Fmoc-based Solid-phase Synthesis Research Articles

Synthesis and properties of PNA containing a dicationic nucleobase based on N4-benzoylated cytosine

We report the synthesis of a peptide nucleic acid (PNA) monomer containing N4-bis(aminomethyl)benzoylated cytosine (BzC2+ base). The BzC2+ monomer was incorporated into PNA oligomers using Fmoc-based solid-phase synthesis. The BzC2+ base in PNA had two positive charges and exhibited greater affinity for DNA G base than the natural C base. The BzC2+ base stabilized PNA-DNA heteroduplexes through electrostatic attractions, even in high salt conditions. The two positive charges on the BzC2+ residue did not compromise the sequence specificity of PNA oligomers. These insights will aid the future design of cationic nucleobases.

Total Synthesis and Bioactivity Evaluation of Hydrophobic Microcionamide-Inspired Peptides.

In this report, we describe the facile synthesis of four microcionamide-inspired peptides where the atypical 2-phenylethylenamine (2-PEA) functional group in the marine natural product, microcionamide A, was replaced with a similarly-aromatic but more easily incorporated tryptophan (Trp) residue. Compounds 1-4 were synthesized using a standard Fmoc-based solid-phase synthesis strategy followed by iodine-mediated on-resin cyclization for disulfide-bridged compounds 1-3. Compound 1 showed antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa, with minimum inhibitory concentrations (MICs) of 9.1 μM and 15 μM, respectively. The inactivity of alanine analogs 2-4 against these pathogens suggests that the N-terminal Val, the cyclic scaffold, the contiguous Ile residues, and consequently, the hydrophobicity of compound 1 are essential for antibacterial activity. Compound 1 also favorably exhibited minimal cytotoxicity against normal mammalian cell lines. In summary, we have synthesized an analog of microcionamide A where replacement of the 2-PEA moiety with a Trp residue retained the antibacterial activity and with favorably low cytotoxicity.

Total synthesis of chaiyaphumines A-D: A case study comparing macrolactonization and macrolactamization approaches

A comparison of macrolactonization and macrolactamization approaches to the synthesis of the pentadepsipeptide chaiyaphumines is outlined. Fmoc-based solid-phase synthesis approaches were used for the synthesis of the requisite precursors. Attempted solution-phase macrolactonization of a seco-acid precursor using Yamaguchi, pivaloyl chloride, or MNBA activation did not give cyclized products. The use of an MNBA / lanthanide(III) triflate mediated macrolactonization protocol led to an isomeric macrocycle. However, the use of a solution-phase macrolactamization approach using EDC·HCl / HOBt was achieved without detectable epimerization. This strategy was successfully applied to the total synthesis of the antimalarial compound chaiyaphumine A, as well as the natural products chaiyaphumine B, C, and D, by using a late-stage acylation of the threonine amino group. An X-ray crystal structure analysis of synthetic chaiyaphumine D reveals the presence of a type II’ beta turn and a type IV4 beta turn motif embedded in the 16-membered depsipeptide ring. This study illustrates potential challenge associated with macrolactonization approaches to depsipeptides compared to macrolactamization.

Total Synthesis and Functional Analysis of Complex Peptidic Natural Products and Their Artificial Analogues

This review focuses on a new solid-phase synthetic strategy for an anticancer natural product yaku'amide B (1) and its target identification and structure-function relationship study using synthetic analogues and probes. To realize the Fmoc-based solid-phase synthesis of 1, we developed new synthetic methods for enamide formation. Namely, modified traceless Staudinger ligation using alkenyl azides and newly designed phosphinophenol esters enabled stereoselective construction of the (E)- and (Z)-ΔIle moieties. Furthermore, resin-cleavage and C-terminus modification were simultaneously achieved with an ester-amide exchange reaction using C-terminal amine and AlMe3, which successfully afforded 1 via a full solid-phase route. The developed strategy was applied to the construction of seven E/Z isomers of 1. In the target identification of 1, fluorescent imaging study and affinity pull-down assay using the synthetic probes revealed that 1 exerts potent cytostatic activity by binding to subunits α and β of mitochondrial FoF1-ATP synthase. On the basis of the mode of action of 1, we conducted biological evaluation of the seven E/Z-isomers of 1. Assessment of growth inhibition activity and the effect on FoF1-ATP synthase indicates that the E/Z-stereochemistry of the three ΔIle residues controls the magnitude of biological functions of 1.

