A coupling reagent

Abstract

The invention discloses a coupling reagent compound, ((2-oxopyridin-l(2H)- yl)oxy)tri(pyrrolidin-l-yl)phosphonium (PyHOPO), which enhances peptide synthesis by increasing efficiency and purity. PyHOPO is characterized by its phosphonium cation linked to a 2-hydroxypyridine-l -oxide moiety, offering high reactivity and solubility in organic solvents. It improves peptide bond formation, reducing by-products and enhancing the yield of desired peptides. Furthermore, integrating anions like hexahalophosphate, tetrahaloborate, trihalomethanesulfonate, and bis(trihalomethylsulfonyl)imide can optimize reaction conditions. This coupling reagent holds promise for research and industrial applications, providing higher- quality peptides with minimal impurities.

Description

[0001] A COUPLING REAGENT

[0002] RELATED APPLICATION

[0003] This application claims the benefit of priority of Israeli Patent No. 318626, filed on January 27, 2025, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention relates to the field of chemical synthesis and, more particularly, to a novel coupling reagent that can be beneficially utilized in the syntheses of various substances and particularly in peptide synthesis.

[0006] The efficient synthesis of peptides, particularly through the formation of amide bonds, is a fundamental aspect of organic and medicinal chemistry. Peptides play a critical role in biological systems and therapeutic applications, with their significance underscored by the increasing number of peptide-based drugs receiving FDA approval. This demand has fueled the development of advanced coupling methodologies, especially for solid-phase peptide synthesis (SPPS).

[0007] Historically, amide bond formation has relied on carbodiimide-mediated coupling with additives, or on stand-alone coupling reagents such as uronium, aminium, and phosphonium salts. Among these, phosphonium-based coupling reagents have gained prominence due to their superior efficiency, reduced side reactions, and compatibility with diverse synthetic conditions. Phosphonium reagents, such as (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and its safer derivative, PyBOP, are extensively used for activating carboxylic acids to form reactive intermediates. These intermediates enable amines to react efficiently, yielding amide bonds. Unlike uronium-based reagents, which may cause guanidination side reactions that terminate peptide chain elongation, phosphonium reagents are free of such complications, making them ideal for fragment condensation and cyclization steps.

[0008] Thus, the presently known peptide coupling reagents are typically limited by their reactivity, side products formed thereby and / or high cost.

[0009] Due to the ongoing developments of peptide-based drugs and the limitations associated with the presently known peptide coupling reagents, there is a widely recognized need, and it would be highly advantageous to have novel, efficient peptide coupling reagent devoid of the above limitations.SUMMARY OF THE INVENTION

[0010] The present disclosure provides a coupling reagent compound which is particularly, but not exclusively, useful in peptide synthesis. The coupling reagent compound described herein demonstrates superior performance in forming amide bonds between various organic acids and bases, particularly in connecting amines and carboxyls. In the context of peptide synthesis, the novel coupling reagent offers significant advantages over existing alternatives. Notably, it facilitates higher yields of the desired peptide products while maintaining stereochemical integrity. Unlike many conventional coupling agents, this compound stands out for its ability to prevent racemization of amino acids during the reaction process. These unique properties make it an invaluable tool for efficient and stereospecific peptide synthesis.

[0011] Thus, according to an aspect of some embodiments of the present invention, there is provided a compound characterized by the formula presented below:

[0012] N

[0013]

[0014] In some or all embodiments, the compound is defined as ((2-oxopyridin-l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium (PyHOPO), and characterized by a phosphonium cation comprising a 2-hydroxypyridine- 1 -oxide (HOPO) moiety bonded to a phosphorus atom via an oxygen atom of the oxyamine functional group.

[0015] In some or all embodiments, the compound further includes a counterion, wherein the counterion is selected from the group consisting of hexahalophosphate, tetrahaloborate, trihalomethanesulfonate, bis(trihalomethylsulfonyl)imide, toluensulfonate and methanesulfonate.

[0016] The crystal structure analysis confirms and validates the molecular formula presented above. This crystallographic evidence unequivocally establishes the structural identity of the compound that is the object of the present invention. Thus, according to another aspect of some embodiments of the present invention, there is provided a crystal structure of the compound provided herein. In embodiments in which the compound is a hexafluorophosphate salt, the crystal structure is characterized by a distorted tetrahedral geometry around the phosphorus atom of the tri(pyrrolidin- l-yl)phosphonium moiety.According to yet another aspect of some embodiments of the present invention, there is provided a process for synthesizing the coupling reagent compound provided herein, which is effected by:

[0017] (a) providing a reaction mixture by contacting 2-hydroxypyridine- 1 -oxide (HOPO) with a base in a solvent to form an intermediate; and

[0018] (b) adding chloro tripyrrolidinophosphonium (PyClOP) to the reaction mixture, to thereby obtain PyHOPO.

[0019] In some embodiments, the process further which includes:

[0020] (c) allowing the reaction to proceed at room temperature for 3-5 hours;

[0021] (d) optionally filtering the reaction mixture to remove unreacted base and solvent; and (e) optionally isolating PyHOPO by precipitation in a mixed solvent system.

[0022] According to yet another aspect of some embodiments of the present invention, there is provided a method for synthesizing a peptide, which includes utilizing the compound provided herein as a coupling reagent in a coupling reaction for forming at least one amide bond, wherein the compound is used in either a pre-activation or in-situ activation protocol.

[0023] In some or all embodiments, utilizing the compound provided herein is effected during solid-phase peptide synthesis (SPPS).

[0024] In some or all embodiments, the coupling reaction results in reduced racemization of chiral peptide building blocks in the peptide. In some embodiments, the peptide synthesis reaction utilizing the compound provided herein is essentially free of racemization. In some or all embodiments, the peptide synthesis reaction utilizing the compound provided herein exhibits reduced or nullified racemization compared to a coupling reaction under similar conditions but utilizing other coupling reagents such as PyBOP and PyOxim.

[0025] In some or all embodiments, the peptide includes at least one residue selected from the group consisting of Ser, His, and Cys.

[0026] In some or all embodiments, the coupling reagent is utilized in a molar ratio of 1: 1: 1 with suitably -protected amino acid and diisopropylethylamine (DIEA) in dimethylformamide (DMF).

[0027] In some or all embodiments, the coupling reaction is for synthesizing peptides containing His and / or Cys and / or Ser residues, exhibits less than 10 % racemization as measured by any industry-acceptable methodology, including HPLC and NMR.

[0028] According to another aspect of some embodiments of the present invention, there is provided a crude peptide composition, which includes:

[0029] the coupling reagent compound provided herein;

[0030] an amount of at least one peptide;an amount of unreacted protected amino acids and side-chain protecting groups; and a solvent.

