids with 42 GalNAc moieties.     

SiO2–poly(amidoamine) dendrimer inorganic/organic hybrids

SiO₂–poly(amidoamine) (PAMAM) dendrimer hybrids were synthesized via (1) a Michael addition reaction between the dendrimer and 3‐(trimethoxysilyl) propyl acrylate, (2) the dissolution of the formed compound in methanol, and (3) the mixing of the latter solution with a methanol solution of partly hydrolyzed tetraethylorthosilicate (TEOS) and its casting on a glass substrate. ¹H NMR indicated that in the first step, 77% of the secondary amines were converted into tertiary amines when the fourth‐generation dendrimer was employed and 46% were converted when the second‐generation dendrimer was used. The final SiO₂–PAMAM dendrimer hybrids were obtained via the hydrolysis and condensation of the compound obtained via the Michael addition and the methanol solution of partly hydrolyzed TEOS. The compartmentalized structure of the hybrids due to the compartments of the dendrimers could be controlled by changing the dendrimer and the amount of TEOS. Scanning electron microscopy and transmission electron microscopy micrographs provided information about the structure of the hybrids. Like the PAMAM dendrimer, the SiO₂–PAMAM dendrimer hybrids exhibited a high metal ion complexing capacity because of the presence of the compartments of the dendrimer; they can be, however, much more easily handled, and, as demonstrated by thermogravimetric experiments, have much higher thermal resistance. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 1443–1449, 2000     

Stereocontrolled synthesis of quinine and quinidine

Disubstituted cyclopentene was prepared from cyclopentene monoacetate and transferred into disubstituted piperidine via oxidative cleavage of the olefin moiety followed by piperidine ring formation. The piperidine was then condensed at the side chain with a quinoline part to afford the olefin precursor of quinine. Finally, the olefin was converted into quinine through the corresponding epoxide. Quinidine was synthesized in a similar way.     

Globular carbohydrate macromolecule “sugar balls”, 2. Synthesis of mono(glycopeptide)-persubstituted dendrimers by polymer reaction with sugar-substituted α-amino acid N-carboxyanhydrides (glycoNCAs)

Sugar-substituted α-amino acid N-carboxyanhydrides (glycoNCAs), i.e., O-(tetra-O-acetyl-β-D -glucopyranosyl)-L -serine NCA (2a ) and O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D -glucopyranosyl)-L -serine NCA (2b ), were successfully used for the introduction of a mono(glycopeptide) unit into each terminal primary amino group of a dendrimer. Well-defined dendrimer-based artificial glycoconjugates, O-(β-D -glucopyranosyl)-L -serine-persubstituted poly(amido amine) (PAMAM) dendrimer (3a ) and O-(2-acetamido-2-deoxy-β-D -glucopyranosyl)-L -serine-persubstituted PAMAM dendrimer (3b ), were synthesized by polymer reaction of PAMAM dendrimer with 2a and 2b , respectively, followed by deacetylation with hydrazine monohydrate.     

Exploring the substrate specificity of OxyB, a phenol coupling P450 enzyme involved in vancomycin biosynthesis

OxyB is a cytochrome P450 enzyme that catalyzes the first oxidative phenol coupling reaction during vancomycin biosynthesis. The preferred substrate is a linear peptide linked as a C-terminal thioester to a peptide carrier protein (PCP) domain of the glycopeptide antibiotic non-ribosomal peptide synthetase. Previous studies have shown that OxyB can efficiently oxidize a model hexapeptide-PCP conjugate (R-Leu(1)-R-Tyr(2)-S-Asn(3)-R-Hpg(4)-R-Hpg(5)-S-Tyr(6)-S-PCP) (Hpg = 4-hydroxyphenylglycine) into a macrocyclic product by phenolic coupling of the aromatic rings in residues-4 and -6. In this work, the substrate specificity of OxyB has been explored using a series of N-terminally truncated peptides related in sequence to this model hexapeptide-PCP conjugate. Deletion of one or three residues from the N-terminus afforded a penta- (Ac-Tyr-Asn-Hpg-Hpg-Tyr-S-PCP) and a tri- (Ac-Hpg-Hpg-Tyr-S-PCP) peptide that were also efficiently transformed into the corresponding macrocyclic cross-linked product by OxyB. The tripeptide, representing the core of the macrocycle in vancomycin created by OxyB, is thus sufficient, as a thioester with the PCP domain, for phenol coupling to occur. The related tetrapeptide-PCP thioester was not cyclized by OxyB, neither was a related model hexapeptide containing tryptophan in place of tyrosine-6, nor were tripeptides (related to the natural product K-13) with the sequence Ac-Tyr-Tyr-Tyr-S-PCP cross-linked by OxyB.