Conventional Solid-phase Peptide Synthesis Research Articles

Preparation, Radiolabeling with 68Ga/177Lu and Preclinical Evaluation of Novel Angiotensin Peptide Analog: A New Class of Peptides for Breast Cancer Targeting.

Angiotensin II (AngII) is known to play a significant part in the development of breast cancer by triggering cell propagation of breast cancer, tumor angiogenesis, and regulating tumor invasion and cell migration. AngII arbitrates its action via two G-protein-coupled receptors, AngII type 1 receptor (AT1) and AngII type 2 receptor (AT2). Overexpression of the AT1 receptor in breast cancer cells seems to promote tumor growth and angiogenesis, thus targeting the AT1 receptor using AngII peptide would facilitate the detection of breast carcinoma. We developed an AngII peptide intending to assess whether the peptide of the renin-angiotensin system holds the ability to target AT1 receptor-overexpressing breast cancer in vivo. DOTA-coupled AngII peptide was synthesized by conventional solid-phase peptide synthesis according to Fmoc/HATU chemistry. 68Ga/177Lu labeled AngII peptide was evaluated for its binding with TNBC MDA-MB-231 and ER+ MCF7 cell lines. Pharmacokinetics was studied in healthy balb/c mice and in vivo tumor targeting in nude mice with MDA-MB-231 tumors xenografts. DOTA-AngII peptide was labeled efficiently with 68Ga/177Lu with high labeling efficiency (≥90%). The stability of the radiopeptide in human plasma was found to be high. The AngII peptide analog showed nanomolar (<40 nM) AT1 receptor-specific binding affinity. The radioactivity internalized into MDA-MBA-231 and MCF7 cells were 14.97% and 11.75%, respectively. In vivo, biodistribution in balb/c mice exhibited efficient clearance of 68Ga/177Lu-DOTA-AngII peptide from the blood and elimination predominantly by the renal system due to its hydrophilic nature. A low amount of radioactivity was seen in the major organs including lungs, liver, stomach, spleen, and intestines (<3% ID/g) except the kidneys. A high renal-urinary excretion was observed for the radiotracer. In the TNBC MDA-MB-231 xenografts model, radiolabeled AngII peptide exhibited specific and effective AT1-based targeting in vivo. A rapid and efficient tumor targeting (2.18% ID/g at 45 min p.i.) together with fast renal excretion (~67% ID) highlights the tumor-targeting potential of the radiotracer. The AT1 receptor specificity of the radiotracer was validated by blocking assays. Furthermore, PET imaging provided sufficient visualization of MDA-MB-231 tumors in nude mice. Our findings suggest that 68Ga/177Lu-DOTA-AngII peptide can be useful for the theranostic application of breast carcinomas. This study suggests the potential of this innovative class of peptides for rapid and efficient targeting of tumors and warrants further evaluation.

Late-stage diversification strategy for the synthesis of peptide acids and amides using hydrazides

Aim: Modification of the C-terminus of a peptide to improve its properties, particularly after constructing the peptide chain, has great promise in the development of peptide therapeutics. This study discusses the development of a late-stage diversification method for synthesizing peptide acids and amides from hydrazides which can serve as a common precursor. Methods: Peptide hydrazides were synthesized solely by using conventional solid-phase peptide synthesis (SPPS). Hydrazides were subjected to oxidation by potassium peroxymonosulfate (Oxone) to afford carboxylic acids. Azidation of hydrazides using sodium nitrite (NaNO2) under acidic conditions, followed by the addition of β-mercaptoethanol (BME), could also be used to generate carboxylic acids. For the preparation of peptide amides, azides that can be prepared from hydrazides were reacted with ammonium acetate (NH4OAc) or tris(2-carboxyethyl)phosphine (TCEP)∙hydrochloride (HCl) to develop the products through ammonolysis or a Staudinger reaction, which produces iminophosphorane from an azide and a phosphine. The antimicrobial activity of modelin-5 derivatives synthesized from the corresponding hydrazides was evaluated by the colony count of Escherichia coli (E. coli) after treatment with the peptides. Results: Oxone oxidation yielded the corresponding acids rapidly although oxidation-prone amino acids were incompatible. Azidation and subsequent treatment with BME afforded peptide acids an acceptable yield even in sequences containing amino acids that are prone to oxidation. Both methods for conversion of hydrazides to amides were found to afford the desired products in good yield and compatibility. The conditions that were developed were adapted to the synthesis of modelin-5 derivatives from the corresponding hydrazides, yielding late-stage production of the desired peptides. The amides of the resulting peptide showed more potent activity against E. coli than the acid form, and the most potent activity was observed from the hydrazide. Conclusions: The developed protocols allow hydrazides to be converted to acids or amides, enabling late-stage diversification of peptide C-terminal residues.