[0031] According to another aspect of some embodiments of the present invention, there is provided a method of reducing racemization during amide bond formation during peptide synthesis, which includes utilizing the coupling reagent compound provided herein as a coupling reagent, wherein the method reduces racemization of amino acids prone to racemization, including His, Cys and Ser by at least 90 % compared to amide bond formation in peptide synthesis utilizing, under similar conditions, other coupling reagents, such as, for a non-limiting example, PyBOP or PyOxim.

[0032] According to another aspect of some embodiments of the present invention, there is provided a kit for peptide synthesis, which includes:

[0033] the coupling reagent compound provided herein;

[0034] a plurality of amino acids suitably protected for peptide synthesis, as known in the art; optionally a resin for solid-phase peptide synthesis; and

[0035] optionally instructions for performing pre-activation and / or in-situ activation protocols.

[0036] DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0037] The present invention relates to the field of chemical synthesis and, more particularly, to a novel coupling reagent that can be beneficially utilized in the syntheses of various substances and particularly in peptide synthesis.

[0038] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The disclosure is meant to encompass other embodiments or of being practiced or carried out in various ways.

[0039] The present invention addresses the problem of inefficient peptide synthesis resulting in low yields of desired peptides and high levels of impurities. The invention provides solutions for the challenges associated with traditional coupling reagents in peptide synthesis, which often lead to the formation of undesired peptide sequences and lower purity of the final product, and more specifically, racemization of the chiral amino acid building block, particularly Cys, His and in some cases, Ser.

[0040] While seeking to improve the yield of peptide synthesis reactions, and in particular, to lower or nullify racemization of amino acids during the coupling reaction, the present inventors have discovered a novel compound that can form a peptide bond while maintaining the chiralityof the amino acid building blocks - the compound referred to herein as PyHOPO, or ((2-oxopyridin- 1 (2H)-yl)oxy )tri(pyrrolidin- 1 -yl)phosphonium.

[0041] The use of the herein provided coupling reagent PyHOPO aims to enhance the efficiency of the coupling process, allowing for a more favorable ratio of desired peptides to impurities. Additionally, the incorporation of specific anions with PyHOPO is proposed to further optimize the synthesis conditions, thereby addressing the need for improved methods that yield higher concentrations of peptides with the desired amino acid sequences while minimizing the presence of undesired byproducts.

[0042] Coupling reagent compound:

[0043] The development of PyHOPO [(2-oxopyridin-l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium] represents a significant leap forward in coupling reagent technology. PyHOPO combines the advantages of phosphonium activation with the specific functional properties of its precursor, 2-hydroxypyridine-N-oxide (HOPO). HOPO, known for its similar reactivity compared to other additives like OxymaPure and HOBt, allows for controlled reactions with minimal racemization. PyHOPO leverages this reduced reactivity while introducing the benefits of phosphonium-based coupling, such as stability, compatibility, and suppression of side reactions.

[0044] The phosphonium chemistry exemplified by PyHOPO not only advances SPPS methodologies but also addresses long-standing challenges in peptide synthesis. PyHOPO's novel approach to reducing racemization while maintaining high coupling efficiency offers a powerful tool for the synthesis of complex peptides, further extending the utility of phosphonium reagents in research and industrial applications.

[0045] Thus, according to some embodiments of the present invention, there is provided a compound represented by the chemical structure:

[0046]

[0047] PyHOPOand having the name the IUPAC name ((2-oxopyridin-l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium, abbreviated and referred to herein as “PyHOPO”. PyHOPO is also characterized by a phosphonium cation comprising a 2-hydroxypyridine- 1 -oxide (HOPO) moiety bonded to a phosphorus atom via an oxygen atom of the oxyamine functional group.

[0048] The coupling reagent, presented herein as a phosphonium cation, further comprises a counter anion. In some or all embodiments of the present invention, the counterion is selected from the group consisting of hexahalophosphate, tetrahaloborate, trihalomethanesulfonate and bis(trihalomethylsulfonyl)imide, as well as toluensulfonate and methanesulfonate. It is noted herein that while in use, the coupling reagent is dissolved in an organic or an aqueous medium allowing the anion to be replaced with other anions, not necessarily the abovementioned counterions.

[0049] As can be seen in the Examples section that follows below, PyHOPO has been crystallized and the crystal structure corroborated the chemical structure and further showed that crystallized PyHOPO is characterized by a distorted tetrahedral geometry around said phosphorus atom. In summary, the compound ((2-oxopyridin-l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium (PyHOPO) is characterized by a unique chemical structure featuring a phosphonium cation. This configuration involves a 2-hydroxypyridine- 1 -oxide (HOPO) moiety that is bonded to a phosphorus atom via the oxygen atom of its oxyamine functional group. The atomic configuration around the phosphorus atom in PyHOPO exhibits a distorted tetrahedral geometry, reflecting its reactive nature in facilitating peptide bond formation. PyHOPO carries a positive charge due to the phosphonium cation, which necessitates the presence of a counterion to balance the charge. Suitable counterions for PyHOPO include options from the group consisting of hexahalophosphate, tetrahaloborate, trihalomethanesulfonate, and bis(trihalomethylsulfonyl)imide, as well as toluensulfonate and methanesulfonate. These counterions not only stabilize PyHOPO but also influence its solubility and reactivity, making it highly effective as a coupling reagent in various peptide synthesis applications.

[0050] Thus, in some embodiments of the present invention, the coupling reagent compound, accompanied with a counter ion, include, without limitation:

[0051]

[0052] ((2-oxopyridin- 1 (2H)-yl)oxy)tri(pyrro lidin- 1 -yl)phosphonium hexafluorophosphate;

[0053]

[0054] ((2-oxopyridin-1(2H)-yl)oxy)tri(pyrrolidin-1-yl)phosphonium hexachlorophosphate;

[0055]

[0056] ((2-oxopyridin- 1 (2H)-yl)oxy)tri(pyrrolidin- 1 -yl)phosphonium tetrafluoroborate;

[0057]

[0058] ((2-oxopyridin- 1 (2H)-yl)oxy)tri(pyrrolidin- 1 -yl)phosphonium tetrachloroborate;

[0059]

[0060] ((2-oxopyridin-1(2H)-yl)oxy)tri(pyrrolidin-1-yl)phosphonium trifluoromethanesulfonate;

[0061]

[0062] ((2-oxopyridin- 1 (2H)-yl)oxy)tri(pyrro lidin- 1 -yl)phosphonium trichloromethanesulfonate;

[0063]

[0064] ((2-oxopyridin- 1 (2H)-yl)oxy)tri(pyrrolidin- 1 -yl)phosphonium bis(trifluoromethylsulfonyl)imide;

[0065]

[0066] ((2-oxopyridin- 1 (2H)-yl)oxy)tri(pyrro lidin- 1 -yl)phosphonium bis(trichloromethylsulfonyl)imide;

[0067]

[0068] ((2-oxopyridin- 1 (2H)-yl)oxy)tri(pyrrolidin- 1 -yl)phosphonium toluensulfonate; and

[0069]

[0070] ((2-oxopyridin- l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium methanesulfonate.

