The potential of N-alkoxymethyl groups as peptide backbone protectants

In this study, the potential of N-alkoxymethyl groups as protectants for the peptide backbone has been investigated. These groups were found to be compatible with the standard conditions of Fmoc/^tBu SPPS, and can be cleaved off from the peptide backbone by acids. Thus, backbone N-alkoxymethyl groups may be useful to prevent undesired side-reactions and/or interchain aggregation during peptide elongation on the solid-phase. However, the main issue for their application as protecting groups is the difficulty to incorporate them into the peptide backbone.     

Synthesis of Cyclic Peptides in SPPS with Npb-OH Photolabile Protecting Group

Significant efforts have been made in recent years to identify more environmentally benign and safe alternatives to side-chain protection and deprotection in solid-phase peptide synthesis (SPPS). Several protecting groups have been endorsed as suitable candidates, but finding a greener protecting group in SPPS has been challenging. Here, based on the 2-(o-nitrophenyl) propan-1-ol (Npp-OH) photolabile protecting group, a structural modification was carried out to synthesize a series of derivatives. Through experimental verification, we found that 3-(o-Nitrophenyl) butan-2-ol (Npb-OH) had a high photo-release rate, high tolerance to the key conditions of Fmoc-SPPS (20% piperidine DMF alkaline solution, and pure TFA acidic solution), and applicability as a carboxyl-protective group in aliphatic and aromatic carboxyl groups. Finally, Npb-OH was successfully applied to the synthesis of head–tail cyclic peptides and side-chain–tail cyclic peptides. Moreover, we found that Npb-OH could effectively resist diketopiperazines (DKP). The α-H of Npb-OH was found to be necessary for its photosensitivity in comparison to 3-(o-Nitrophenyl)but-3-en-2-ol (Npbe-OH) during photolysis-rate verification.     

Solid phase synthesis of two muramyl pentapeptide derivatives

Solid phase peptide synthesis (SPPS) of two selected muramyl pentapeptide derivatives is described. The simplicity of removing the protecting groups via one-step deprotection and cleavage from the resin is the biggest advantage of SPPS. Using this method, two muramyl pentapeptide derivatives, D-MurN₃-L-Ala-D-iGlu-L-Lys-D-Ala-D-Ser (5) and D-MurN₃-L-Ala-D-iGlu-L-Lys-D-Ala-D-Ala (6), were obtained. Their chemical structures were confirmed by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy. To determine the absolute configuration of the carbon atom in the side chain of the muramic acid derivative, single-crystal X-ray diffraction measurements were recorded.     

Nin-cyclohexyloxycarbonyl group as a new protecting group for tryptophan

Several new protecting groups were introduced at the Nⁱⁿ-position of tryptophan, and their reactivities were examined under the conditions used for peptide synthesis by Boc-strategy. Among them, the cyclohexyloxycarbonyl (Hoc) group was found to be the most suitable in terms of stability during elongation of the peptide chain and removability at the final HF reaction without resorting to the use of thiols.     

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.     

A new protecting group for tryptophan in solid-phase peptide synthesis which protects against acid-catalyzed side reactions and facilitates purification by HPLC

The indole nucleus of Z-Trp-OBzl is modified by acylation of the indole nitrogen using Boc-N-methyl butyric acid followed by catalytic hydrogenation and introduction of the Fmoc group. The resulting derivative, Fmoc-Trp(Boc-Nmbu)-OH, is incorporated into peptide chains via solid-phase peptide synthesis (SPPS). After assembly of the peptide chain, the Boc group is cleaved by treatment with TFA. The peptide is isolated with the tryptophan residue modified with a cationic 4-(N-methylamino) butanoyl group, which improves the solubility of the peptide during HPLC purification. On treatment of the purified peptide at pH 9.5, the Nmbu group undergoes an intramolecular cyclization reaction; this results in the fully deprotected peptide and N-methylpyrrolidone.     