Suppression of alpha-carbon racemization in peptide synthesis based on a thiol-labile amino protecting group

In conventional solid-phase peptide synthesis (SPPS), α-amino groups are protected with alkoxycarbonyl groups (e.g., 9-fluorenylmethoxycarbonyl [Fmoc]). However, during SPPS, inherent side reactions of the protected amino acids (e.g., α-C racemization and aspartimide formation) generate by-products that are hard to remove. Herein, we report a thiol-labile amino protecting group for SPPS, the 2,4-dinitro-6-phenyl-benzene sulfenyl (DNPBS) group, which is attached to the α-amino group via a S–N bond and can be quantitatively removed in minutes under nearly neutral conditions (1 M p-toluenethiol/pyridine). The use of DNPBS greatly suppresses the main side reactions observed during conventional SPPS. Although DNPBS SPPS is not as efficient as Fmoc SPPS, especially for synthesis of long peptides, DNPBS and Fmoc are orthogonal protecting groups; and thus DNPBS SPPS and Fmoc SPPS can be combined to synthesize peptides that are otherwise difficult to obtain.

ApoE Mimetic Peptide COG1410 Exhibits Strong Additive Interaction with Antibiotics Against Mycobacterium smegmatis.

Drug-resistant tuberculosis (TB) is an emerging threat to public health worldwide. Antimicrobial peptide (AMP) is a promising solution to solve the antimicrobial resistance crisis. The apolipoprotein E mimetic peptide COG1410 has been confirmed to simultaneously have neuroprotective, anti-inflammatory, and antibacterial activity. However, whether it is effective to inhibit growth of mycobacteria has not been investigated yet. The peptide COG1410 was synthesized with conventional solid-phase peptide synthesis and qualified by HPLC and mass spectrometry. Micro-dilution method was used to determine the minimal inhibitory concentration. A time-kill assay was used to determine the bactericidal dynamics of antimicrobial peptide and relative antibiotics. Static biofilm formation was conducted in 24-well plate and the biofilm was separated from planktonic cells and collected. The mechanism of action of COG1410 was explored by TEM observation and ATP leak assay. The localization of COG1410 was observed by confocal laser scan microscopy. The drug-drug interaction was determined by a checkerboard assay. COG1410 was a potent bactericidal agent against M. smegmatis in vitro and within the macrophages with MIC 16 μg/mL, but invalid against M. abscess and M. tuberculosis. A time-kill assay showed that COG1410 killed M. smegmatis as potent as clarithromycin, but faster than LL-37, another short synthetic cationic peptide. 1× MIC COG1410 almost reduced 90% biofilm formation of M. smegmatis. Additionally, COG1410 was able to penetrate the cell membrane of macrophage and inhibit intracellular M. smegmatis growth. TEM observation and ATP leak assay found that COG1410 disrupted cell membrane and caused release of cell contents. Confocal fluorescence microscopy showed that FITC-COG1410 aggregated around cell membrane instead of entering the cytoplasm. Although COG1410 had relative high cytotoxicity, it exhibited strong additive interaction with regular anti-TB antibiotics, which reduced the working concentration of COG1410 and expanding safety window. After 30 passages, there was no induced drug resistance for COG1410. COG1410 was a novel and potent AMP against M. smegmatis by disrupting the integrity of cell membrane.

