Despite advances in solid phase peptide synthesis and peptide ligation, challenges remain in the assembly of polypeptides through coupling of peptide fragments. Herein we describe a new method for peptide fragment coupling employing the Ag(i)-promoted transformation of peptide thioamides. This process proceeds via an isoimide-tethered intermediate, which undergoes an O-N acyl transfer to generate the polypeptide. This method is applicable to both solution- and solid-phase coupling.

Solid-phase peptide synthesis has become an indispensable technique for the routine preparation of linear peptides of up to approximately 40 amino acids in length. However, the solid-phase approach is still hampered by chain insolubility and aggregation, which reduces synthetic yields. Moreover, many of the deletion impurities that can form are often chromatographically inseparable from the target sequence, which diminishes final product purity. The use of backbone N-protecting groups can ameliorate this synthetic inefficiency by increasing peptide chain solubility and suppressing aggregation. Backbone protection is also useful for promoting peptide macrocyclization, suppressing common side reactions in peptide chemistry, and improving solution-phase handling. Commercially available precursors containing benzyl-based groups and pseudoprolines have found widespread use, in academic laboratories and industry. A range of other strategies have also been investigated in a bid to increase the utility of backbone protecting groups and to develop more efficient methods for their introduction and removal. This review provides a comprehensive account of the state of the art, and includes detailed synthetic methods relating to the use of backbone protection, and its application to "difficult" peptides and proteins of biological significance. The strengths and weaknesses of each approach are analyzed, and a commentary on future directions is provided.

Fmoc solid-phase peptide synthesis has been indispensable for the efficient manufacture of research grade peptides and proteins, and peptide APIs. However, the solid-phase approach is still hampered by solubility issues and aggregation of the resin-bound peptide chain, which limits routine access to peptides > 40 amino acids in length. The use of backbone amide protecting groups, such as through the introduction of N -benzyl-based moieties and pseudoproline dipeptides, ameliorates this synthetic inefficiency somewhat. But benzyl groups can be difficult to remove postassembly, and pseudoprolines are limited to serine, threonine, and cysteine-rich peptide segments. To enhance the utility of backbone protection, we have evaluated the tetrahydropyranyl (Thp) group as a more acid labile alternative to benzyl protection. The Thp group can be efficiently introduced to the resin-bound peptide as a protected dipeptide and is readily cleaved and scavenged postsynthesis. A drastic improvement in the solid-phase assembly of aggregation-prone amyloid-β and prion-derived peptide fragments is observed using Thp as a backbone protecting group. We envisage that Thp-protected dipeptides will become useful building blocks for peptide manufacturing, complementing existing backbone protecting group strategies.

Palladium(II)-catalyzed C-H functionalization has attracted considerable attention as a pathway to late-stage modification of peptides. Herein, we report the Pd-catalyzed C(sp3)-H arylation of peptides directed by an amidoxime ether, which can be easily incorporated into peptides at any amide bond. Site- and stereoselective arylation of peptides has been achieved, including unprecedented functionalization of internal residues of native peptides. Removal of the amidoxime ether was achieved to generate the parent amide and facilitate a traceless C-H functionalization process.

The Plasmodium falciparum cytoplasmic tyrosine tRNA synthetase (PfTyrRS) is an attractive drug target that is susceptible to reaction-hijacking by AMP-mimicking nucleoside sulfamates. We previously identified an exemplar pyrazolopyrimidine ribose sulfamate, ML901, as a potent reaction hijacking inhibitor of PfTyrRS. Here we examined the stage specificity of action of ML901, showing very good activity against the schizont stage, but lower trophozoite stage activity. We explored a series of ML901 analogues and identified ML471, which exhibits improved potency against trophozoites and enhanced selectivity against a human cell line. Additionally, it has no inhibitory activity against human ubiquitin-activating enzyme (UAE) in vitro. ML471 exhibits low nanomolar activity against asexual blood stage P. falciparum and potent activity against liver stage parasites, gametocytes and transmissible gametes. It is fast-acting and exhibits a long in vivo half-life. ML471 is well-tolerated and shows single dose oral efficacy in the SCID mouse model of P. falciparum malaria. We confirm that ML471 is a reaction hijacking inhibitor that is converted into a tight binding Tyr-ML471 conjugate by the PfTyrRS enzyme. A crystal structure of the PfTyrRS/ Tyr-ML471 complex offers insights into improved potency, while molecular docking into UAE provides a rationale for improved selectivity.