Solid-Phase Total Synthesis of Yaku'amide B Enabled by Traceless Staudinger Ligation.

We report a solid-phase strategy for total synthesis of the peptidic natural product yaku'amide B (1), which exhibits antiproliferative activity against various cancer cells. Its linear tridecapeptide sequence bears four β,β-dialkylated α,β-dehydroamino acid residues and is capped with an N-terminal acyl group (NTA) and a C-terminal amine (CTA). To realize the Fmoc-based solid-phase synthesis of this complex structure, we developed new methods for enamide formation, enamide deprotection, and C-terminal modification. First, traceless Staudinger ligation enabled enamide formation between sterically encumbered alkenyl azides and newly designed phosphinophenol esters. Second, application of Eu(OTf)3 led to chemoselective removal of the enamide Boc groups without detaching the resin linker. Finally, resin-cleavage and C-terminus modification were simultaneously achieved with an ester-amide exchange reaction using CTA and AlMe3 to deliver 1 in 9.1 % overall yield (24 steps from the resin).

A single Asp isomer substitution in an αA-crystallin-derived peptide induces a large change in peptide properties

The eye lens is mainly composed of crystallins, which undergo modifications such as oxidation, deamidation and isomerization with aging. Asp58, Asp76, Asp84, and Asp151 residues of αA-crystallin are site-specifically isomerized to L-iso, D-, and D-iso isomers in aged-related cataract lenses. In addition, an αA66–80 peptide, corresponding to the 66–80 (66SDRDKFVIFLDVKHF80) fragment of human αA-crystallin, is detected in aged lens. This peptide induces protein aggregation and causes loss of the chaperone function of α-crystallin. The αA66–80 peptide contains Asp76, but it is not known whether isomerization of Asp76 in αA66–80 specifically induces protein aggregation or affects α-crystallin function. Using Fmoc-based solid-phase synthesis, here we synthesized four αA66–80 peptides, each containing L-, L-iso, D-, or D-isoAsp at position 76, and compared their structures and properties. Normal αA66–80 peptide containing the L-Asp76 isomer increased the EDTA-induced aggregation of ADH protein, DTT-induced aggregation of insulin, and heat-induced aggregation of βL-crystallin. αA66–80 peptide containing D- or D-isoAsp76 had similar or no effects on the aggregation of these proteins. By contrast, αA66–80 peptide containing L-isoAsp76 inhibited the aggregation of all three proteins, indicating that it has chaperone activity. With regard to secondary structure, αA66–80 peptide containing the L-, D-, or D-isoAsp76 isomer had random-coil structure, whereas αA66–80 peptide containing L-isoAsp76 had β-sheet like structure. A Thioflavin T (ThT) assay indicated that only the L-isoAsp-containing αA66–80 peptide has β-sheet structure and generates amyloid fibrils. Collectively, these observations indicate that isomerization of Aps76 to the Lβ isomer endows β-sheet structure and chaperone function on this peptide.

Emerging Peptide Science in Canada

Emerging Peptide Science in Canada

Properties of novel surfactin-derived biosurfactants obtained through solid-phase synthesis.

Eight molecules, four peptides (SPs) and four lipopeptides (LPs) derived by rational design from surfactin, a well-known secreted biosurfactant from Bacillus subtilis, were produced employing Fmoc-based solid-phase synthesis. These new peptides were tested to evaluate their potential biosurfactant and biological activities, aiming at possible applications in industrial, biological, pharmaceutical, and medical use. Five molecules (SP1, SP2, SP4, LP5, and LP8) presented potential for medical uses, mainly due to their drug delivery properties as suggested by their synergistic activity with the antibiotic vancomycin against Staphylococcus aureus. All synthetic peptides showed low toxicity against Vero cell cultures, in assays of hemolysis, and in different cytotoxicity assays. In addition, we found that three peptides (SP1, LP6, and LP7) had potential technological and industrial use because of their emulsifying capacity, low toxicity, and ability to physically stabilize solutions. These novel molecules retained some properties of the parental molecule (surfactin, which was originally obtained through nonribosomal synthesis in Bacillus subtilis) but have the advantage of being linear peptides, which can be produced at large scales through the use of conventional heterologous protein expression protocols.