[0071] Process for synthesizing PyHOPO:

[0072] A detailed description of an exemplary synthetic procedure for affording PyHOPO is presented in the Examples section that follows below. Generally, the process for synthesizing ((2-oxopyridin-l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium (PyHOPO) involves the following steps:

[0073] Preparation of Reaction Mixture: Begin by preparing a reaction mixture by contacting 2-hydroxypyridine- 1 -oxide (HOPO) with an appropriate base within a solvent. The choice of solventmay include typical organic solvents that facilitate dissolution and reaction of the HOPO and base. This reaction forms an intermediate suitable for further processing.

[0074] Addition of Chlorotripyrrolidinophosphonium:

[0075] Introduce chlorotripyrrolidinophosphonium (PyClOP) into the reaction mixture. The addition of PyClOP initiates a reaction with the intermediate, leading to the formation of PyHOPO. It is preferable to maintain conditions that are conducive to efficient coupling, such as appropriate temperature and stirring, to ensure complete reaction.

[0076] Reaction Conditions: Allow the reaction to proceed at room temperature for a duration of approximately four hours. These conditions are chosen to optimize the formation of PyHOPO while minimizing any potential side reactions or decomposition of reactive species.

[0077] Post-Reaction Processing: Optionally, filter the reaction mixture to remove any unreacted base and solvents, which could interfere with subsequent applications of the product. Filtration ensures that the final product is free from impurities that might affect its performance as a coupling reagent.

[0078] Isolation of PyHOPO: Conclude the synthesis process by isolating PyHOPO through precipitation in a mixed solvent system. This step helps in purifying PyHOPO, leading to a homogeneous product that delivers consistent performance in peptide coupling reactions.

[0079] By adhering to this detailed process, a scalable and cost-effective production of PyHOPO can be achieved, suitable for both research and industrial applications. The methodology emphasizes green chemistry principles by potentially minimizing hazardous waste generation, aligning with sustainable practices in chemical synthesis.

[0080] Peptide synthesis using PyHOPO:

[0081] The method for synthesizing a peptide using ((2-oxopyridin-l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium (PyHOPO) as a coupling reagent is designed to enhance the efficiency and purity of peptide synthesis. This method can be applied either using a pre-activation or in-situ activation protocol, providing flexibility in tailoring the process to specific needs.

[0082] In SPPS, PyHOPO is especially effective. Here, amino acids are added sequentially to a peptide chain that is anchored to a solid resin. This solid support facilitates easier handling and purification. One advantage of using PyHOPO in this process is its ability to significantly reduce the racemization of chiral peptide building blocks, an area where it outperforms conventional reagents such as PyBOP and PyOxim. This is particularly beneficial when synthesizing peptides containing sensitive amino acid residues like cysteine (Cys), histidine (His), and to some extent serine (Ser), which are prone to side reactions.In the context of the present invention, the term "racemization" is defined as the chemical process by which an optically active compound, typically an amino acid or peptide, converts into an equal mixture of its two enantiomers, resulting in a loss of optical activity. This process involves the interconversion of the stereocenters within the molecule, leading to the formation of both left-handed (L) and right-handed (D) isomers. Racemization is a critical consideration in peptide synthesis as it can affect the biological activity and structural integrity of peptides and proteins, impacting their desired functional properties.

[0083] The coupling reaction using PyHOPO generally involves a molar ratio of 1:1:1 with PyHOPO, an Fmoc-protected amino acid, and diisopropylethylamine (DIEA) dissolved in a solvent such as dimethylformamide (DMF). It is noted that other amino acid protection groups and / or alternative organic bases are also contemplated within the scope of the present invention. This ratio affects coupling efficiency and consistent reaction outcomes. PyHOPO effectively minimizes racemization during the synthesis, achieving less than 10 %, less than 5 % and less than 1 % racemization for peptides containing at leasy one Cys, His, and / or Ser residues. This low level of racemization is verified using industry-standard methods like high performance liquid chromatography (HPLC), ensuring the integrity and functionality of the synthesized peptides.

[0084] Overall, this method offers a robust approach to peptide synthesis, yielding high-purity peptides with minimal side reactions and impurities. It is well-suited for both research and therapeutic applications where precision and quality are critical.

[0085] The method for synthesizing a peptide using ((2-oxopyridin-l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium (PyHOPO) as a coupling reagent involves several steps, tailored to enhance efficiency and purity in peptide synthesis:

[0086] Selection of Activation Protocol: PyHOPO can be employed either in a pre-activation or in-situ activation protocol. This flexibility allows for adaptation to specific synthesis requirements and optimizes the coupling conditions for forming one or more amide bonds.

[0087] Solid-Phase Peptide Synthesis (SPPS): The method is particularly effective when applied to SPPS, where amino acids are sequentially added to a growing peptide chain anchored to a solid resin. The solid support facilitates handling and purification processes.

[0088] Reaction Conditions: The coupling reaction typically employs a molar ratio of 1:1:1, involving PyHOPO, an Fmoc-protected amino acid, and diisopropylethylamine (DIEA) in a solvent such as dimethylformamide (DMF). This precise stoichiometry ensures optimal coupling efficiency and reaction consistency.Through these steps, the method optimizes peptide synthesis, yielding high-purity peptides with minimal side reactions and impurities, making it ideal for research and therapeutic applications where precision and quality are paramount.

[0089] As discussed hereinabove, one advantage of using PyHOPO is the significant reduction of racemization in chiral peptide building blocks. When compared to conventional reagents like PyBOP and PyOxim, PyHOPO exhibits superior control, preserving the chirality of sensitive residues. This method is particularly advantageous for synthesizing peptides containing amino acid residues such as serine (Ser), histidine (His), and cysteine (Cys), which are known for their susceptibility to side reactions during synthesis. Racemization levels can be monitored as described in the Examples section that follows below.

[0090] It is noteworthy that coupling reagents that are useful in peptide synthesis, such as the coupling reagent compound provided herein, can also be used in other organic syntheses that require activation of a carboxylic moiety. Such syntheses can be used to produce organic compounds of biological interest such as, for example, small molecules containing simple amide / esters bonds, peptoids, oligocarbamates, oligoamides, β-lactams, esters, polyenamides, benzodiazepines, diketopiperazines, and hydantoins.

[0091] A protocol for assessing racemization:

[0092] In the context of the resent invention, "racemization" refers to the process by which chiral amino acids within a synthetic peptide undergo conversion from their native L-configuration to the non-native D-configuration, or vice versa, resulting in a mixture of both enantiomers. This alteration of stereochemistry can occur during peptide bond formation and is of particular concern in peptide synthesis because it can affect the structural and functional integrity of the peptide. Assessing the level or degree of racemization involves quantifying the proportion of D-amino acids present in the synthesized peptide compared to the L-amino acids, typically through analytical techniques such as chiral high-performance liquid chromatography (HPLC). This assessment is crucial for evaluating the fidelity of the synthetic process and ensuring the resultant peptide's efficacy and safety in its intended application.