Synthesis, antiproliferative and antimicrobial activities of (KLAKLAK)2-NH2 analogue containing nor-Leu and its conjugates with a second pharmacophore

Peptides are a promising alternative of conventional medical drugs for the treatment of different diseases because they have no or have very few side effects owing to the natural mechanisms for their elimination. There are a lot of examples of drugs on the pharmaceutical market based on modified amino acids and peptides. Herein, we report the synthesis and studies on the antimicrobial peptide (KLAKLAK)2-NH2 where Leu is replaced by the unnatural amino acid nor-Leu. In addition, a second pharmacophore with well proven anticancer properties is introduced to the peptide moiety. All structures were synthesized by conventional solid phase peptide synthesis. The antiproliferative and antimicrobial activities were studied using MTT-dye reduction assay and disk-diffusion test, respectively. Biological activity assays showed that the introduction of nor-Leu in the primary structure of the parent compound does not lead to an increase in the antiproliferative activity. However, the combination with the second pharmacophore 1,8-naphtalimide in a hybrid structure 1,8-NphtG-(KNleAKNleAK)2-NH2 leads to a significant increase in the antiproliferative properties. The antimicrobial tests showed that all tested compounds exhibit antimicrobial activity. The peptide and the second pharmacophore had a synergistic effect. In combination with complete hydrolytic stability for 72 h in model systems, the compound 1,8-NphtG-(KNleAKNleAK)2-NH2 is the best candidate for a medical drug in the treatment of mammary gland type A adenocarcinoma (MCF-7) in combination with antimicrobial properties.

Chemoselective Solution- and Solid-Phase Synthesis of Disulfide-Linked Glycopeptides.

Glycosylation of peptides and proteins is a widely employed strategy to mimic important post-translational modifications or to modulate the physicochemical properties of peptides to enhance their delivery. Furthermore, glycosylation via a sulfur atom imparts increased chemical and metabolic stability to the resulting glycoconjugates. Herein, we report a simple and chemoselective procedure to prepare disulfide-linked glycopeptides. Acetate-protected glycosylsulfenyl hydrazines are shown to be highly reactive with the thiol group of cysteine residues within peptides, both in solution and as part of conventional solid-phase peptide synthesis protocols. The efficiency of this glycosylation methodology with unprotected carbohydrates is also demonstrated, which avoids the need for deprotection steps and further extends its utility, with disulfide-linked glycopeptides produced in excellent yields. Given the importance of glycosylated peptides in structural glycobiology, pharmacology, and therapeutics, the methodology outlined provides easy access to disulfide-linked glycopeptides as molecules with multiple biological applications.

Synthesis and evaluation of a para-carboxylated benzyl-DOTA for labeling peptides and polypeptides

IntroductionRadiolabeled peptides and low-molecular-weight (LMW) polypeptides show high and persistent radioactivity levels in the kidney. To develop a DOTA-based bifunctional chelating agent that provides a radiometabolite with a rapid elimination rate from the kidney, a para-carboxyl Bn-DOTA (p-COOH-Bn-DOTA) was designed, synthesized, and evaluated. MethodsA precursor compound, p-COOH-Bn-DOTA(tBu)4, was synthesized in 9 steps using N-Boc-p-iodo-L-phenylalanine as the starting material. A synthetic somatostatin analog (TOC) was used as a representative peptide metabolized in the renal lysosomes. p-COOH-Bn-DOTA-conjugated TOC (DOTA-Bn-TOC) was synthesized by the conventional solid-phase peptide synthesis using p-COOH-Bn-DOTA(tBu)4. DOTA-tris(tBu ester) was also conjugated with TOC to prepare DOTATOC. 111In-labeling of the peptides was conducted under similar conditions. The radiochemical conversions, stability against apo-transferrin (apoTf), and in vivo behaviors were compared. Results[111In]In-DOTA-Bn-TOC was obtained with higher radiochemical conversions than [111In]In-DOTATOC. Both 111In-labeled TOC derivatives remained stable against apoTf. In biodistribution studies, [111In]In-DOTA-Bn-TOC exhibited higher initial uptake in the kidney, followed by a faster elimination rate of radioactivity into the urine than [111In]In-DOTATOC. The metabolic studies showed that the shorter residence time of the radiometabolite from [111In]In-DOTA-Bn-TOC was responsible for the renal radioactivity decline. Conclusionp-COOH-Bn-DOTA provides stable 111In-labeled peptides in high yields at low peptide concentrations. p-COOH-Bn-DOTA also provides a radiometabolite with a short residence time in the kidney. Such characteristics would render p-COOH-Bn-DOTA useful to the future application to radiolabeled LMW polypeptides with low renal radioactivity levels.

