Full orthogonality between Dde and Fmoc: the direct synthesis of PNA--peptide conjugates

New deprotection conditions for the Dde amine protecting group that are fully orthogonal to Fmoc are described and successfully applied to the dual synthesis of PNA-peptide conjugates.     

Synthesis and cellular uptake of cell delivering PNA-peptide conjugates

The synthesis and cellular uptake of fluorescently labelled PNA-peptide conjugates is described; Dde/Mmt protected PNA monomers, fully orthogonal to Fmoc chemistry, were used to develop a flexible strategy to give Peptide Nucleic Acids conjugated to tri- and hepta-arginine and the short basic Tat(48-57) peptide as examples of cellular penetrating peptides, thereby allowing efficient cellular delivery of PNA into cells.     

Rapid and high-yield solution-phase synthesis of DOTA-Tyr-octreotide and DOTA-Tyr-octreotate using unprotected DOTA

An improved method for the solution-phase derivatization of Tyr³-Lys⁵(Dde)-octreotide (TOC(Dde)) and Tyr³-Lys⁵(Dde)-octreotate (TATE(Dde)) with the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-N′,N″,N?,N?-tetraacetic acid) has been developed. The fully protected parent peptides were assembled via solid-phase peptide synthesis (SPPS) using Fmoc-strategy. After cleavage from the solid support, disulfide bond formation was carried out using H₂O₂. Both TOC(Dde) and TATE(Dde) were successfully coupled with DOTA in the presence of NHS, EDCI and DIPEA in a water/DMF solvent system. Yields of the coupling reaction were >98% within only 2 h with no detectable formation of sideproducts. This method for the preparation of DOTATOC, DOTATATE and other DOTA-peptide conjugates is therefore a rapid and economic alternative to the currently used methods.     

Solid-phase synthesis of asymmetrically branched sequence-defined poly/oligo(amidoamines)

We present for the first time the synthesis of asymmetrically branched sequence-defined poly/oligo(amidoamines) (PAAs) using solid-phase synthesis with the capability of introducing diversity at the side chains. We introduce two new versatile (diethylenetriamine) building blocks for solid-phase synthesis bearing Fmoc/Boc and Fmoc/Alloc protecting groups expanding recently used Fmoc/Boc protecting group strategy for linear PAAs to an Fmoc/Alloc/Boc strategy. This allows for orthogonal on-resin cleavage of Fmoc and Alloc protecting groups during solid-phase synthesis of PAAs with backbones differing in chain length and sequence. With these structures we then demonstrate the potential for generating asymmetrical branching by automated multiple on-resin cleavage of Alloc protecting groups as well as the introduction of side chains varying in length and number. Such systems have high potential as nonviral vectors for gene delivery and will allow for more detailed studies on the correlation between the degree of branching of PAAs and their resulting biological properties.     

Hydrazine hydrate: A new reagent for Fmoc group removal in solid phase peptide synthesis

In solid-phase peptide synthesis using the Fmoc/tBu strategy (SPPS-Fmoc/tBu), an orthogonal protection scheme of amino acids is used; specifically, the alpha-amine group is protected by the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which is removed by weak bases, while side chains are protected by groups that are acid labile. We demonstrated that hydrazine hydrate is an efficient reagent for eliminating the Fmoc group in SPPS-Fmoc/tBu. First, experimental conditions were established for Fmoc group removal from Fmoc-Val-OH in solution. It was determined that the Fmoc group was completely removed with 16% hydrazine hydrate in DMF after 60?min at rt. Second, SPPS-Fmoc/tBu using hydrazine hydrate for Fmoc group removal was standardized. The Fmoc group removal was completed using 16% hydrazine hydrate in DMF for 10?min at rt (twice). When the reaction of Fmoc group removal was microwave-assisted, the reaction only required 30?s to efficiently remove the Fmoc group in SPPS-Fmoc/tBu. The method reported here can be routinely used, and it is equivalent to conventional SPPS-Fmoc/tBu methodologies where 4-methylpiperidine or piperidine is used.     