[0093] A method for assaying the degree of racemization in synthetic peptides comprises a series of steps aimed at quantifying the proportion of D-amino acids relative to the naturally occurring L-amino acids. This method involves the preparation, hydrolysis, derivatization, and analysis of the peptide sample and includes necessary controls to ensure reliability and reproducibility.

[0094] A typical yet non-limiting example of a racemization assessment protocol may include: Synthesize the peptide of interest using established synthetic protocols, such as SPPS. Confirm the peptide's identity and purity using mass spectrometry, high-performance liquidchromatography (HPLC), or a combination of both. These steps ensure that the assay targets the desired peptide and minimizes interference from impurities or incomplete synthesis. To determine the extent of racemization, a comparative analysis can be performed using HPLC. This involves comparing the HPLC profile of the synthesized peptide with that of a control sample containing the enantiomer of the amino acid under investigation. The resulting data will reveal the degree of racemization that occurred during the synthesis process, providing valuable insights into the stereochemical integrity of the peptide product.

[0095] While the described HPLC-based racemization assay provides a reliable method for evaluating stereochemical purity, it is important to note that this approach is not the only available technique for assessing racemization in peptide synthesis. Skilled artisans in the field would be familiar with and could readily employ various alternative analytical methods to achieve similar results. These may include, but are not limited to, chiral gas chromatography, nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD) spectroscopy, or mass spectrometrybased techniques. The choice of method may depend on factors such as the specific amino acids involved, the complexity of the peptide sequence, available instrumentation, and the desired level of sensitivity. Researchers in the field would be expected to select and adapt the most appropriate technique based on their specific needs and resources, always with the goal of accurately determining the extent of racemization in their synthesized peptides.

[0096] A crude reaction mixture:

[0097] According to an aspect of the present invention, there is provided a crude composition or mixture, comprising,arkers of PyHOPO. In some embodiments, the crude mixture is a crude peptide composition. The crude peptide composition in question can be a mid-process mixture used during peptide synthesis, specifically leveraging the coupling reagent provided herein, namely PyHOPO. This composition embodies a blend of ingredients that are expected to be found in the synthesis reaction mixture. It primarily includes the components 2-hydroxypyridine-1-oxide and / or tri(pyrrolidin-1-yl)phosphane, and tri-pyrrolidyn phosphorotriamide and / or 1-hydroxypyridin-2(1H)-one, which are markers for verifying the use of PyHOPO. These components are byproducts of the process, released during the formation of peptide bonds by activating amino acids for condensation. The presence of these markers in the composition are indicative to the method employed and provided herein, utilizing PyHOPO. Additionally, the crude mixture can also encompass at least one peptide, which is the product of the synthesis, alongside unreacted protected or unprotected amino acids that have not yet participated in bond formation. Lastly, the composition may include a solvent, which acts as a medium to dissolve these components, facilitate the reaction, and allow the mixing and transfer of reactants. Thiscombination of 2-hydroxypyridine-1-oxide and / or tri(pyrrolidin-1-yl)phosphane, tri-pyrrolidyn phosphoro triamide and / or 1-hydroxypyridin-2(1H)-one, and optionally some peptides, some remnants of unreacted amino acids, and solvent constitutes an exemplary crude peptide composition that is indicative of a peptide synthesis process utilizing PyHOPO as a coupling reagent. Identification of such crude composition may serve as a key component for enforcement of the present invention.

[0098] A kit:

[0099] The method for synthesizing a peptide using PyHOPO as part of a peptide synthesis kit is designed to streamline the peptide production process while ensuring high levels of purity and efficiency. The kit includes the coupling reagent PyHOPO, a selection of Fmoc-protected, or otherwise protected amino acids, a resin suitable for SPPS, and detailed instructions for conducting both pre-activation and in-situ activation protocols.

[0100] Using this kit, the peptide synthesis begins by attaching the first protected amino acid to the resin, which serves as a solid support. The solid-phase allows for easy washing and removal of side products after each coupling step, facilitating a cleaner synthesis process. Once the initial amino acid is immobilized, subsequent amino acids are sequentially added to the growing peptide chain. This process is mediated by PyHOPO, the chosen coupling reagent, noted for its ability to enhance the efficiency of amide bond formation, which is vital for constructing polypeptide chains.

[0101] The kit’s instructions provide guidance on how to utilize PyHOPO in two different activation protocols. Pre-activation involves preparing a reactive intermediate before the coupling reaction, while in-situ activation allows for the simultaneous activation and coupling of the amino acids. Both methods offer differing advantages in terms of speed and control over the reaction conditions.

[0102] Notably, the use of PyHOPO in this process helps reduce racemization, preserving the stereochemical integrity of chiral amino acids within the peptide. This characteristic is crucial when synthesizing peptides with functional residues that are sensitive to racemization.

[0103] The inclusion of a comprehensive selection of Fmoc-protected amino acids in the kit ensures that users have access to a wide variety of building blocks to construct diverse peptide sequences. Overall, this method promotes an efficient, high-fidelity process for peptide synthesis, making it suitable for applications in both research and therapeutic contexts.

[0104] Definitions and Abbreviations:

[0105] As used herein, the term "coupling reagent compound" refers to a compound that is capable of sequentially coupling two or more amino acids together in the presence of PyHOPO.As used herein, the term "crude composition" refers to a mixture of peptides, reactants, carriers / solvents and impurities.

[0106] As used herein, the term "peptide" encompasses a biomolecule that comprises a plurality of amino acid residues, linked one to another via a peptide bond or a modification thereof. A peptide includes at least 2 amino acid residues and up to 200 amino acid residues and even more. This term, as used herein, encompasses also polypeptides, peptidomimetics, as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.

[0107] As used herein in the specification and in the claims section below the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.

[0108] Abbreviations used herein for amino acids and the designations of peptides follow the rules d the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972, 247, 977-983.

[0109] Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-conventional, modified and unnatural amino acids (Table 2) which can be used in the context of the present invention.

