Colorimetric monitoring of solid-phase aldehydes using 2,4-dinitrophenylhydrazine

A simple and rapid method to achieve colorimetric monitoring of resin-bound aldehydes, based on ambient temperature reaction with 2,4-dinitrophenylhydrazine (DNPH) in the presence of dilute acid, has been developed as an adjunct to solid-phase organic synthesis and combinatorial chemistry. By this test, the presence of aldehydes is indicated by a red to dark-orange appearance, within a minute. Alternatively, resins that are free of aldehydes or in which aldehyde functions have reacted completely retain their original color. The DNPH test was demonstrated for poly(ethylene glycol)-polystyrene (PEG-PS), aminomethyl polystyrene (AMP), cross-linked ethoxylate acrylate resin (CLEAR), and acryloylated O,O'-bis(2-aminopropyl)poly(ethylene glycol) (PEGA) supports and gave results visible to the naked eye at levels as low as 18 micromol of aldehyde per gram of resin.     

Solid-Phase Total Synthesis of Bacitracin A

An efficient solid-phase method for the total synthesis of bacitracin A is reported. This work was undertaken in order to provide a general means of probing the intriguing mode of action of the bacitracins and exploring their potential for use against emerging drug-resistant pathogens. The synthetic approach to bacitracin A involves three key features: (1) linkage to the solid support through the side chain of the L-asparaginyl residue at position 12 (L-Asn(12)), (2) cyclization through amide bond formation between the alpha-carboxyl of L-Asn(12) and the side chain amino group of L-Lys(8), and (3) postcyclization addition of the N-terminal thiazoline dipeptide as a single unit. To initiate the synthesis, Fmoc L-Asp(OH)-OAllyl was attached to a PAL resin. The chain of bacitracin A was elaborated in the C-to-N direction by sequential piperidine deprotection/HBTU-mediated coupling cycles with Fmoc D-Asp(OtBu)-OH, Fmoc L-His(Trt)-OH, Fmoc D-Phe-OH, Fmoc L-Ile-OH, Fmoc D-Orn(Boc)-OH, Fmoc L-Lys(Aloc)-OH, Fmoc L-Ile-OH, Fmoc D-Glu(OtBu)-OH, and Fmoc L-Leu-OH. The allyl ester and allyl carbamate protecting groups of L-Asn(12) and L-Lys(8), respectively, were simultaneously and selectively removed by treating the peptide-resin with palladium tetrakis(triphenylphosphine), acetic acid, and triethylamine. Cyclization was effected by PyBOP/HOBT under the pseudo high-dilution conditions afforded by attachment to the solid support. After removal of the N-terminal Fmoc group, the cyclized peptide was coupled with 2-[1'(S)-(tert-butyloxycarbonylamino)-2'(R)-methylbutyl]-4(R)-carboxy-Delta(2)-thiazoline (1). The synthetic peptide was deprotected and cleaved from the solid support under acidic conditions and then purified by reverse-phase HPLC. The synthetic material exhibited an ion in the FAB-MS at m/z 1422.7, consistent with the molecular weight calculated for the parent ion of bacitracin A (MH(+) = C(73)H(84)N(10)O(23)Cl(2), 1422.7 g/mol). It was also indistinguishable from authentic bacitracin A by high-field (1)H NMR and displayed antibacterial activity equal to that of the natural product, thus confirming its identity as bacitracin A. The overall yield for the solid-phase synthesis was 24%.     

Tartric acid-based linker for the solid-phase synthesis of C-terminal peptide alpha-oxo aldehydes

A novel linker, based on the anchoring of (+)-dimethyl 2,3-O-isopropylidene-D-tartrate to PEGA or PEG-PS solid supports, was developed for the solid-phase synthesis of C-terminal peptide alpha-oxo aldehydes. Peptide elongation was performed using the 9-fluorenylmethoxycarbonyl/t-Bu chemistry. The peptide and the 1,2-diol were deprotected on the solid phase. Then, a periodic oxidation of the fully deprotected peptidyl-resin led to the simultaneous cleavage of the product from the solid support and to the generation of the alpha-oxo aldehyde moiety. The methodology allowed the distance between the alpha-oxo aldehyde and the peptide to be easily modulated. The C-terminal peptide alpha-oxo aldehydes synthesized in this study were found to be useful partners in hydrazone, thiazolidine, and oxime chemical ligations.     