Synthesis of Amino Acid α-Thioethers and Late-Stage Incorporation into Peptides

The accelerating discovery of structurally distinct peptide natural products bearing α-thioether cross-links, such as the family of sactipeptide natural products, highlights the need for strategies to synthesize this underexplored functional motif. Herein, we describe the preparation of orthogonally protected, cross-linked amino acid α-thioether building blocks and probe their stability toward conventional solid-phase peptide synthesis. We overcome challenges with linkage lability by developing a late-stage, on-resin approach to α-thioethers, providing important proof-of-principle for sactipeptide synthesis.

Combining flavin photocatalysis with parallel synthesis: a general platform to optimize peptides with non-proteinogenic amino acids.

Most peptide drugs contain non-proteinogenic amino acids (NPAAs), born out through extensive structure–activity relationship (SAR) studies using solid-phase peptide synthesis (SPPS). Synthetically laborious and expensive to manufacture, NPAAs also can have poor coupling efficiencies allowing only a small fraction to be sampled by conventional SPPS. To gain general access to NPAA-containing peptides, we developed a first-generation platform that merges contemporary flavin photocatalysis with parallel synthesis to simultaneously make, purify, quantify, and even test up to 96 single-NPAA peptide variants via the unique combination of boronic acids and a dehydroalanine residue in a peptide. We showcase the power of our newly minted platform to introduce NPAAs of diverse chemotypes-aliphatic, aromatic, heteroaromatic-directly into peptides, including 15 entirely new residues, and to evolve a simple proteinogenic peptide into an unnatural inhibitor of thrombin by non-classical peptide SAR.

Synthesis and Application of a Long-Circulating Radiolabeled Peptide for Targeting of Osteosarcoma.

The small peptide TMTP1 (NVVRQ) has been proved to target a series of highly metastatic tumor cells. The aim of this study was to develop a new agent based on TMTP1 conjugated with Evans blue (EB), to increase tumor uptake and modify the pharmacokinetic characteristics of the resulting radiolabeled agent. DOTA-EB-TMTP1 was prepared through conventional solid-phase peptide synthesis chemistry. Then, it was successfully labeled with Cu-64 to obtain [64Cu]DOTA-EB-TMTP1. The tumor targeting properties were evaluated in vivo using 143B xenografts. DOTA-EB-TMTP1 was successfully labeled with Cu-64 in a yield of 87.3 ± 5.2%. In a small animal positron emission tomography/X-ray computed tomography (PET/CT) study in osteosarcoma 143B xenograft mice, [64Cu]DOTA-EB-TMTP1 was found to rapidly accumulate in the tumor tissue. The tumor uptake increased over time and reached a plateau of 6.50 ± 0.88% ID/g 8h after tail vein injection. The radioactivity remained in the tumor tissue 48h postinjection with a negligible decrease. Overall, the introduction of the EB motif to TMTP1 significantly changed its pharmacokinetics in vivo, and this strategy fulfills the purpose of prolonging the blood circulation and enhancing the tumor uptake. [64Cu]DOTA-EB-TMTP1 is a promising agent for osteosarcoma targeting. Moreover, our study highlights that DOTA-EB-TMTP1 is a good candidate for labeling with different radionuclides for potential theranostic applications.