Photoprotected Peptide α‐Ketoacids and Hydroxylamines for Iterative and One‐Pot KAHA Ligations: Synthesis of NEDD8

The convergent synthesis of proteins by multiple ligations requires segments protected at the N‐ and/or C‐terminus with masking groups that are orthogonal to the acid‐ and base‐labile protecting groups used in Fmoc‐SPPS. They must be stable to solid‐phase peptide synthesis, HPLC purification, and ligation conditions and easily removed in the presence of unprotected side chains. In this report, we document photolabile protecting groups for both α‐ketoacids and hydroxylamines, the key functional groups employed in the α‐ketoacid–hydroxylamine (KAHA) ligation. The novel photoprotected α‐ketoacid is easily installed onto numerous different C‐terminal peptide α‐ketoacids and removed by UV light under aqueous conditions. These advances were applied to the one‐pot synthesis of NEDD8, an important modifier protein, by three different convergent routes. These new protecting groups provide greater flexibility on the order of fragment assembly and reduce the number of reaction and purification steps needed for protein synthesis with the KAHA ligation.     

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.     

Amino acid bromides: their utilization for difficult couplings in solid-phase peptide synthesis

Use of N-protected-α-amino acid bromides for facile solid-phase synthesis of peptides (SPPS) containing extremely sterically hindered non-proteinogenic amino acids is presented. Amino acid bromides (Aaa-Br), generated in situ, were used for the synthesis of long chain homopeptides containing up to eight successive α-MeVal or Aib residues. SPPS of a heteropeptide containing a very bulky amino acid building block is also described. The choice of suitable N-protections is discussed.     

Quaternary β2,2‐Amino Acids: Catalytic Asymmetric Synthesis and Incorporation into Peptides by Fmoc‐Based Solid‐Phase Peptide Synthesis

β‐Amino acid incorporation has emerged as a promising approach to enhance the stability of parent peptides and to improve their biological activity. Owing to the lack of reliable access to β^2,2‐amino acids in a setting suitable for peptide synthesis, most contemporary research efforts focus on the use of β³‐ and certain β^2,3‐amino acids. Herein, we report the catalytic asymmetric synthesis of β^2,2‐amino acids and their incorporation into peptides by Fmoc‐based solid‐phase peptide synthesis (Fmoc‐SPPS). A quaternary carbon center was constructed by the palladium‐catalyzed decarboxylative allylation of 4‐substituted isoxazolidin‐5‐ones. The N?O bond in the products not only acts as a traceless protecting group for β‐amino acids but also undergoes amide formation with α‐ketoacids derived from Fmoc‐protected α‐amino acids, thus providing expeditious access to α‐β^2,2‐dipeptides ready for Fmoc‐SPPS.     

Novel diphenylmethyl-Derived Amide Protecting Group for Efficient Liquid-Phase Peptide Synthesis: AJIPHASE

An efficient method for the synthesis of peptides bearing an amide at the C-terminal is described. This method involves the attachment of a C-terminal protecting group bearing long aliphatic chains, followed by the repetition of simple reaction and precipitation steps with the combined advantages of liquid-phase peptide synthesis (LPPS) and solid-phase peptide synthesis (SPPS). Using this method, a hydrophobic peptide was successfully synthesized in good yield and high purity, which cannot be obtained satisfactorily by SPPS.     

An efficient synthesis of a novel analog of octreotide with an unnatural l-lysine-like tetrazolyl amino acid

A new cyclic octreotide-like octapeptide was prepared by incorporation of an unnatural tetrazolyl amino acid, an analog of Fmoc-l-lysine, into the peptide chain. The new tetrazolyl amino acid, (S)-2-{[2-(9H-fluoren-9-yl)acetoxy]amino}-6-(1H-tetrazol-1-yl)hexanoic acid, was obtained by azidation of Fmoc-l-lysine trifluoroacetate with sodium azide in the presence of triethyl orthoformate in glacial acetic acid. The linear peptide sequence was prepared using efficient Fmoc/t-Bu solid-phase peptide synthesis (SPPS). Cyclization of the octapeptide was carried out via oxidation with iodine. The structure and purity of the cyclic octapeptide were confirmed by LC–MS, MALDI-TOF MS/MS analysis as well as 1D/2D ¹H and ¹³C NMR spectroscopy.     