[0110] Table 1

[0111] Amino Acid Three-Letter One-letter

[0112] Abbreviation Symbol

[0113] alanine Ala A

[0114] Arginine Arg R

[0115] Asparagine Asn N

[0116] Aspartic acid Asp D

[0117] Cysteine Cys C

[0118] Glutamine Gln Q

[0119] Glutamic Acid Glu E

[0120] glycine Gly G

[0121] Histidine His H isoleucine Ile I leucine Leu L

[0122] Lysine Lys K

[0123] Methionine Met M phenylalanine Phe F

[0124] Proline Pro P

[0125] Serine Ser S

[0126] Threonine Thr T tryptophane Trp W

[0127]

[0128] tyrosine Tyr YValine Val V

[0129]

[0130] Any amino acid as above Xaa X

[0131] Table 2

[0132] N on-conventional amino acid Code N on-conventional amino acid Code α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgin carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D -phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcyclopentylalanine Mcpen D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap D-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu OC-napthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec Dmval N-cyclododeclglycine Ncdod

[0133]

[0134] D-OC-methylvalineD-α-methylalnine Dnmala N -cyclooctylglycine Ncoct D-α-methylarginine Dnmarg N -cyclopropylglycine Ncpro D-α-methylasparagine Dnmasn N -cycloundecylglycine Ncund D-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-α-methylcysteine Dnmcys N-(3, 3 -diphenylpropyl)glycine Nbhe D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylomithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Ngly D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-( 1 -methylethyl)glycine Nva D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine Nhis D-N-methylleucine Dnmleu N-(3 -indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylomithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-( 1 -methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys

[0135]

[0136] L-α-methylmethionine Mmet L-α-methylnorleucine Mnle

[0137] L-α-methylnorvaline Mnva L-α-methylornithine Morn L-α-methylphenylalanine Mphe L-α-methylproline Mpro

[0138] L-α-methylserine mser L-α-methylthreonine Mthr L-α-methylvaline Mtrp L-α-methyltyrosine Mtyr L-α-methylleucine Mval Nnbhm L-N -methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl)

[0139] carbamylmethyl-glycine Nnbhm carbamylmethyl(1)glycine Nnbhe

[0140] 1-carboxy-1-(2,2-diphenyl Nmbc

[0141] ethylamino)cyclopropane

[0142]

[0143] Herein throughout, the following abbreviations are used:

[0144] BOP for Benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate;

[0145] Cl-HOBt for 6-chloro-1-hydroxybenzotriazole;

[0146] DCC for N, N'-dicyclohexylcarbodiimide, dicyclohexylcarbodiimide;

[0147] DIBOC for di-t-butyl dicarbonate;

[0148] DIC for 1,3-Diisopropylcarbodiimide; or N, N’ -Diisopropylcarbodiimide;

[0149] DIEA for N, N-Diisopropylethylamine;

[0150] DIP or DIPCDI DIC for N, N'-diisopropylcarbodiimide;

[0151] DMAP for 4-Dimethylaminopyridine;

[0152] DMF for N,N-dimethylformamide;

[0153] EDC for 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide;

[0154] EDC·HCl for 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride;

[0155] Fmoc for 9-fluorenylmethoxycarbonyl;

[0156] HATU for N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide;

[0157] HCTU for N-[(1H-6-chlorobenzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide;

[0158] HAUT for 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;

[0159] HBTU for 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; N-HBTU for N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide;

[0160] HCTU for 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate;

[0161] HOAt for 1-hydroxy-7-azabenzotriazole;

[0162] HOBt for 1-hydroxybenzotriazole;

[0163] HPTU for 2-(2-oxo-1(2H)-pyridyl-1,1,3,3-tetramethyluronium hexafluorophosphate;PyAOP for 7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate;

[0164] PyBOP for benzotriazol- l-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate; TBTU for N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide;

[0165] TCTU for N-[(1H-6-chlorobenzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide;

[0166] TFA for trifluoroacetic acid;

[0167] TFFH for Fluoro-N, N, N", N"-tetramethylformamidinium hexafluorophosphate; and TNBSA for trinitrobenzenesulfonic acid.

[0168] General chemistry:

[0169] Definitions of specific functional groups, chemical terms, and general terms used throughout the specification are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987.

[0170] As used herein, the terms “amine” or ’’amino”, describe both a -NR’R” end group and a -NR'- linking moiety, wherein R’ and R" are each independently hydrogen, alkyl, cycloalkyl, aryl, as these terms are defined hereinbelow.

[0171] The amine group can therefore be a primary amine, where both R’ and R” are hydrogen, a secondary amine, where R’ is hydrogen and R” is alkyl, cycloalkyl or aryl, or a tertiary amine, where each of R’ and R” is independently alkyl, cycloalkyl or aryl.

[0172] Alternatively, R' and R" can each independently be hydrogen, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, carbonyl, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine, as these terms are defined herein.The term "alkyl" describes a saturated aliphatic hydrocarbon including straight chain (unbranched) and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., "1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. Substituted alkyl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine.

[0173] The alkyl group can be an end group, as this phrase is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking moiety, as this phrase is defined hereinabove, which connects two or more moieties via at least two carbons in its chain. When an alkyl is a linking moiety, it is also referred to herein as “alkylene”, e.g., methylene, ethylene, propylene, etc.

[0174] The term "alkenyl" describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be substituted or unsubstituted by one or more substituents, as described for alkyl hereinabove.

[0175] The terms "alkynyl" or "alkyne", as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

[0176] The term "cycloalkyl" describes an all-carbon monocyclic or fused ring (i.e., rings that share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted. Substituted cycloalkyl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The cycloalkyl group can be an end group, as this phrase is defined hereinabove, wherein it is attached to a singleadjacent atom, or a linking moiety, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof.

[0177] The term "hetero alicyclic" describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic may be substituted or unsubstituted. Substituted heteroalicyclic may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The heteroalicyclic group can be an end group, as this phrase is defined hereinabove, where it is attached to a single adjacent atom, or a linking moiety, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.

[0178] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The aryl group may be substituted or unsubstituted. Substituted aryl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The aryl group can be an end group, as this term is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking moiety, as this term is defined hereinabove, connecting two or more moieties at two or more positions thereof. Preferably, the aryl is phenyl.

[0179] The term "heteroaryl" describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted. Substituted heteroaryl may have one ormore substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The heteroaryl group can be an end group, as this phrase is defined hereinabove, where it is attached to a single adjacent atom, or a linking moiety, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof. Representative examples are pyridine, pyrrole, oxazole, indole, purine and the like.

[0180] The term “alkaryl” describes an alkyl, as defined herein, which is substituted by one or more aryl or heteroaryl groups. An example of alkaryl is benzyl.

[0181] The term "amine-oxide” describes a -N(OR’)(R”) or a -N(OR')- group, where R’ and R” are as defined herein. This term refers to a -N(OR')(R") group in cases where the amine-oxide is an end group, as this phrase is defined hereinabove, and to a -N(OR')- group in cases where the amine-oxime is an end group, as this phrase is defined hereinabove.