A carbamoyl-protective group for tyrosine that facilitates purification of hydrophobic synthetic peptides

The Boc-N-methyl-N-[2-(methylamino)ethyl]carbamoyl group (Boc-Nmec) is reported as a new side chain-protective group for tyrosine in Fmoc solid-phase peptide synthesis. Tyrosine is incorporated into the peptide as Fmoc-Tyr(Boc-Nmec)-OH by standard coupling methods. During the cleavage of the peptide from the resin with TFA the Boc group is simultaneously cleaved while the cationic N-methyl-N-[2-(methylamino)ethyl]carbamoyl group remains attached to the tyrosine residue, thereby increasing the solubility of the peptide. After purification of the peptide, the Nmec protective group can be cleaved under neutral or mild alkaline conditions via an intramolecular cyclization reaction.     

Chemical Synthesis of Proteins with Base-Labile Posttranslational Modifications Enabled by a Boc-SPPS Based General Strategy Towards Peptide C-Terminal Salicylaldehyde Esters

Chemical synthesis of proteins bearing base-labile post-translational modifications (PTMs) is a challenging task. For instance, O-acetylation and S-palmitoylation PTMs cannot survive Fmoc removal conditions during Fmoc-solid phase peptide synthesis (SPPS). In this work, we developed a new Boc-SPPS-based strategy for the synthesis of peptide C-terminal salicylaldehyde (SAL) esters, which are the key reaction partner in Ser/Thr ligation and Cys/Pen ligation. The strategy utilized the semicarbazone-modified aminomethyl (AM) resin, which could support the Boc-SPPS and release the peptide SAL ester upon treatment with TFA/H₂O and pyruvic acid. The non-oxidative aldehyde regeneration was fully compatible with all the canonical amino acids. Armed with this strategy, we finished the syntheses of the O-acetylated protein histone H3(S10ac, T22ac) and the hydrophobic S-palmitoylated peptide derived from caveolin-1.     

Synthesis of cyclic peptides via O-N-acyl migration

We describe here a novel and convenient synthesis of head-to-tail cyclic peptide avoiding racemization. Linear depsipeptides including a serine residue as the key element for ester bond formation and acyl transfer were synthesized on 2-chlorotrityl chloride resin. After cleavage from the resin, intramolecular head-to-tail cyclization was performed in solution by C-terminal activation of urethane protected O-acyl serine residue. After removal of the N^α-serine protecting group, the final step consisted in O-N-acyl migration reaction on the ‘switch’ or ‘click’ element to restore native cyclic peptides.     

Peptide‐Stabilized,Fluorescent Silver Nanoclusters: Solid‐Phase Synthesis and Screening

Few‐atom silver nanoclusters (AgNCs) can exhibit strong fluorescence; however, they require ligands to prevent aggregation into larger nanoparticles. Fluorescent AgNCs in biopolymer scaffolds have so far mainly been synthesized in solution, and peptides have only found limited use compared to DNA. Herein, we demonstrate how solid‐phase methods can increase throughput dramatically in peptide ligand screening and in initial evaluation of fluorescence intensity and chemical stability of peptide‐stabilized AgNCs (P‐AgNCs). 9‐Fluorenylmethyloxycarbonyl (Fmoc) solid‐phase peptide synthesis on a hydroxymethyl‐benzoic acid (HMBA) polyethylene glycol polyacrylamide copolymer (PEGA) resin enabled on‐resin screening and evaluation of a peptide library, leading to identification of novel peptide‐stabilized, fluorescent AgNCs. Using systematic amino acid substitutions, we synthesized and screened a 144‐member library. This allowed us to evaluate the effect of length, charge, and Cys content in peptides used as ligands for AgNC stabilization. The results of this study will enable future spectroscopic studies of these peptide‐stabilized AgNCs for bioimaging and other applications.     

Fmoc solid-phase synthesis of peptide thioesters by masking as trithioortho esters

[reaction: see text] Total chemical synthesis of proteins by chemoselective ligation relies on C-terminal peptide thioesters as building blocks. Their preparation by standard Fmoc solid-phase peptide synthesis is made difficult by the lability of thioesters to aminolysis by the secondary amines used for removal of the Fmoc group. Here we present a novel backbone amide linker (BAL) strategy for their synthesis in which the thioester functionality is masked as a trithioortho ester throughout the synthesis.     