High-pressure-promoted Fmoc-aminoacylation of N-ethylcysteine: preparation of key devices for the solid-phase synthesis of peptide thioesters

In this study synthesis of Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteine, a new N→S acyl migratory device for the preparation of peptide thioesters by Fmoc solid-phase peptide synthesis (SPPS) is described. Condensation of Fmoc-aminoacyl fluoride and N-ethyl-S-triphenylmethylcysteine allyl ester, readily prepared from known S-triphenylmethylcysteine allyl ester, was efficiently promoted in CH₂Cl₂ under high-pressure (800 MPa). When the reaction was performed with the additive DIEA, considerable epimerization at the chiral centers occurred, affording a mixture of diastereomers. When the preparation procedure for N-ethyl-S-triphenylmethylcysteine allyl ester was changed and the additive DIEA in the high-pressure reaction was excluded, Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteines was obtained as a single stereoisomer without epimerization. The Fmoc-l-leucine adduct thus prepared was deallylated and used for the SPPS of a known decapeptide. A remarkable increase (44%) in the overall yield of the decapeptidethioester was achieved, compared to the 7% obtained by the stepwise on-resin Leu-Cys condensation method.     

Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for ¹H and ¹³C (¹H→¹³C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for ¹H and 13.9±0.5 for ¹³C (direct polarization).     

An Optimized Protocol for the Synthesis of Peptides Containing trans-Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes

Despite the great advances in solid-phase peptide synthesis (SPPS), the incorporation of certain functional groups into peptide sequences is restricted by the compatibility of the building blocks with conditions used during SPPS. In particular, the introduction of highly reactive groups used in modern bioorthogonal reactions into peptides remains elusive. Here, we present an optimized synthetic protocol enabling installation of two strained dienophiles, trans-cyclooctene (TCO) and bicyclononyne (BCN), into different peptide sequences. The two groups enable fast and modular post-synthetic functionalization of peptides, as we demonstrate in preparation of peptide-peptide and peptide-drug conjugates. Due to the excellent biocompatibility, the click-functionalization of the peptides can be performed directly in live cells. We further show that the introduction of both clickable groups into peptides enables construction of smart, multifunctional probes that can streamline complex chemical biology experiments such as visualization and pull-down of metabolically labeled glycoconjugates. The presented strategy will find utility in construction of peptides for diverse applications, where high reactivity, efficiency and biocompatibility of the modification step is critical.     

An N-protection free ligation of the peptide thioester and the peptide with an N-alkoxy- or N-aryloxyamino group at its N-terminus

Peptides with an N-alkoxy or N-aryloxy amino acid at their N-terminus were synthesized and successfully ligated with a peptide thioester by silver ion activation under a slightly acidic condition without requiring protection of the side chain amino groups. The N-methoxy group was easily cleaved by the SmI₂ reduction in CH₃OH aq. to obtain the desired peptide with a native peptide bond. This method was successfully applied to the synthesis of the human atrial natriuretic peptide showing the efficiency of the novel ligation.     

SUBPOL: a novel SUcrose-Based Polymer support for solid-phase peptide synthesis and affinity chromatography applications