[0182] As used herein, the term “acyl” refers to a group having the general formula -C(=O)R’, -C(=O)OR’, -C(=O)-O-C(=O)R’, -C(=O)SR’, -C(=O)N(R’)2, -C(=S)R’, -C(= S)N(R’)2, and -C(=S)S(R’), -C(=NR’)R”, -C(=NR’)OR”, -C(=NR’)SR”, and -C(=NR’)N(R”)2, wherein R’ and R” are each independently hydrogen, halo, substituted or unsubstituted hydroxyl, substituted or unsubstituted thiol, substituted or unsubstituted amine, substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic, cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic, cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, hetero aryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphaticamino, mono-or di-heteroaliphaticamino, mono- or di- alkylamino, mono- or di-heteroalkylamino, mono- or diarylamino, or mono- or di-heteroarylamino; or two RX1groups taken together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-CO2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thioxo, cyano, isocyano, amino, azido, nitro,hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, hetero aryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0183] As used herein, the term “aliphatic” or “aliphatic group” denotes an optionally substituted hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (“carbocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-12 carbon atoms. In some embodiments, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms, and in yet other embodiments aliphatic groups contain 1-3 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0184] As used herein, the terms “heteroaliphatic” or “heteroaliphatic group”, denote an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, heteroaliphatic groups contain 1-6 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups.

[0185] The term “halo” describes fluorine, chlorine, bromine or iodine substituent.

[0186] The term "halide" describes an anion of a halogen atom, namely F“, Cl’ Br“ and T.

[0187] The term “haloalkyl” describes an alkyl group as defined above, further substituted by one or more halide.

[0188] The term “sulfate” describes a -O–S(=O)2–OR’ end group, as this term is defined hereinabove, or an –O-S(=O)2-O– linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.The term “thiosulfate” describes a -O-S(=S)(=O)-OR’ end group or a -O-S(=S)(=O)-O-linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0189] The term “sulfite” describes an -O-S(=O)-O-R’ end group or a -O-S(=O)-O- group linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0190] The term “thiosulfite” describes a -O-S(=S)-O-R’ end group or an -O-S(=S)-O- group linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0191] The term “sulfinate” or “sulfinyl” describes a -S(=O)-OR’ end group or an -S(=O)-O-group linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0192] The terms “solfoxide” or “sulfinyl” describe a -S(=O)R’ end group or an -S(=O)- linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0193] The term "sulfonate” or “sulfonyl” describes a –S(=O)2–R’ end group or an –S(=O)2–linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0194] The term “S-sulfonamide” describes a –S(=O)2–NR’R” end group or a –S(=O)2–NR’-linking moiety, as these phrases are defined hereinabove, with R’ and R’ ’ as defined herein.

[0195] The term " N-sulfonamide" describes an R’S(=O)2-NR”- end group or a –S(=O)2–NR’-linking moiety, as these phrases are defined hereinabove, where R’ and R’ ’ are as defined herein.

[0196] The term “disulfide” refers to a -S-SR’ end group or a -S-S- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0197] The term “phosphate” describes an -O-P(=O)2(OR’) end or reactive group or a -O-P(=O)2(O)- linking moiety, as these phrases are defined hereinabove, with R’ as defined herein.

[0198] The term “phosphonate” describes a -P(=O)(OR’)(OR”) end or reactive group or a -P(=O)(OR’)(O)- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0199] The term “thiophosphonate” describes a -P(=S)(OR’)(OR”) end group or a -P(=S)(OR’)(O)- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0200] The term "carbonyl" or "carbonate" as used herein, describes a -C(=O)-R’ end group or a -C(=O)- linking moiety, as these phrases are defined hereinabove, with R’ as defined herein.

[0201] The term "thiocarbonyl" as used herein, describes a -C(=S)-R’ end group or a -C(=S)-linking moiety, as these phrases are defined hereinabove, with R’ as defined herein.

[0202] The term “oxo” as used herein, described a =0 end group.

[0203] The term “thioxo” as used herein, described a =S end group.The term “oxime” describes a =N-OH end group or a =N-O- linking moiety, as these phrases are defined hereinabove.

[0204] The term “hydroxyl” describes a -OH group.

[0205] As used herein, the term “aldehyde” refers to an -C(=O)-H group.

[0206] The term “acyl halide” describes a -(C=O)R"" group wherein R"" is halo, as defined hereinabove.

[0207] The term “alkoxy” as used herein describes an -O-alkyl, an -O-cycloalkyl, as defined hereinabove. The ether group -O- is also a possible linking moiety.

[0208] The term "aryloxy" describes both an -O-aryl and an -O-heteroaryl group, as defined herein.

[0209] The term “disulfide” as used herein describes an -S-S- linking moiety, which in some cases forms between two thiohydroxyl groups.

[0210] The terms “thio”, "sulfhydryl" or "thiohydroxyl" as used herein describe an -SH group. The term "thioalkoxy" or “thioether” describes both a -S-alkyl group, and a -S-cycloalkyl group, as defined herein. The thioether group -S- is also a possible linking moiety.

[0211] The term "thioaryloxy" describes both a -S-aryl and a -S-heteroaryl group, as defined herein. The thioarylether group -S-aryl- is also a possible linking moiety.

[0212] The term "cyano" or “nitrile” describes a -C=N group.

[0213] The term “isocyanate” describes an -N=C=O group.

[0214] The term "nitro" describes an -NO2group.

[0215] The term “carboxylate” or "ester", as used herein encompasses C-carboxylate and O-carboxylate.

[0216] The term “C-carboxylate” describes a -C(=O)-OR’ end group or a -C(=O)-O- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0217] The term “O-carboxylate” describes a -OC(=O)R’ end group or a -OC(=O)- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0218] The term “thiocarboxylate” as used herein encompasses “C-thiocarboxylate and O-thiocarboxylate.

[0219] The term “C-thiocarboxylate” describes a -C(=S)-OR’ end group or a -C(=S)-O- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0220] The term “O-thiocarboxylate” describes a -OC(=S)R’ end group or a -OC(=S)- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0221] The term “carbamate” as used herein encompasses N-carbamate and O-carbamate.The term “N-carbamate” describes an R”OC(=O)-NR’- end group or a -OC(=O)-NR’-linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0222] The term “O-carbamate” describes an -OC(=O)-NR’R” end group or an -OC(=O)-NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0223] The term “thiocarbamate” as used herein encompasses N-thiocarbamate and O-thiocarbamate.

[0224] The term “O-thiocarbamate” describes a -OC(=S)-NR’R” end group or a -OC(=S)-NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0225] The term “N-thiocarbamate” describes an R”OC(=S)NR’- end group or a -OC(=S)NR’-linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0226] The term “dithiocarbamate” as used herein encompasses N-dithiocarbamate and S-dithiocarbamate.

[0227] The term “S -dithiocarbamate” describes a -SC(=S)-NR’R” end group or a -SC(=S)NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0228] The term “N-dithiocarbamate” describes an R”SC(=S)NR’- end group or a -SC(=S)NR’-linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0229] The term "urea", which is also referred to herein as “ureido”, describes a -NR’C(=O)-NR”R’” end group or a -NR’C(=O)-NR”- linking moiety, as these phrases are defined hereinabove, where R’ and R” are as defined herein and R'" is as defined herein for R' and R".

[0230] The term “thiourea”, which is also referred to herein as “thioureido”, describes a -NR’-C(=S)-NR”R”’ end group or a -NR’-C(=S)-NR”- linking moiety, with R’, R” and R”’ as defined herein.

