Solid-phase Peptide Synthesis Research Articles

Efficient Solution Phase Synthesis of PPII Helix Mimicking Ena/VASP EVH1 Inhibitors from Proline‐Derived Modules (ProMs)

AbstractIn the search for efficient inhibitors for the enabled/vasodilator‐stimulated phosphoprotein homology 1 (EVH1) domain to reduce cell motility in metastatic cancer, we previously developed a toolkit of proline‐derived modules (ProMs), which mimic the PPII helix found in the natural −FPPPP− binding motif of EVH1. In this work, we describe the modular assembly of these ProM‐based pentapeptidic EVH1 ligands through liquid phase peptide synthesis. We initially used pentafluorophenyl (Pfp) active esters for amide bond formation and built up the growing peptide chain from the C‐ to the N‐terminus. Switching to HATU/DIPEA coupling conditions and changing the directionality of the synthesis from the N‐ to the C‐terminus afforded the target ligands with improved overall yields and purity. Employing a Fmoc‐protected (instead of the N‐acetylated) phenylalanine derivative as N‐terminal building block significantly reduced epimerization. In contrast to the originally used solid phase peptide synthesis (SPPS), the developed solution phase method allowed for a facile alteration of the C‐terminal ProM unit and the production of various pentapeptidic ligands in an efficient fashion even on a multigram scale.

Exploiting Chemical Protein Synthesis to Study the Role of Tyrosine Sulfation on Anticoagulants from Hematophagous Organisms.

ConspectusTyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech Hirudo medicinalis, whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown. We hypothesized that the intrinsic instability of the tyrosine sulfate functionality, particularly under the acidic conditions used to isolate and analyze peptides and proteins, has led to poor detection during the isolation and/or expression of these molecules.Herein, we summarize our efforts to interrogate the functional role of tyrosine sulfation in the thrombin inhibitory and anticoagulant activity of salivary peptides and proteins from a range of different blood feeding organisms, including leeches, ticks, mosquitoes, and flies. Specifically, we have harnessed synthetic chemistry to efficiently generate homogeneously sulfated peptides and proteins for detailed structure-function studies both in vitro and in vivo.Our studies began with the leech protein hirudin P6 (from Hirudinaria manillensis), which is both sulfated on tyrosine and O-glycosylated at a nearby threonine residue. Synthetically, this was achieved through solid-phase peptide synthesis (SPPS) with a late-stage on-resin sulfation, followed by native chemical ligation and a folding step to generate six differentially modified variants of hirudin P6 to assess the functional interplay between O-glycosylation and tyrosine sulfation. A one-pot, kinetically controlled ligation of three peptide fragments was used to assemble homogeneously sulfoforms of madanin-1 and chimadanin from the tick Haemaphysalis longicornis. Dual tyrosine sulfation at two distinct sites was shown to increase the thrombin inhibitory activity by up to 3 orders of magnitude through a novel interaction with exosite II of thrombin. The diselenide-selenoester ligation developed by our lab provided us with a means to rapidly assemble a library of different sulfated tick anticoagulant proteins: the andersonins, hyalomins, madanin-like proteins, and hemeathrins, thus enabling the generation of key structure-activity data on this family of proteins. We have also confirmed the presence of tyrosine sulfation in the anticoagulant proteins of Anopheles mosquitoes (anophelins) and the Tsetse fly (TTI) via insect expression and mass spectrometric analysis. These molecules were subsequently synthesized and assessed for thrombin inhibitory and anticoagulant activity. Activity was significantly improved by the addition of tyrosine sulfate modifications and led to molecules with potent antithrombotic activity in an in vivo murine thrombosis model.The Account concludes with our most recent work on the design of trivalent hybrids that tandemly occupy the active site and both exosites (I and II) of α-thrombin, with a TTI-anophelin hybrid (Ki = 20 fM against α-thrombin) being one of the most potent protease inhibitors and anticoagulants ever generated. Taken together, this Account highlights the importance of the tyrosine sulfate post-translational modification within salivary proteins from blood feeding organisms for enhancing anticoagulant activity. This work lays the foundation for exploiting native or engineered variants as therapeutic leads for thrombotic disorders in the future.