Efficient Chemical Protein Synthesis using Fmoc-Masked N-Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one-pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N-masking group of the N-terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o-aminoanilide. The ready availability of Fmoc-Cys(Trt)-OH, which is routinely used in Fmoc solid-phase peptide synthesis, where the Fmoc group is pre-installed on cysteine residue, minimizes additional steps required for the temporary protection of the N-terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.     

Synthesis of spirohydantoins and spiro-2,5-diketopiperazines via resin-bound cyclic alpha,alpha-disubstituted alpha-amino esters

A seven-step solid-phase synthesis of spirohydantoins and an eight-step solid-phase synthesis of spiro-2,5-diketopiperazines is reported. Key intermediate in the synthesis of both compound libraries is the resin-bound cyclic alpha,alpha-disubstituted alpha-amino ester, which can be obtained after selective homogeneous reduction of the aliphatic nitro ester using tin(II) chloride dihydrate. Nitro ester, in turn, is synthesized by a high-pressure-assisted [4 + 2] cycloaddition of resin-bound nitro alkene and butadiene, whereas nitro alkene is obtained by a Knoevenagel condensation of resin-bound nitro acetate with an imine. Novel spirohydantoins are obtained by isocyanate coupling with the resin-bound amino ester 5, followed by cyclization cleavage using a base. Novel spiro-2,5-diketopiperazines are obtained by PyBOP coupling of a Fmoc-protected amino acid with resin-bound amino ester, followed by Fmoc deprotection and an acid-assisted cyclization cleavage. After preparation of seven different resin-bound alpha,alpha-disubstituted alpha-amino esters, a 7 x 8 compound library of spirohydantoins was synthesized using eight different isocyanates, and a 7 x 8 compound library of spiro-2,5-diketopiperazines was synthesized using eight different Fmoc amino acids.     

Synthesis of monofunctionalized gold nanoparticles by fmoc solid-phase reactions

In this communication, solid-phase reactions for the synthesis of Lys-monofunctionalized gold nanoparticles are described. A controlled and selective fabrication of linear nanoparticle arrays can be achieved through peptide linkage systems, and therefore it is essential to prepare Fmoc amino acid nanoparticle building blocks susceptible to Fmoc solid-phase peptide synthesis. Gold nanoparticles containing carboxylic acids (2) in the organic shell were covalently ligated to Lys on solid supports through amide bond coupling reactions. We employed Fmoc-Lys-substituted polymer resins such as Fmoc-Lys-Wang or Fmoc-Lys-HMPA-PEGA. The low density of Lys on the matrix enabled 2 nm-sized gold nanoparticles to react with Lys in a 1:1 ratio. Subsequent cleavage reactions using 60% TFA reagent resulted in Lys transfer from the solid matrix to gold nanoparticles, and the Fmoc-Lys-monofunctionalized gold nanoparticles (5) were obtained with 3-15% yield. Synthesis using HMPA-PEGA resin increased productivity due to the superior swelling properties of PEGA resin in DMF. Monofunctionalization of nanoparticles was microscopically characterized using TEM for the ethylenediamine-bridged nanoparticle dimers (6). By counting the number of 6, we found that at least 60% of cleaved nanoparticles were monofunctionalized by Lys. This method is highly selective and efficient for the preparation of monofunctionalized nanoparticles.     

4-(9-fluorenylmethyloxycarbonyl)phenylhydrazine (FmPH): a new chromophoric reagent for quantitative monitoring of solid-phase aldehydes(1-3)

A direct method for quantifying solid-phase aldehydes has been developed, using a new reagent, 4-(9-fluorenylmethyloxycarbonyl)phenylhydrazine (FmPH). The FmPH reagent was synthesized in three steps (24% overall yield) from commercially available p-hydrazinobenzoic acid. Resin-bound aldehydes reacted quantitatively with FmPH, in the presence of trimethylorthoformate (TMOF) as a dehydrating agent, to form a highly conjugated, immobilized FmPH-hydrazone. Next, mild treatment of the hydrazone with an excess of piperidine-N,N-dimethylformamide (1:1) released the piperidine-dibenzofulvene adduct chromophore (epsilon(301nm) = 7800 M(-1) cm(-1)) from the support. FmPH quantitation of aldehydes proved to be a straightforward, sensitive, and reproducible technique for monitoring resin-bound aldehydes [albeit insufficiently reactive to allow reliable quantification of ketones]. The FmPH aldehyde assay is applicable to a range of solid supports, as demonstrated specifically for poly(ethylene glycol)-polystyrene (PEG-PS), aminomethylpolystyrene (AMP), and cross-linked ethoxylate acrylate resin (CLEAR).     