[0231] The term “amide” as used herein encompasses C-amide and N-amide.

[0232] The term “C-amide” describes a -C(=O)-NR’R” end group or a -C(=O)-NR’- linking moiety, as these phrases are defined hereinabove, where R’ and R” are as defined herein.

[0233] The term “N-amide” describes a R’C(=O)-NR”- end group or a R’C(=O)-N- linking moiety, as these phrases are defined hereinabove, where R’ and R” are as defined herein.

[0234] The term “imine”, which is also referred to in the art interchangeably as “Schiff-base”, describes a -N=CR'- linking moiety, with R' as defined herein or hydrogen. As is well known in the art, Schiff bases are typically formed by reacting an aldehyde or a ketone and an amine-containing moiety such as amine, hydrazine, hydrazide and the like, as these terms are definedherein. The term “aldimine" refers to a -CH=N- imine which is derived from an aldehyde. The term “ketimine" refers to a -CR-N- imine which is derived from a ketone.

[0235] The term “hydrazone" refers to a -R'C=N-NR”- linking moiety, wherein R’ and R” are as defined herein.

[0236] The term “semicarbazone" refers to a linking moiety which forms in a condensation reaction between an aldehyde or ketone and semicarbazide. A semicarbazone linking moiety stemming from a ketone is a -R'C=NNR" C(=O)NR"'-, and a linking moiety stemming from an aldehyde is a -CR'=NNR" C(=O)NR"'-, wherein R’ and R” are as defined herein and R'" or as defined for R’.

[0237] As used herein, the term "lactone" refers to a cyclic ester, namely the intra-condensation product of an alcohol group -OH and a carboxylic acid group -COOH in the same molecule.

[0238] As used herein, the term "lactam" refers to a cyclic amide, as this term is defined herein. A lactam with two carbon atoms beside the carbonyl and four ring atoms in total is referred to as a P-lactam, a lactam with three carbon atoms beside the carbonyl and five ring atoms in total is referred to as a y-lactam, a lactam with four carbon atoms beside the carbonyl and six ring atoms in total is referred to as a 5-lactam, and so on.

[0239] The term “guanyl” describes a R’R”NC(=N)- end group or a -R’NC(=N)- linking moiety, as these phrases are defined hereinabove, where R’ and R” are as defined herein.

[0240] The term “guanidine” describes a -R’NC(=N)-NR”R”’ end group or a -R’NC(=N)- NR”- linking moiety, as these phrases are defined hereinabove, where R’, R" and R'" are as defined herein.

[0241] The term “hydrazine” describes a -NR’-NR”R”’ end group or a -NR’-NR”- linking moiety, as these phrases are defined hereinabove, with R’, R”, and R'" as defined herein.

[0242] As used herein, the term “hydrazide” describes a -C(=O)-NR’-NR”R”’ end group or a -C(=O)-NR’-NR”- linking moiety, as these phrases are defined hereinabove, where R’, R” and R’” are as defined herein.

[0243] The term "hydroxylamine", as used herein, refers to either a -NHOH group or a -ONH2. As used herein, the terms “azo” or “diazo” describe a -N=N-R’ end group or a -N=N-linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0244] As used herein, the term “azido” described a -N=N+=N“ (-N3) end group.

[0245] The term “triazine" refers to a heterocyclic ring, analogous to the six-membered benzene ring but with three carbons replaced by nitrogen atoms. The three isomers of triazine are distinguished from each other by the positions of their nitrogen atoms, and are referred to as 1,2,3-triazine, 1,2,4-triazine, and 1,3,5-triazine. Other aromatic nitrogen heterocycles include pyridineswith 1 ring nitrogen atom, diazines with 2 nitrogen atoms in the ring and tetrazines with 4 ring nitrogen atoms.

[0246] The term "triazole" refers to either one of a pair of isomeric chemical compounds with molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms, namely 1,2,3-triazoles and 1,2,4-triazoles.

[0247] The term “aziridine", as used herein, refers to a reactive group which is a three membered heterocycle with one amine group and two methylene groups, having a molecular formula of -C2H3NH.

[0248] As used herein, the term “thiohydrazide” describes a -C(=S)-NR’-NR”R”’ end group or a -C(=S)-NR’-NR”- linking moiety, as these phrases are defined hereinabove, where R’, R” and R’” are as defined herein.

[0249] As used herein, the term “methyleneamine” describes an -NR’-CH2-CH=CR”R”’ end group or a -NR’-CH2-CH=CR”- linking moiety, as these phrases are defined hereinabove, where R’, R” and R’” are as defined herein.

[0250] The term "diene", as used herein, refers to a -CR'=CR"-CR"'=CR""- group, wherein R’ as defined hereinabove, and R", R'" and R"" are as defined for R'.

[0251] The term "dienophile", as used herein, refers to a reactive group that reacts with a diene, typically in a Diels-Alder reaction mechanism, hence a dienophile is typically a double bond or an alkenyl.

[0252] The term “epoxy", as used herein, refers to a reactive group which is a three membered heterocycle with one oxygen and two methylene groups, having a molecular formula of -C2H3O.

[0253] The phrase "covalent bond", as used herein, refers to one or more pairs of electrons that are shared between atoms in a form of chemical bonding.

[0254] General Definitions:

[0255] As used herein the term “about” or “approximately,” refers to ±10 %. For example, the term “about 100 units” encompasses the value 100 units, as well as the values 90 units, 91 units, 92 units, 93 units, 94 units, 95 units, 96 units, 97 units, 98 units, 98 units, 99 units, 100 units, 101 units, 102 units, 103 units, 104 units, 105 units, 106 units, 107 units, 108 units, 109 units, and 110 units.

[0256] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to"; namely, as used herein, these terms are intended to be open-ended and not limiting. They indicate that the presence of the listed elements does not preclude the inclusion of additional, unrecited elements or method steps.

[0257] The term “consisting of’ means “including and limited to”.The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0258] The phrase “one or more” as used herein includes one, two, three, or more of the described elements or components and does not exclude any combinations or sub-combinations thereof.

[0259] The terms “preferred” or “preferably” indicate an example or embodiment that is more suitable or favorable under certain circumstances, but these terms are not intended to limit the scope of the invention or to suggest that other variations are excluded.

[0260] As used herein, the phrase “selected from the group consisting of’ includes all members of the recited group, each member of the recited group, and all possible combinations. For example, selected from the group consisting of A, B, and C, includes A, only, as well as B, only, as well as C, only, as well as A and B, as well as A and C, as well as B and C, and as well as A, B, and C.

[0261] The term “substantially,” when used in reference to a characteristic or parameter, means that the characteristic or parameter need not be absolute but is close enough to the specified value or condition so as to achieve the intended purpose or effect.