Peptide receptor radionuclide therapy (PRRT) is a promising form of systemic radiation therapy designed to eradicate cancer. Cholecystokinin-2 receptor (CCK2R) is an important molecular target that is highly expressed in a range of cancers. This study describes the synthesis and in vivo characterization of a novel series of 177Lu-labeled peptides ([177Lu]Lu-2b-4b) in comparison with the reference CCK2R-targeting peptide CP04 ([177Lu]Lu-1b). [177Lu]Lu-1b-4b showed high chemical purity (HPLC ≥ 94%), low Log D7.4 (-4.09 to -4.55) with strong binding affinity to CCK2R (KD 0.097-1.61 nM), and relatively high protein binding (55.6-80.2%) and internalization (40-67%). Biodistribution studies of the novel 177Lu-labeled peptides in tumors (AR42J and A431-CCK2R) showed uptake one- to eight-fold greater than the reference compound CP04 at 1, 24, and 48 h. Rapid clearance and high tumor uptake and retention were established for [177Lu]Lu-2b-4b, making these compounds excellent candidates for theranostic applications against CCK2R-expressing tumors.

(1) Background: [18F]Flumazenil 1 ([18F]FMZ) is an established positron emission tomography (PET) radiotracer for the imaging of the gamma-aminobutyric acid (GABA) receptor subtype, GABAA in the brain. The production of [18F]FMZ 1 for its clinical use has proven to be challenging, requiring harsh radiochemical conditions, while affording low radiochemical yields. Fully characterized, new methods for the improved production of [18F]FMZ 1 are needed. (2) Methods: We investigate the use of late-stage copper-mediated radiofluorination of aryl stannanes to improve the production of [18F]FMZ 1 that is suitable for clinical use. Mass spectrometry was used to identify the chemical by-products that were produced under the reaction conditions. (3) Results: The radiosynthesis of [18F]FMZ 1 was fully automated using the iPhase FlexLab radiochemistry module, affording a 22.2 ± 2.7% (n = 5) decay-corrected yield after 80 min. [18F]FMZ 1 was obtained with a high radiochemical purity (>98%) and molar activity (247.9 ± 25.9 GBq/µmol). (4) Conclusions: The copper-mediated radiofluorination of the stannyl precursor is an effective strategy for the production of clinically suitable [18F]FMZ 1.

A novel method for lactam stapling of Asp/Lys-containing peptides has been developed that does not require coupling agents. A backbone thioamide is incorporated at the N-terminal side of the aspartate residue. Ag(I)-promoted activation of the thioamide in the vicinity of the Asp carboxylate generates a cyclic isoimide intermediate that is trapped by the Lys amine to generate the macrolactam. This method is suitable for generation of i,i+2, i,i+3, and i,i+4-spaced lactam-bridged peptides.

A site-specific method for the preparation of N-glycosylated peptides is described. Incorporation of a peptide backbone thioamide linkage adjacent to an Asp residue facilitates a AgI -promoted, site-specific conversion to N-glycosylated Asn residues in peptides.

Peptide modifications can unlock a variety of compounds with structural diversity and abundant biological activity. In nature, peptide modifications, such as functionalisation at the side-chain position of amino acids, are performed using post-translational modification enzymes or incorporation of unnatural amino acids. However, accessing these modifications remains a challenge for organic chemists. During the past decades, selective C-H activation/functionalisation has attracted considerable attention in synthetic organic chemistry as a pathway to peptide modification. Various directing group strategies have been discovered that assist selective C-H activation. In particular, bidentate directing groups that enable tuneable and reversible coordination are now recognised as one of the most efficient methods for the site-selective C-H activation and functionalisation of numerous families of organic compounds. Synthetic peptide chemists have harnessed bidentate directing group strategies for selective functionalisation of the β- and γ-positions of amino acids. This method has been expanded and recognised as an effective device for the late stage macrocyclisation and total synthesis of complex peptide natural products. In this review, we discuss various β-, γ-, and δ-C(sp3)-H bond functionalisation reactions of amino acids for the formation of C-X bonds with the aid of directing groups and their application in late-stage macrocyclisation and the total synthesis of complex peptide natural products.