Strategy for the Biosynthesis of Short Oligopeptides: Green and Sustainable Chemistry.

Short oligopeptides are some of the most promising and functionally important amide bond-containing components, with widespread applications. Biosynthesis of these oligopeptides may potentially become the ultimate strategy because it has better cost efficiency and environmental-friendliness than conventional solid phase peptide synthesis and chemo-enzymatic synthesis. To successfully apply this strategy for the biosynthesis of structurally diverse amide bond-containing components, the identification and selection of specific biocatalysts is extremely important. Given that perspective, this review focuses on the current knowledge about the typical enzymes that might be potentially used for the synthesis of short oligopeptides. Moreover, novel enzymatic methods of producing desired peptides via metabolic engineering are highlighted. It is believed that this review will be helpful for technological innovation in the production of desired peptides.

Synthesis of a GlcNAcylated arginine building block for the solid phase synthesis of death domain glycopeptide fragments

Herein we describe the synthesis of glycopeptide fragments from the death domains of TRADD and FADD bearing the recently discovered Nω-GlcNAc-β-arginine post-translational modification. TRADD and FADD glycopeptides were accessed through the use of a suitably protected synthetic glycosylamino acid ‘cassette’ that could be directly incorporated into conventional solid phase peptide synthesis (SPPS) protocols.

Synthesis of neurosecretory protein GM composed of 88 amino acid residues by native chemical ligation

Recently, we found a novel neuropeptide and named it neurosecretory protein GM (NPGM). Rat NPGM is composed of 88 amino acid residues and has an amidated C-terminus and an intramolecular disulfide bond. In our preliminary experiment, NPGM production using conventional solid-phase peptide synthesis was impossible due to its length and hydrophobicity. In the present study, NPGM was produced by native chemical ligation and a disulfide bond was formed by potassium ferricyanide.

Hydrogen Bonded Squaramide-Based Foldable Module Induces Both β- and α-Turns in Hairpin Structures of α-Peptides in Water

A novel tertiary squaramido-based reverse-turn module SQ is reported, and its conformational properties are evaluated. This module is easily incorporated into a α-peptide sequence by conventional solid-phase peptide synthesis. The structure characterization of the hybrid squaramido-peptide 4 is described, showing that the turn segment induces the formation of hairpin structures in water through the formation of both αSQ- and βSQ-turns.

Lanthanide-Mediated Dephosphorylation Used for Peptide Cleavage during Solid Phase Peptide Synthesis

Lanthanide(III) ions can accelerate the hydrolysis of phosphomonoesters and phosphodiesters in neutral aqueous solution. In this paper, lanthanide-mediated dephosphorylation has been applied in aqueous media as an orthogonal cleavage condition that can be employed in conventional solid phase peptide synthesis (SPPS). A phosphorylated polymeric support for SPPS was developed using Boc chemistry. The cleavage of resin-bound phosphates was investigated with the addition of Eu(III), Yb(III), acid or base, a mixture of solvents or different temperatures. To demonstrate the utility of this approach for SPPS, a peptide sequence was synthesized on a phosphorylated polymeric support and quantitatively cleaved with lanthanide ions in neutral aqueous media. The protecting groups for side chains were retained during peptide cleavage using lanthanide ions. This new methodology provides a mild orthogonal cleavage condition of phosphoester as a linker during SPPS.