Synthesis of S-Carbamidomethyl Cysteine and Its Use for Quantification of Cysteinyl Peptides by Targeted Proteomics.

Proteomics has played a central role in the identification of reliable disease biomarkers, which are the basis of precision medicine, a promising approach for tackling recalcitrant diseases such as cancer, that elude conventional treatments. Among proteomic methodologies, targeted proteomics employing stable isotope-labeled (SIL) internal standards is particularly suited for the clinical translation of biomarker information owing to its high throughput and accuracy in the quantitative analysis of patient-derived proteomes. Using SIL internal standards ensures the utmost level of confidence in detection and precision in targeted MS experiments. For successfully establishing assays based on targeted proteomics, it is crucial to secure broad coverage when selecting the SIL standard peptide panel. However, cysteinyl peptides have often been excluded because of cysteine's high chemical reactivity. To address this limitation, a new cysteine building block was developed by incorporating a sulfhydryl group configured with an S-carbamidomethyl group, which is commonly used in proteome sampling. This compound was found to be chemically stable and applicable to a variety of solid-phase peptide synthesis (SPPS) campaigns. Furthermore, a direct comparison of the synthesized SIL peptides and tryptic endogenous peptides demonstrated the potential utility of an SPPS flow based on the new cysteine building block for improving the success of targeted proteomic applications.

Modular synthetic strategy for N/C-terminal protected amyloidogenic peptides.

N/C-terminal protected amyloidogenic peptides are valuable biomaterials. Optimization of the protective structures at both termini is, however, synthetically laborious because a linear sequence of solid-phase peptide synthesis protocol (on-resin peptide assembly/peptide removal from resin/high-performance liquid chromatography purification) is required for the peptides each time the protective group is modified. In this study, we demonstrate a modular synthetic strategy for the purpose of rapidly deriving the N/C-terminal structures of amyloidogenic peptides. The precursor sequences that can be easily synthesized due to a non-amyloidogenic property were stocked as the synthetic intermediates. Condensation of the intermediates with N/C-terminal units in a liquid phase followed by high-performance liquid chromatography purification gave the desired peptides P1-P8. The amyloidogenic peptides that have various N/C-terminal protective structures were therefore synthesized in a labor-effective manner. This method is suggested to be useful for synthesizing amyloidogenic peptides possessing divergent protective structures at the N/C-terminus.

In-house standards derived from doping peptides: Enzymatic and serum stability and degradation profile of GHRP and GHRH-related peptides.

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.

Improving 2-Chlorotrityl Chloride (2-CTC) Resin Activation.

Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl2) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl2 as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl2. For instance, the use of 2% SOCl2 in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl2 in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases.

Development of a Mechanistic Amino Acid Activation Reaction Kinetics Model for Safe and Efficient Solid Phase Peptide Synthesis

Development of a Mechanistic Amino Acid Activation Reaction Kinetics Model for Safe and Efficient Solid Phase Peptide Synthesis

Trifluoromethylthiolation of Tryptophan and Tyrosine Derivatives: A Tool for Enhancing the Local Hydrophobicity of Peptides.

The incorporation of fluorinated groups into peptides significantly affects their biophysical properties. We report herein the synthesis of Fmoc-protected trifluoromethylthiolated tyrosine (CF3S-Tyr) and tryptophan (CF3S-Trp) analogues on a gram scale (77-93% yield) and demonstrate their use as highly hydrophobic fluorinated building blocks for peptide chemistry. The developed methodology was successfully applied to the late-stage regioselective trifluoromethylthiolation of Trp residues in short peptides (66-80% yield) and the synthesis of various CF3S-analogues of biologically active monoamines. To prove the concept, Fmoc-(CF3S)Tyr and -Trp were incorporated into the endomorphin-1 chain (EM-1) and into model tripeptides by solid-phase peptide synthesis. A remarkable enhancement of the local hydrophobicity of the trifluoromethylthiolated peptides was quantified by the chromatographic hydrophobicity index determination method, demonstrating the high potential of CF3S-containing amino acids for the rational design of bioactive peptides.