Background: Circulating autoantibodies and sex-dependent discrepancy in prevalence are unexplained phenomena of Alzheimer’s disease (AD). Using the 3xTg-AD mouse model, we reported that adult males show early manifestations of systemic autoimmunity, increased emotional reactivity, enhanced expression of the histone variant macroH2A1 in the cerebral cortex, and loss of plaque/tangle pathology. Conversely, adult females display less severe autoimmunity and retain their AD-like phenotype. This study examines the link between immunity and other traits of the current 3xTg-AD model. Methods: Young 3xTg-AD and wild-type mice drank a sucrose-laced 0.4 mg/ml solution of the immunosuppressant cyclophosphamide on weekends for 5 months. After behavioral phenotyping at 2 and 6 months of age, we assessed organ mass, serologic markers of autoimmunity, molecular markers of early AD pathology and expression of genes associated with neurodegeneration. Results : Chronic immunosuppression prevented hematocrit drop and reduced soluble Aβ in 3xTg-AD males while normalizing the expression of histone variant macroH2A1 in 3xTg-AD females. This treatment also reduced hepatosplenomegaly, lowered autoantibody levels, and increased the effector T cell population while decreasing the proportion of regulatory T cells in both sexes. Exposure to cyclophosphamide, however, neither prevented reduced brain mass and BDNF expression nor normalized increased tau and anxiety-related behaviors. Conclusion : The results suggest that systemic autoimmunity increases soluble Aβ production and affects transcriptional regulation of macroH2A1 in a sex-related manner. Despite the complexity of multisystem interactions, 3xTg-AD mice can be a useful in vivo model for exploring the regulatory role of autoimmunity in the etiology of AD-like neurodegenerative disorders.

Proteins adopt unique folded secondary and tertiary structures that are responsible for their remarkable biological properties. This structural complexity is key in designing efficacious peptides that can mimic the three-dimensional structure needed for biological function. In this study, we employ different chemical strategies to induce and stabilize a β-hairpin fold of peptides targeting cholecystokinin-2 receptors for theranostic application (combination of a targeted therapeutic and a diagnostic companion). The newly developed peptides exhibited enhanced folding capacity as demonstrated by circular dichroism (CD) spectroscopy, ion-mobility spectrometry-mass spectrometry, and two-dimensional (2D) NMR experiments. Enhanced folding characteristics of the peptides led to increased biological potency, affording four optimal Ga-68 labeled radiotracers ([68Ga]Ga-4b, [68Ga]Ga-11b-13b) targeting CCK-2R. In particular, [68Ga]Ga-12b and [68Ga]Ga-13b presented improved metabolic stability, enhanced cell internalization, and up to 6 fold increase in tumor uptake. These peptides hold great promise as next-generation theranostic radiopharmaceuticals.

The AgI -promoted reaction of thiolactams with N-Boc amino acids yields an N-(α-aminoacyl) lactam that can rearrange through an acyl transfer process. Boc-deprotection results in convergence to the ring-expanded adduct, thereby facilitating an overall insertion of an amino acid into the thioamide bond to generate medium-sized heterocycles. Application to the site-specific insertion of amino acids into cyclic peptides is demonstrated.

Many cancers of neuroendocrine origin overexpress cholecystokinin-2 receptors (CCK-2R) including medullary thyroid cancer, small cell lung cancer and other lung carcinoids. Fluorine-18 labelled peptides targeting CCK-2R enable direct visualization and quantification of this receptor in vivo using positron emission tomography imaging. CP04 1 and MG11 2 are two previously described truncated peptides derived from the native CCK-2R hormone ligand, gastrin. The N-terminus of the MG11 2 octopeptide was chemically modified with various fluorine containing aromatic (4-fluorobenzoate), heterocyclic (6-fluoronicotinate) and aliphatic (2-fluoropropionate) moieties. To assess the impact these modifications had on CCK-2R binding, ligand-binding assays were conducted using A431 cells overexpressing human CCK-2R. MG11 2 modified by 4-fluorobenzoate (FB-MG11 3) demonstrated the highest binding affinity (0.20 nM) followed by MG11 2 modified by 6-fluoronicotinate (FNic-MG11 4; 0.74 nM) and 2-fluoropropionate (FP-MG11 5; 1.80 nM), respectively. Whilst indirect labelling of MG11 2 using fluorine-18 labelled activated esters of fluorobenzoate and 6-fluoronicotinate was unsuccessful, direct fluorine-18 labelling at the N-terminus modified with 6-nitronicotinate afforded a 47.6% radiochemical yield of [18F]FNic-MG11. Unfortunately, [18F]FNic-MG11 4 was chemically unstable, decomposing slowly through defluorination, thereby impeding any further work with this radiotracer.

Macrolactonisation of peptides to generate cyclic depsipeptides is often challenging due to the low nucleophilicity of hydroxyl groups, epimerisation, cyclodimerisation, and potential acyl transfer reactions of the ester. Herein, we report a novel macrolactonisation strategy employing a Ag(i)-promoted conversion of peptide thioamides to isoimide intermediates, which undergo site-selective intramolecular acyl transfer to serine/threonine side chains to generate the macrolactone.