A convenient route to N-[2-(Fmoc)aminoethyl]glycine esters and PNA oligomerization using a Bis-N-Boc nucleobase protecting group strategy

A simple and practical synthesis of the benzyl, allyl, and 4-nitrobenzyl esters of N-[2-(Fmoc)aminoethyl]glycine is described starting from the known N-(2-aminoethyl)glycine. These esters are stored as stable hydrochloride salts and were used in the synthesis of peptide nucleic acid monomers possessing bis-N-Boc-protected nucleobase moieties on the exocyclic amino groups of ethyl cytosin-1-ylacetate, ethyl adenin-9-ylacetate and ethyl (O(6)-benzylguanin-9-yl)acetate. Upon ester hydrolysis, the corresponding nucleobase acetic acids were coupled to N-[2-(Fmoc)aminoethyl]glycine benzyl ester or to N-[2-(Fmoc)aminoethyl]glycine allyl ester in order to retain the O(6) benzyl ether protecting group of guanine. The Fmoc/bis-N-Boc-protected monomers were successfully used in the Fmoc-mediated solid-phase peptide synthesis of mixed sequence 10-mer PNA oligomers and are shown to be a viable alternative to the currently most widely used Fmoc/Bhoc-protected peptide nucleic acid monomers.     

An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis

An amine-derivatized DOTA has been used to modify the surface of a polymeric support for conventional solid phase peptide synthesis (SPPS) following standard Fmoc chemistry methods. This methodology was used to synthesize a peptide-DOTA conjugate that was demonstrated to be a PARACEST MRI contrast agent. Therefore, this synthesis methodology can facilitate Fmoc SPPS of molecular imaging contrast agents.     

Facile synthesis of cyclopentapeptide,cyclo[Arg(NO2)-Gly-Asp(OBn)-D-Phe-Lys(Fmoc)], and its application in synthesis of integrin-targeting anticancer conjugate

An efficient approach for integrin-targeting cRGDfK conjugate synthesis has been developed using a new protected cyclopentapeptide, cR(NO₂)GD(Bn)fK(Fmoc), as the key intermediate. cR(NO₂)GD(Bn)fK(Fmoc) was conveniently prepared in high yield. The Fmoc group of this cyclopentapeptide was selectively removed under mild conditions which makes it an ideal intermediate for cRGDfK conjugate synthesis as was well demonstrated in this paper by the synthesis of cRGDfK chlorambucil conjugate.     

Practical and Efficient Synthesis of Orthogonally Protected α‐2,3‐Diaminopropionic Acid (2,3‐Dap), 2,4‐Diaminobutanoic Acid (2,4‐Dab), and their N‐Methylated Derivatives

The synthesis of orthogonally protected Fmoc‐Dap/Dab (Boc/Z/Alloc)‐OH starting from Fmoc‐Asp/Glu has been described. The salient features of our synthetic strategy involved formation of Fmoc‐Asp/Glu‐5‐oxazolidinone acids, conversion of acid function to acyl azides, Curtius rearrangement, and hydrolysis of the oxazolidinone group.     

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%.     

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.     

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 and characterization of (R)-miniPEG-containing chiral γ-peptide nucleic acids using the Fmoc strategy

Diethylene glycol (miniPEG)-containing chiral γPNA is considered to be one of the best PNA derivatives. Its preparation is mainly based on the Boc strategy for solid phase peptide synthesis (SPPS), requiring the repeated use of trifluoroacetic acid TFA, which is not suitable for the in situ synthesis of PNA arrays and some other applications under mild conditions. Herein, Fmoc/Cbz orthogonal protected miniPEG-containing chiral γPNA monomers were synthesized, and a 15mer γPNA was prepared using the Fmoc strategy under mild conditions.     

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.     