Effects of Conjugation of Ferrocene and Gallic Acid On desCys11/Lys12/Lys13-(p-BthTX-I)2K Peptide: Structure, Permeabilization and Antibacterial Activity.

Antimicrobial resistance is an emerging global health challenge that has led researchers to study alternatives to conventional antibiotics. A promising alternative is antimicrobial peptides (AMPs), produced as the first line of defense by almost all living organisms. To improve its biological activity, the conjugation of AMPs is a promising approach. In this study, we evaluated the N-terminal conjugation of p-Bt (a peptide derived from Bothrops Jararacuçu`s venom) with ferrocene (Fc) and gallic acid (GA). Acetylated and linear versions of p-Bt were also synthesized to evaluate the importance of N-terminal charge and dimeric structure. The compounds were obtained using solid-phase peptide synthesis. Circular dichroism, vesicle permeabilization, antimicrobial activity, and cytotoxicity studies were conducted. No increase in antibacterial activity against Escherichia coli was observed by adding either Fc or GA to p-Bt. However, Fc-p-Bt and GA-p-Bt exhibited improved activity against Staphylococcus aureus. No cytotoxicity upon fibroblast was observed for GA-p-Bt. On the other hand, conjugation with Fc increased cytotoxicity. This toxicity may be related to the membrane permeabilization capacity of this bioconjugate, which showed the highest carboxyfluorescein leakage in vesicle permeabilization experiments. Considering these observations, our findings highlight the importance of adding bioactive organic compounds in the N-terminal position as a tool to modulate the activity of AMPs.

Development of New Leishmanicidal Compounds via Bioconjugation of Antimicrobial Peptides and Antileishmanial Guanidines.

Leishmaniasis refers to a collection of diseases caused by protozoa from the Leishmania genus. These diseases, along with other parasitic afflictions, pose a significant public health issue, particularly given the escalating number of at-risk patients. This group includes immunocompromised individuals and those residing in impoverished conditions. The treatment of leishmaniasis is crucial, particularly in light of the mortality rate associated with nontreatment, which stands at 20-30,000 deaths per year globally. However, the therapeutic options currently available are limited, often ineffective, and potentially toxic. Consequently, the pursuit of new therapeutic alternatives is warranted. This study aims to design, synthesize, and evaluate the leishmanicidal activity of antimicrobial peptides functionalized with guanidine compounds and identify those with enhanced potency and selectivity against the parasite. Accordingly, three bioconjugates were obtained by using the solid-phase peptide synthesis protocol. Each proved to be more potent against intracellular amastigotes than their respective peptide or guanidine compounds alone and demonstrated higher selectivity to the parasites than to the host cells. Thus, the conjugation strategy employed with these compounds effectively contributes to the development of new molecules with leishmanicidal activity.

Sintesis Senyawa Heksapeptida Siklik Analog Pipecolisporin (Pro-Lys-Pip-Trp-

Pipecolisporin compounds isolated from the fungus Nigrospora oryzae from theroots of Triticum sp. is one of the compounds that has potential as an antimalarial with its activity against Plasmodium falciparum of 3.21 μM. The modification of the amino acid sequence to Pro-Lys-Pip-Trp-β-Ala-Phe aims to determine the best arrangement of pipecolisporin analogues as antimalarials. Analogues of pipecolisporin linear hexapeptide compounds were synthesized using the solid phase peptide synthesis (SPPS) method with the use of buffers in the form of 2-chlorotrityl chloride resins, Fmoc and Boc protective groups, and DIC, Oxyma, Dipea coupling reagent solutions. The synthesis results were obtained as much as 9 mg and then characterized using mass spectroscopy with a peak m/z of 959.5246 [M]. Analogues of linear pipecolisporin hexapetide compounds were successfully cyclized using the solution phase peptide synthesis (LPPS) method with the use of DIPEA, HATU, DCM reagent solutions. The synthesis results were characterized using mass spectroscopy with an m/z peak of 743.78 [M+H]+.
 Senyawa pipecolisporin hasil isolasi jamur Nigrospora oryzae dari akar Triticum sp. merupakan salah satu senyawa yang berpotensi sebagai antimalaria dengan aktivitasnya terhadap Plasmodium falciparum sebesar 3,21 μM. Modifikasi urutan asam amino menjadi Pro-Lys-Pip-Trp-

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.