Improving the Imaging Contrast of 68Ga-PSMA-11 by Targeted Linker Design: Charged Spacer Moieties Enhance the Pharmacokinetic Properties.

68Ga-Glu-urea-Lys-(Ahx)-HBED-CC (68Ga-PSMA-11) represents a successful radiopharmaceutical for PET/CT imaging of prostate cancer. Further optimization of the tumor-to-background contrast might significantly enhance the sensitivity of PET/CT imaging and the probability of detecting recurrent prostate cancer at low PSA values. This study describes the advantage of histidine (H)/glutamic acid (E) and tryptophan (W)/glutamic acid (E) containing linkers on the pharmacokinetic properties of 68Ga-PSMA-11. The tracers were obtained by a combination of standard Fmoc-based solid-phase synthesis and copper(I)-catalyzed azide-alkyne cycloaddition. Their 68Ga complexes were compared to the clinical reference 68Ga-PSMA-11 with respect to cell binding, effective internalization, and tumor targeting properties in LNCaP-bearing balb/c nu/nu mice. The introduction of (HE)i (i = 1-3) or (WE)i (i = 1-3) into PSMA-11 resulted in a significantly changed biodistribution profile. The uptake values in kidneys, spleen, liver, and other background organs were reduced for (HE)3 while the tumor uptake was not affected. For (HE)1 the tumor uptake was significantly increased. The introduction of tryptophan-containing linkers also modulated the organ distribution profile. The results clearly indicate that histidine is of essential impact in order to improve the tumor-to-organ contrast. Hence, the histidine/glutamic acid linker modifications considerably improved the pharmacokinetic properties of 68Ga-PSMA-11 leading to a reduced uptake in dose limiting organs and a significantly enhanced tumor-to-background contrast. Glu-urea-Lys-(HE)3-HBED-CC represents a promising 68Ga complex ligand for PET/CT-imaging of prostate cancer.

PNA monomers fully compatible with standard Fmoc-based solid-phase synthesis of pseudocomplementary PNA

Here we report the synthesis of new PNA monomers for pseudocomplementary PNA (pcPNA) that are fully compatible with standard Fmoc chemistry. The thiocarbonyl group of the 2-thiouracil (sU) monomer was protected with the 4-methoxy-2-methybenzyl group (MMPM), while the exocyclic amino groups of diaminopurine (D) were protected with Boc groups. The newly synthesized monomers were incorporated into a 10-mer PNA oligomer using standard Fmoc chemistry for solid-phase synthesis. Oligomerization proceeded smoothly and the HPLC and MALDI-TOF MS analyses indicated that there was no remaining MMPM on the sU nucleobase. The new PNA monomers reported here would facilitate a wide range of applications, such as antigene PNAs and DNA nanotechnologies.

Native Chemical Ligation Strategy to Overcome Side Reactions during Fmoc-Based Synthesis of C-Terminal Cysteine-Containing Peptides.

The Fmoc-based solid phase synthesis of C-terminal cysteine-containing peptides is problematic, due to side reactions provoked by the pronounced acidity of the Cα proton of cysteine esters. We herein describe a general strategy consisting of the postsynthetic introduction of the C-terminal Cys through a key chemoselective native chemical ligation reaction with N-Hnb-Cys peptide crypto-thioesters. This method was successfully applied to the demanding peptide sequences of two natural products of biological interest, giving remarkably high overall yields compared to that of a state of the art strategy.

Solid phase synthesis of ω-aspartic thioacid containing peptides

The Fmoc-based solid phase synthesis of unprotected ω-aspartic thioacid containing peptides is demonstrated. The method involves the novel use of a silyl ester as a carboxylate surrogate for mild peptide bond formation in the presence of a reactive ω-aspartyl thioester. The resulting peptide thioacids may be used in N-glycoligation and other thioacid-mediated conjugation reactions.

ChemInform Abstract: Backbone Amide Linker Strategy: Protocols for the Synthesis of C‐Terminal Peptide Aldehydes

AbstractReview: 22 refs.