Facile synthesis of C-terminal peptide hydrazide and thioester of NY-ESO-1 (A39-A68) from an Fmoc-hydrazine 2-chlorotrityl chloride resin

The NY-ESO-1 (A39-A68) peptide hydrazide was prepared through 9-fluorenyl-methoxycarbonyl solid-phase peptide synthesis (Fmoc SPPS) from a new 9-fluorenyl-methoxycarbonyl hydrazine 2-chlorotrityl chloride (Fmoc-hydrazine 2CTC) resin. The new resin was ideal for long-term storage and usage in Fmoc SPPS. Besides, the title peptide hydrazide could be transformed nearly quantitatively into the corresponding peptide thioester, which was both isolable and usable directly in native chemical ligation (NCL).     

On-resin peptide ligation via C-terminus benzyl ester

Here, we reported a new approach of on-resin peptide ligation using C-terminal benzyl ester as the stabilized precursor of thioester, which enables both N-terminal elongation and C-terminal peptide ligation on a Rink Amide resin.     

On-resin peptide ligation via C-terminus benzvl ester

Here, we report a new approach of on-resin peptide ligation using C-terminal benzyl ester as the stabilized precursor of thioester, which enables both N-terminal elongation and C-terminal peptide ligation on a Rink Amide resin. On-resin native chemical ligation and auxiliary-assisted peptide ligation were successfully achieved. This method is compatible to both protected and unprotected peptide fragments and has potential application in poor water-soluble peptide ligation.     

Efficient Chemical Protein Synthesis using Fmoc‐Masked N‐Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one‐pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N‐masking group of the N‐terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o‐aminoanilide. The ready availability of Fmoc‐Cys(Trt)‐OH, which is routinely used in Fmoc solid‐phase peptide synthesis, where the Fmoc group is pre‐installed on cysteine residue, minimizes additional steps required for the temporary protection of the N‐terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.     

半胱氨酸肽片段连接法合成ω-芋螺毒素MVIIA

ω-芋螺毒素MVIIA是已上市的镇痛药Ziconotide的有效成分.采用标准Fmoc保护策略在聚苯乙烯树脂上合成ω-MVIIA比较困难,是固相合成中的"困难肽".本研究将ω-MVIIA分为N-端15肽硫酯和C-端10肽两个片段采用标准Fmoc保护策略分别合成,再通过半胱氨酸肽片段连接得到全长的ω-芋螺毒素MVIIA肽链.该方法提高了合成ω-芋螺毒素MVIIA产率.该研究为"困难肽"的合成提供了较好的参考方法.     

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).     

Synthesis of cyclic peptides through hydroxyl side-chain anchoring

A general method was developed for the synthesis of serine or threonine containing cyclic peptides utilizing the β-hydroxyl side-chain of these residues as an anchor point to Wang resin. The peptide chain was assembled by conventional Fmoc/tBu solid-phase chemistry followed by palladium catalyzed exposure of the allyl protected C-terminus group and on-resin cyclization. The cyclic heptapeptide stylostatin 1 was prepared to demonstrate the utility of this technique.     

A new approach of characterizing the hydrolytic degradation of poly(ethylene terephthalate) by MALDI-MS

The hydrolytic degradation of technical poly(ethylene terephthalate) (PET) was investigated by means of different methods such as size-exclusion chromatography (SEC), viscometry, light-scattering, thin-layer chromatography, end-group titration, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The long-term degradation was simulated by exposing PET filament yarns to aqueous neutral conditions at 90°C for up to 18 weeks. By means of MALDI-MS and thin-layer chromatography, the formation of different oligomers was obtained during polymer degradation. As expected, an ester scission process was found generating acid terminated oligomers (H-[GT]_m-OH) and T-[GT]_m-OH and ethylene glycol terminated oligomers (H-[GT]_m-G), where G is an ethylene glycol unit and T is a terephthalic acid unit. Additionally, the scission of the ester bonds during the chemical treatment led to a strong decrease in the number of cyclic oligomers ([GT]_m). The occurrence of di-acid terminated species demonstrated a high degree of degradation. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35 : 2183–2192, 1997