Microwave-Assisted Solid Phase Synthesis of Different Peptide Bonds: Recent Advancements

Abstract: Peptides are important as drugs and biologically active molecules. The synthesis of pep-tides has gathered considerable attention in recent years due to their various attractive properties. Conventional peptide synthesis is tedious and requires hazardous reagents and solvents. Micro-wave-assisted solid-phase peptide synthesis has several advantages compared with conventional batch synthesis. Herein, we have discussed various microwave-assisted solid-phase peptide bond synthesis methods developed over the last five years. Peptides are categorized into four groups - small, medium, large, and cyclic based on their length and structural characteristics to make it easier to understand. This review article also discusses the scope and limitations of microwave-assisted solid-phase peptide synthesis.

Abstract P320: Novel Alpha-calcitonin Gene Related Peptide Agonist Analogs In The Treatment Of Cardiovascular Disease

Alpha-calcitonin gene related peptide (α-CGRP) is a potent vasodilator and protects against heart failure and hypertension in various animal models; however, rapid clearance of the peptide from the circulation makes it difficult to use as a therapeutic agent. Recently, we have synthesized two α-CGRP agonist analogs linked with two n-methoxyethylglycine (NMEG) peptoid molecules at either end: 1)- α-CGRP with NMEG molecules at the N-terminal end (N-ter NMEG), and 2)- α-CGRP with NMEG molecules at the C-terminal end (C-ter NMEG). Bioactivity and toxicity of modified peptides were evaluated in in vitro and in vivo conditions. Peptide-peptoid hybrids, N-ter NMEG and C-ter NMEG, were synthesized using a solid-phase peptide synthesis method with a >98% purity. To test the in vitro cell viability of the analogs, rat H9C2 cardiac myocyte cells were treated with 1 μM, 3 μM, and 10 μM concentrations of N-ter NMEG and C-ter NMEG peptides for 4 days followed by an MTT assay. Our in vitro results demonstrated that neither α-CGRP analogs were toxic to H9C2 cells. To test the bioactivity of these peptides, blood pressure (BP) was measured in wild-type C57BL6 mice using a tail-cuff BP analysis system. A bolus dose of 1.2, 3.6, and 12 (in mg/kg b.wt./mouse) of either peptide was injected subcutaneously (n= 4 mice/dose) followed by BP measurement at increasing time points. Our results demonstrate that there is a variance in the bioactivity of the two peptides. Subcutaneous delivery of N-ter NMEG reduced BP similar to the native peptide. After ten minutes of N-ter NMEG delivery, the BP (in mmHg ±SD) at a dose of 1.2 mg/kg was 76 ±5 (baseline BP= 118 ±15), at a dose of 3.6 mg/kg was 79 ±7 (baseline BP= 113 ±9), and at a dose of 12 mg/mg was 83 ±12 (baseline BP= 104 ±6). In contrast, C-ter NMEG administration did not reduce BP at any peptide concentration tested. In summary, in this study we determined that the α-CGRP analog N-ter NMEG, but not C-ter NMEG, is bioactive. Furthermore, N-ter NMEG showed no toxic effects on H9C2 cardiac myocytes in an in vitro viability assay. These results demonstrate that the analog N-ter NMEG is an effective α-CGRP agonist and a promising candidate molecule to treat cardiac diseases.

Advances in screening, synthesis, modification, and biomedical applications of peptides and peptide aptamers.