Incorporation of pseudoproline derivatives allows the facile synthesis of human IAPP, a highly amyloidogenic and aggregation-prone polypeptide

The efficient Fmoc solid-phase peptide synthesis of the 37-residue human Amylin and its amyloidogenic 8-37 fragment was achieved using pseudoproline (oxazolidine) dipeptide derivatives. Syntheses of hAmylin(8-37) using Fmoc amino acids produced only traces of the desired peptide. Incorporation of pseudoproline dipeptides produced the desired product with high yield and allowed for the synthesis of the full length peptide. The crude material was pure enough to allow formation of the Cys-2 to Cys-7 disulfide by air oxidation. [Structure: see text]     

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.     

One‐Pot/Sequential Native Chemical Ligation Using N‐Sulfanylethylanilide Peptide

N‐Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide thioesters by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N‐terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three‐fragment or unprecedented four‐fragment ligation for efficient one‐pot peptide/protein synthesis. Furthermore, dual‐kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto‐peptide thioesters. DOI 10.1002/tcr.201200007     

Enantioselective Solid‐Phase Peptide Synthesis Using Traceless Chiral Coupling Reagents and Racemic Amino Acids

The enantioselective condensing reagent 4,6‐dimethoxy‐1,3,5‐triazine (DMT)/strychnine/BF$\rm{{_{4}^{-}}}$ was obtained by treatment of 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) with strychnine tetrafluoroborate. The reagent was useful under typical conditions of solid‐phase peptide synthesis (SPPS) with enantiomerically homogeneous substrates. By SPPS, desired dipeptides were obtained in 84–94% yield using 4 equiv. of racemic Fmoc‐Ala, Fmoc‐Phe, and/or Fmoc‐Tyr for 1 equiv. of Wang resin loaded with Gly, Ala, Leu, Phe, Glu(^tBu), and/or Pro, respectively. For all three Fmoc‐protected amino acids, the configuration of the enantiomer preferred under SPPS conditions was independent of the structure of the acylated component and identical to that established in condensations proceeding in solution. In all cases, the enantiomer ratios L /D (er) were in a similar range, and varied from 9 : 92 to 2 : 98 for alanine, and from 90 : 10 to 100 : 0 for aromatic amino acids. The synthesis of Ac‐L ‐Lys(Ac)‐D ‐Ala‐D ‐Ala‐OH from racemic Fmoc‐Ala gave an L /D ratio of 10 : 90 for the esterification of Wang resin, and 0 : 100 for the formation of peptide bonds.     

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).     

Self‐Assembled Peptide‐Based Hydrogels as Scaffolds for Proliferation and Multi‐Differentiation of Mesenchymal Stem Cells

Fluorenyl‐9‐methoxycarbonyl (Fmoc)‐diphenylalanine (Fmoc‐FF) and Fmoc‐arginine‐glycine‐­aspartate (Fmoc‐RGD) peptides self‐assemble to form a 3D network of supramolecular hydrogel (Fmoc‐FF/Fmoc‐RGD), which provides a nanofibrous network that uniquely presents bioactive ligands at the fiber surface for cell attachment. In the present study, mesenchymal stem cells (MSCs) in Fmoc‐FF/Fmoc‐RGD hydrogel increase in proliferation and survival compared to those in Fmoc‐FF/Fmoc‐RGE hydrogel. Moreover, MSCs encapsulated in Fmoc‐FF/Fmoc‐RGD hydrogel and induced in each defined induction medium undergo in vitro osteogenic, adipogenic, and chondrogenic differentiation. For in vivo differentiation, MSCs encapsulated in hydrogel are induced in each defined medium for one week, followed by injection into gelatin sponges and transplantation into immunodeficient mice for four weeks. MSCs in Fmoc‐FF/Fmoc‐RGD hydrogel increase in differentiation into osteogenic, adipogenic, and chondrogenic differentiation, compared to those in Fmoc‐FF/Fmoc‐RGE hydrogel. This study concludes that nanofibers formed by the self‐assembly of Fmoc‐FF and Fmoc‐RGD are suitable for the attachment, proliferation, and multi‐differentiation of MSCs, and can be applied in musculoskeletal tissue engineering.