Facile preparation of photodegradable hydrogels by photopolymerization

Photodegradable hydrogels have emerged as a powerful material platform for studying and directing cell behaviors, as well as for delivering drugs. The premise of this technique is to use a cytocompatible light source to cleave linkers within a hydrogel, thus causing reduction of matrix stiffness or liberation of matrix-tethered biomolecules in a spatial-temporally controlled manner. The most commonly used photodegradable units are molecules containing nitrobenzyl moieties that absorb light in the ultraviolet (UV) to lower visible wavelengths (∼280–450 nm). Because photodegradable linkers and hydrogels reported in the literature thus far are all sensitive to UV light, highly efficient UV-mediated photopolymerizations are less likely to be used as the method to prepare these hydrogels. As a result, currently available photodegradable hydrogels are formed by redox-mediated radical polymerizations, emulsion polymerizations, Michael-type addition reactions, or orthogonal click chemistries. Here, we report the first photodegradable poly(ethylene glycol)-based hydrogel system prepared by step-growth photopolymerization. The model photolabile peptide cross-linkers, synthesized by conventional solid phase peptide synthesis, contained terminal cysteines for step-growth thiol-ene photo-click reactions and a UV-sensitive 2-nitrophenylalanine residue in the peptide backbone for photo-cleavage. Photolysis of this peptide was achieved through adjusting UV light exposure time and intensity. Photopolymerization of photodegradable hydrogels containing photolabile peptide cross-linkers was made possible via a highly efficient visible light-mediated thiol-ene photo-click reaction using a non-cleavage type photoinitiator eosin-Y. Rapid gelation was confirmed by in situ photo-rheometry. Flood UV irradiation at controlled wavelength and intensity was used to demonstrate the photodegradability of these photopolymerized hydrogels.

Bidirectional solid phase synthesis of a model oligoglycine bolaamphiphile and purification by rapid self-assembly

We utilised a simple bidirectional (N→C and C→N) solid phase synthesis strategy entailing conventional solid phase peptide synthesis and fragment condensation with a water-soluble carbodiimide to synthesise a model anionic glycylglycine bolaamphiphile containing a suberic acid linker moiety, namely N,N'-suberoyldiglycylglycine. The synthetic suberoyldiglycylglycine was purified using its inherent ability to rapidly self-assemble in an aqueous acidic solution (0.1% trifluoroacetic acid). Monitoring of the rapid assembly process corroborated our visual observation and confirmed packing-directed self-assembly rather than non-specific aggregation or precipitation. The progress of suberoyldiglycylglycine self-assembly was observed to be via the formation of oligomers in the solution, which then self-assembled to form layered β-sheet type macrostructures. Within 24 h, nanotubes grew from these macrostructures and eventually combined to formed microtubes, which we isolated after 5-7 days.

A novel 18F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor

Two-helix scaffold proteins (~ 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an (18)F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors. An aminooxy-functionalized two-helix peptide (AO-MUT-DS) with high HER2 binding affinity was synthesized through conventional solid phase peptide synthesis. The purified linear peptide was cyclized by I(2) oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-(18)F-fluorobenzyl aldehyde ((18)F-FBA), through the aminooxy functional group at the peptide N terminus (30% yield, non-decay corrected). The binding affinities of the peptides were analyzed by Biacore analysis. Cell uptake assay of the resulting PET probe, (18)F-FBO-MUT-DS, was performed at 37°C. (18)F-FBO-MUT-DS with high specific activity (20-32 MBq/nmol, 88-140 μCi/μg, end of synthesis) was injected into mice xenograft model bearing SKOV3 tumor. MicroPET and biodistribution and metabolic stability studies were then conducted. Cell uptake assays showed high and specific cell uptake (~12% applied activity at 1 h) by incubation of (18)F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (K(D)) of AO-MUT-DS and FBO-MUT-DS as tested by Biacore analysis were 2 and 1 nM, respectively. In vivo small animal PET demonstrated fast tumor targeting, high tumor accumulation, and good tumor to normal tissue contrast of (18)F-FBO-MUT-DS. Biodistribution studies further revealed that the probe had excellent tumor uptake (6.9%ID/g at 1 h post-injection) and was cleared through both liver and kidneys. Co-injection of the probe with 500 μg of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p < 0.05). F-FBO-MUT-DS displays excellent HER2 targeting ability and tumor PET imaging quality. The two-helix scaffold proteins are suitable for development of (18)F-based PET probes.