Peptides and peptide aptamers have emerged as promising molecules for a wide range of biomedical applications due to their unique properties and versatile functionalities. The screening strategies for identifying peptides and peptide aptamers with desired properties are discussed, including high-throughput screening, display screening technology, and in silico design approaches. The synthesis methods for the efficient production of peptides and peptide aptamers, such as solid-phase peptide synthesis and biosynthesis technology, are described, along with their advantages and limitations. Moreover, various modification techniques are explored to enhance the stability, specificity, and pharmacokinetic properties of peptides and peptide aptamers. This includes chemical modifications, enzymatic modifications, biomodifications, genetic engineering modifications, and physical modifications. Furthermore, the review highlights the diverse biomedical applications of peptides and peptide aptamers, including targeted drug delivery, diagnostics, and therapeutic. This review provides valuable insights into the advancements in screening, synthesis, modification, and biomedical applications of peptides and peptide aptamers. A comprehensive understanding of these aspects will aid researchers in the development of novel peptide-based therapeutics and diagnostic tools for various biomedical challenges.

Antimicrobial peptides: Design, chemical synthesis, activity evaluation, and application

Antimicrobial peptides (AMPs) are active short peptides that exist in microorganisms, insects, amphibians, plants, and mammals. Some naturally occurring AMPs have low antimicrobial activity, high haemolysis, potential toxicity toward mammalian cells, and high susceptibility to proteolytic degradation, which limit their practical application. In recent years, many efforts have been made to design and modify AMPs to improve their properties. The present review focuses on site-directed mutation, truncation, hybridisation, capping, and cyclisation of AMPs. The review further introduces the application of solid-phase peptide synthesis technology for AMPs, and summarises the methods for evaluating the antimicrobial activity of AMPs. The in-depth research on AMPs is expected to play an essential role in the fields of agriculture, animal husbandry, food industry, and medicine.

Tryptophan Substitution in CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) Introduces δ-Opioid Receptor Antagonism, Preventing Antinociceptive Tolerance and Stress-Induced Reinstatement of Extinguished Cocaine-Conditioned Place Preference.

The macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibit kappa opioid receptor (KOR) antagonism which prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference. Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides' opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [35S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2'-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.

Merging Solid-Phase Peptide Synthesis and Automated Glycan Assembly to Prepare Lipid-Peptide-Glycan Chimeras.

Biomaterials with improved biological features can be obtained by conjugating glycans to nanostructured peptides. Creating peptide-glycan chimeras requires superb chemoselectivity. We expedite access to such chimeras by merging peptide and glycan solid-phase syntheses employing a bifunctional monosaccharide. The concept was explored in the context of the on-resin generation of a model α(1→6)tetramannoside linked to peptides, lipids, steroids, and adamantane. Chimeras containing a β(1→6)tetraglucoside and self-assembling peptides such as FF, FFKLVFF, and the amphiphile palmitoyl-VVVAAAKKK were prepared in a fully automated manner. The robust synthetic protocol requires a single purification step to obtain overall yields of about 20 %. The β(1→6)tetraglucoside FFKLVFF chimera produces micelles rather than nanofibers formed by the peptide alone as judged by microscopy and circular dichroism. The peptide amphiphile-glycan chimera forms a disperse fiber network, creating opportunities for new glycan-based nanomaterials.

Morpholine, a strong contender for Fmoc removal in solid-phase peptide synthesis.

Morpholine, which scores 7.5 in terms of greenness and is not a regulated substance, could be considered a strong contender for Fmoc removal in solid-phase peptide synthesis (SPPS). Morpholine in dimethylformamide (DMF) (50%-60%) efficiently removes Fmoc in SPPS, minimizes the formation of diketopiperazine, and almost avoids the aspartimide formation. As a proof of concept, somatostatin has been synthesized using 50% morpholine in DMF with the same purity as when using 20% piperidine-DMF.

Integration of DOTA as a bridging unit during solid-phase peptide synthesis

A method for the introduction of bifunctionalized 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA) into the bridging unit of peptide-based radiopharmaceuticals has been developed. The method is executed in the context of Fmoc-based solid-phase peptide synthesis (SPPS) using standard coupling reagents (HOAt, TBTU, DIPEA) and was optimized to maximize yields (>99% conversion) and suppress the formation of side products (<25%). The bifunctionalized DOTA chelator can be used to integrate radioactive or non-radioactive Ga3+-isotopes. This facilitates novel and structurally simple PET tracer designs and the combination of other functional moieties, such as dyes, with Ga-DOTA-chelates in one hybrid tracer.