A novel SUcrose-Based Polymer support (SUBPOL) with tailored morphology suitable for the use in solid-phase peptide synthesis (SPPS) is described, and its application as a hydrophilic affinity matrix for the specific removal of fibrinogen from human plasma is demonstrated. After suspension polymerization of partly methacrylated 2,1':4,6-di-O-isopropylidene sucrose and subsequent removal of the protecting groups, hydrophilic spherical polymer beads were obtained. The morphology of the resulting resin was controlled by variation of the porogen as well as the average degree of substitution with respect to the methacryloyl groups of the monomer mixture. After introduction of amino groups for a permanent attachment of immobilized peptide ligands, prevention of unintended esterification during SPPS was achieved by silylation of remaining hydroxy groups. Alternatively, a Rink amide linker was introduced prior to SPPS to allow cleavage and subsequent analysis of the peptide assembled on the SUBPOL resins. Two hexapeptides of sequence kwiivw and hffflw, consisting of d-amino acids, as well as a 19-mer peptide corresponding to the sequence GSGVRGDFGSLAPRVARQL of the VP1 protein from the foot-and-mouse disease virus (FMDV) were successfully prepared both manually or in a semi-automated process on SUBPOL resins according to the Fmoc/tBu strategy. Yields and purities were comparable to peptides prepared on commercially available polystyrene resins. A specific affinity adsorbent containing the fibrinogen-binding pentapeptide GPRPK was prepared by SPPS on SUBPOL resins of different morphology, and the strong impact of the affinity matrix on adsorption performance was demonstrated.     

Synthesis of fluorescein-labelled O-mannosylated peptides as components for synthetic vaccines: comparison of two synthetic strategies

Mannose-binding proteins on the surface of antigen-presenting cells (APCs) are capable of recognizing and internalizing foreign agents in the early stages of immune response. These receptors offer a potential target for synthetic vaccines, especially vaccines designed to stimulate T cells. We set out to synthesize a series of fluorescein-labelled O-mannosylated peptides using manual solid phase peptide synthesis (SPPS) on pre-loaded Wang resin, in order to test their ability to bind mannose receptors on human APCs in vitro. A flexible and reliable method for the synthesis of fluorescein-labelled O-mannosylated glycopeptides was desired in order to study their lectin-binding properties using flow cell cytometry. Two synthetic strategies were investigated: incorporation of a fluorescein label into the peptide chain via a lysine side chain epsilon-amino group at the final stage of standard Fmoc solid phase peptide synthesis or attachment of the fluorescein label to the N(alpha)-amino group of a lysine with further incorporation of a mannosylated peptide unit through the side chain N(epsilon)-amino group. The latter strategy proved more effective in that it facilitated SPPS by positioning the growing mannosylated peptide chain further removed from the fluorescein label.     

A novel strategy for the solid-phase synthesis of cyclic lipodepsipeptides

A rapid and efficient Fmoc solid-phase synthesis of cyclic lipodepsipeptide analogue 1 to antibiotic fusaricidin A is described. Our synthetic approach includes resin attachment of the first amino acid via side chain, successful use of combination of four quasi-orthogonal removable protecting groups, stepwise solid-phase synthesis of linear peptide analogue, lipid tail attachment followed by depsipeptide bond formation and on-resin head-to-tail cyclization. Undesired O→N acyl shift, which may occur during Fmoc removal, was successfully avoided by the incorporation of the lipid tail into the linear peptide precursor prior to on-resin depsipeptide bond formation and the ring closure.     

Solid-phase synthesis of core 3 and core 6 O-glycan-linked glycopeptides by benzyl-protection method

Core 3 and core 6 O-glycoamino acids were prepared in a protected form suited for Fmoc solid-phase peptide synthesis (SPPS). An N-trichloroacetyllactosamine derivative (2) was used as a highly β-selective glycosyl donor in 3-O-glycosylation of acceptors 3/4 and in 6-O-glycosylation of acceptors 5/6. Zn reduction of trisaccharides 7/8 and 13/14 was followed by acetylation to readily transform trichloroacetamido and azido groups to acetamido groups. Selective deprotection by Pd(0)-catalysis afforded core 3 O-glycan building blocks 11/12 and core 6 O-glycan building blocks 17/18. Usefulness of these building blocks for SPPS was demonstrated by the syntheses of the core 3-linked MUC2 tandem repeat glycopeptide and the core 6-linked glycopeptide segment of MUC6. The synthetic glycopeptides detached from the resin were debenzylated under the ‘low-acidity TfOH’ conditions.