Development of a Sensitive Bead-Based Assay for Enhanced Monoclonal Antibody Detection

ABSTRACTMonoclonal antibodies are increasingly used in the treatment of cancer due to their enhanced targeting and immune system stimulation properties. Dosage guidelines typically do not account for personal cancer load or metabolism, thereby possibly affecting treatment outcome or causing unwanted side effects. The requirement for an assay that can quickly and precisely measure the concentration of the monoclonal antibody in a serum sample of a patient during therapy is unmet. A bead-based assay with peptide antigen mimetics has been developed to rapidly determine the concentration of antibody drug present in serum specimens with high sensitivity. Alemtuzumab (anti-CD52) and rituximab (anti-CD20) antigen mimetic peptides, as discovered by phage display, were synthesized on 10 um TentaGel resin beads using conventional solid phase peptide synthesis techniques. The beads were modified to allow for multiplexing and microfluidic handling via fluorescent labeling and magnetic functionalization. The antigen-displaying fluoromagnetic particles were incubated with spiked serum samples which allowed free antibody to be captured. Primary antibody detection was performed on alemtuzumab while rituximab detection was used to compensate for non-specific serum binding to the beads. After washing, the beads were incubated with a fluorescently tagged secondary label for detection by flow cytometry. (Results) A fast, low cost, specific assay has been developed with several key techniques which allows detection at low concentration (0.1ug/ml) of spiked samples. Primary to achieving this detection limit was the implementation of a compensation scheme where two antigen mimetic peptides behave linearly (R2=0.996) which enables the calculation of the zero response of the antigen mimetic peptide of interest (alemtuzumab antigen mimetic) while measuring the zero response of the compensatory antigen mimetic peptide (rituximab antigen mimetic) during primary assay measurement. This reduces fluorescence response variation due to variations present due to sample preparation, storage and different patients because of the equivalent interactions these effects have on the compensatory beads. The developed assay is therefore robust against serum variation and enables a lower limit of detection.

Radiofluorinated Rhenium Cyclized α-MSH Analogues for PET Imaging of Melanocortin Receptor 1

In order to accomplish in vivo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R), several α-melanocyte-stimulating hormone (α-MSH) analogues have been labeled with N-succinimidyl-4-¹⁸F-fluorobenzoate (¹⁸)F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. To further pursue a radiofluorinated α-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl 2-¹⁸F-fluoropropionate (¹⁸F-NFP) to radiofluorinate the transition metal rhenium cyclized α-MSH metallopeptides for PET imaging of MC1R positive malignant melanoma. Metallopeptides Ac-d,Lys-ReCCMSH(Arg¹¹) (two isomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesis chemistry and rhenium cyclization reaction. The two isomers were then conjugated with ¹⁹F-NFP or ¹⁸F-NFP. The resulting cold or radiofluorinated metallopeptides, (¹⁸/¹⁹)F-FP-RMSH-1 and (¹⁸/¹⁹)F-FP-RMSH-2, were further evaluated for their in vitro receptor binding affinities, in vivo biodistribution, and small-animal PET imaging properties. The binding affinities of ¹⁹F-FP-RMSH-1 and ¹⁹F-FP-RMSH-2 were determined to be within low nanomolar range. In vivo studies revealed that both F-labeled metallopeptides possessed good tumor uptake in the B16F10 murine model with high MC1R expression, while possessing much lower uptake in A375M human melanoma xenografts. Moreover, ¹⁸F-FP-RMSH-1 displayed more favorable in vivo performance in terms of higher tumor uptake and much lower accumulation in the kidney and liver, when compared to that of ¹⁸F-FP-RMSH-2 at 2 h postinjection (p.i.). ¹⁸F-FP-RMSH-1 also displayed lower liver and lung uptake when compared with that of the same peptide labeled with ¹⁸F-SFB (named as ¹⁸F-FB-RMSH-1). Small animal PET imaging of ¹⁸F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptake and poorer tumor/normal organ contrast were observed for A375M model compared to those of the B16F10 model. ¹⁸F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retention when compared with ¹⁸F-FB-RMSH-1. ¹⁸F-FP-RMSH-1 demonstrates significant advantages over ¹⁸F-FB-RMSH-1 and ¹⁸F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1R expression in vivo.