Interplay among Subunit Identity, Subunit Proportion, Chain Length, and Stereochemistry in the Activity Profile of Sequence-Random Peptide Mixtures

Fmoc-based solid-phase synthesis methodology was used to prepare peptide mixtures containing one type of hydrophobic residue and one type of cationic residue. Each mixture was random in terms of sequence but highly controlled in terms of length. Analysis of the antibacterial and hemolytic properties of these mixtures revealed that selective antibacterial activity can be achieved with heterochiral binary mixtures but not homochiral binary mixture, if the proper amino acid residues are used.

Enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin

BackgroundMicrobial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity. MethodsUsing the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured. ResultsA peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26−Α29), and K4N Δ(K12-Q18; Ν26−Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25μM and 5 to 25μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900μM for their EC50, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells. ConclusionsThese peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections. General significanceOur results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.

Synthesis of a protected derivative of (2R,3R)-β-hydroxyaspartic acid suitable for Fmoc-based solid phase synthesis

Synthesis of d-threo-hydroxyaspartic acid, orthogonally protected and compatible with an Fmoc solid-phase peptide synthesis strategy is reported. This synthetic procedure starting from (2R,3R)-dimethyltartrate is adaptable to a multi-gram scale. 2,2-Dimethyl-5-oxo-1,3-dioxazolane formation between the β-hydroxy alcohol and the β-carboxylic functions constitutes a key step of the differentiation of the two acidic functions allowing the preparation of (2R,3R)-Fmoc-β-hydroxyaspartic α-allyl ester.

Fmoc-based solid-phase synthesis of adenylylated peptides using diester-type adenylylated amino acid derivatives

Phosphodiester-type adenylylated (AMPylated) Ser, Thr, and Tyr derivatives were developed for Fmoc solid phase peptide synthesis of AMPylated peptides. One-pot/sequential reaction consisting of condensation of an N-nonprotected adenosine derivative and Fmoc-Ser/Thr/Tyr-OAllyl using allyl-N,N-diisopropylchlorophosphoramidite and subsequent oxidation with m-chloroperbenzoic acid gave phosphotriester-type AMPylated Ser/Thr/Tyr derivatives. After Pd(0)-mediated deprotection of allyl groups, the resulting phosphodiester-type AMPylated Ser/Thr/Tyr derivatives were successfully incorporated into peptides by standard Fmoc solid phase peptide synthesis without significant side reactions including dehydroalanine formation.

A stereocontrolled construction of 2-azido-2-deoxy-1,2-cis-α-galactosidic linkages utilizing 2-azido-4,6-O-benzylidene-2-deoxygalactopyranosyl diphenyl phosphates: stereoselective synthesis of mucin core 5 and core 7 structures

TMSOTf-promoted glycosidation of 2-azido-4,6-O-benzylidene-2-deoxygalactosyl diphenyl phosphates with fluorenylmethoxycarbonyl (Fmoc)-protected serine and threonine derivatives in THF/Et2O (1:1) gave glycosyl amino acids in high yields and with excellent levels of α-selectivity (α/β=94:6–95:5). The synthetic utility of the present glycosidation method was demonstrated by a stereoselective synthesis of mucin-type glycopeptide core 5 and core 7 building blocks, which are suitable for Fmoc-based solid-phase synthesis of O-glycopeptides.

ChemInform Abstract: 9‐Fluorenylmethoxycarbonyl‐Based Solid‐Phase Synthesis of Peptide α‐Thioesters

AbstractReview: ca. 80 refs.

Fmoc-SPPS chemistry compatible approach for the generation of (glyco)peptide aryl thioesters

A new approach is described for the general Fmoc-based solid-phase synthesis of (glyco)peptide aryl thioesters. A peptide alkyl oxoester obtained by standard Fmoc-based chain elongation undergoes an O-to- S acyl shift, and is followed by alkyl thioester exchanges with a large excess of aryl thiol, affording the corresponding peptide aryl thioester. The newly developed methodology is useful for the chemical synthesis of post-translationally modified proteins because of its compatibility with standard Fmoc-SPPS conditions. In addition, the peptide aryl thioesters are essential intermediates for chemical synthesis of proteins by kinetically controlled convergent strategy.