[0262] As used herein, the phrases "substantially devoid of" and / or "essentially devoid of" in the context of a certain substance, refer to a composition that is totally devoid of this substance or includes less than about 5, 1, 0.5 or 0.1 percent of the substance by total weight or volume of the composition. Alternatively, the phrases "substantially devoid of" and / or "essentially devoid of" in the context of a process, a method, a property or a characteristic, refer to a process, a composition, a structure or an article that is totally devoid of a certain process / method step, or a certain property or a certain characteristic, or a process / method wherein the certain process / method step is effected at less than about 5, 1, 0.5 or 0.1 percent compared to a given standard process / method, or property or a characteristic characterized by less than about 5, 1, 0.5 or 0.1 percent of the property or characteristic, compared to a given standard. Further alternatively, the terms "substantially" and / or "essentially " in the context of a characterizing property, means that the characterizing property is expressed to at least 99 %, at least 95 %, at least 90 % of its full or complete expression. For example, the phrase “the elements are maintained substantially in a certain configuration” should be read as “at least 99 % of the elements are maintained in the certain configuration.”

[0263] As used herein, the phrases "substantially devoid of" and / or "essentially devoid of" in the context of a certain substance, refer to a composition that is totally devoid of this substance or includes less than about 5, 1, 0.5 or 0.1 percent of the substance by total weight or volume of the composition. Alternatively, the phrases "substantially devoid of" and / or "essentially devoid of" inthe context of a process, a method, a property or a characteristic, refer to a process, a composition, a structure or an article that is totally devoid of a certain process / method step, or a certain property or a certain characteristic, or a process / method wherein the certain process / method step is effected at less than about 5, 1, 0.5 or 0.1 percent compared to a given standard process / method, or property or a characteristic characterized by less than about 5, 1, 0.5 or 0.1 percent of the property or characteristic, compared to a given standard.

[0264] When applied to an original property, or a desired property, or an afforded property of an object or a composition, the term “substantially maintaining”, as used herein, means that the property has not change by more than 20 %, 10 % or more than 5 % in the processed object or composition.

[0265] The term “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

[0266] The words “optionally” or “alternatively” are used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

[0267] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0268] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0269] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.As used herein the terms “process” and "method" refer to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, material, mechanical, computational and digital arts.

[0270] Terms used in the singular form shall also include the plural, and vice versa, unless context clearly indicates otherwise. Furthermore, words of any gender include all genders and are intended to cover all corresponding terms.

[0271] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0272] It is expected that during the life of a patent maturing from this application many relevant coupling reagents will be developed and the scope of the phrase "coupling reagent" or “coupling agent” is intended to include all such new technologies a priori.

[0273] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0274] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0275] EXAMPLES

[0276] Reference is now made to the following examples, which together with the above descriptions, illustrate some embodiments of the invention in a non-limiting fashion.EXAMPLE 1

[0277] Synthesis of PyHOPO

[0278] All reagents and solvents were purchased from commercial suppliers and used without further purification. Fmoc amino acids, Fmoc-RinkAmide-AM-PS resin (loading 0.69 mmol / g), were purchased from Iris Biotech. HOPO, PyBOP and PyOxim were gifted from Luxembourg Biotech. Organic solvents, dimethylformamide (DMF) and HPLC quality acetonitrile (CH3CN), were purchased from Merck. Milli-Q water was used for RP-HPLC.

[0279] Analytical HPLC was performed on an Agilent 1100 system using a Phenomenex AerisTMC18 (3.6 pm, 4.6 * 150 mm) column, with a flow rate of 1.0 mL / min and UV detection at 220 nm. Chemstation software was used for data processing. Buffer A: 0.1% TFA in H₂O; buffer B: 0.1% TFA in CH₃CN.

[0280] LC-MS was performed on a Thermo Fisher Scientific UltiMate 3000 UHPLC-ISQTM EC single quadrupole mass spectrometer in positive ion mode using a Phenomenex AerisTM C18 (3.6 pm, 4.6 x 150 mm) column. Buffer A: 0.1% formic acid in H₂O; buffer B: 0.1% formic acid in CH₃CN. Method: 5-60% B into A in 15 min.

[0281] In a round bottom flask, 2-hydroxypyridine- 1 -oxide (HOPO) (1.0 g, 9.0 mmol, leq.) was dissolved in ACN (20 mL). K₂CO₃ (0.6 g, 4.5 mmol, 0.5 eq.) was added, and the reaction was stirred for 10 mins at rt. Chloro tripyrrolidinophosphonium hexafluorophosphate (PyClOP) (3.8 g, 9.0 mmol, 1 eq.) was added to the stirring reaction mixture and was allowed to react for 4 h at rt. The completion of the reaction was monitored by TLC using n-Hex: EtOAc (7:3). The reaction mixture was then filtered to remove unreacted K2CO3 and concentrated the filtrate under high vacuum. The residue was further washed with ACN (10 mL). The filtrate was concentrated as the residue was dissolved in DCM:n-Hex (1:3) (50 mL) and placed the mixture in ice bath undisturbed for precipitation. After precipitation the residue was filtered and dried to afford PyHOPO 4.2 g (94 % yield). HPLC [5-95% of ACN (0.1% TFA / H2O (0.1% TFA) over 15 min] tR= 6.05 min; ’H NMR (600 MHz, CDCI3): 5=7.70 (d, J= 7.2 Hz; ArH), 7.43 (dd, J= 7.1 Hz, ArH), 6.68 (dd, J= 9.2 Hz, ArH), 6.45 (dd, J= 6.9 Hz, ArH), 3.38 (m, 12H), 1.97 (m, 12H),13C NMR (150 MHz, CDCl₃): 157.4, 140.6, 136.1, 122.3, 107.3, 48.2, 48.1, 26.1, 26.0.

[0282] Synthesis of PyHOPO was performed using HOPO wherein later was reacted with a base initially followed by reaction with chloro tripyrrolidinophosphonium hexafluorophosphate (PyClOP). The desired product was obtained as a white powder in excellent yield (94 %) and purity (>99%) as monitored by HPLC.Scheme 1

[0283] i. K2CO3, ACN, 10 mins, rt

[0284] "■ Phosphonium salt, 4h, rt

[0285]

[0286] HOPO

[0287]

[0288] PyHOPO

[0289] EXAMPLE 2

[0290] Crystal Structure of PyHOPO

[0291] Colorless trapezoid- shaped crystals of PyHOPO were obtained by slow evaporation technique of a methanolic solution at ambient temperatures. The X-ray crystallographic investigations were recorded on a Bruker Smart APEXII diffractometer with a MoKa radiation source.11The crystal was kept at 293(2) K during data collection. Using Olex2, the structure was solved with the SHELXS structure solution program using Direct Methods and refined with the SHELXL refinement package using Least Squares minimisation.12 15The crystallographic data and structure refinement details are summarized in Table 3. The disordered PF₆ and pyrrolidine moieties were modelled using PART instructions with the major component having site occupancy of 58 %. Table 3 presents the crystal data and structure refinement details for compound PyHOPO.

[0292] Table 3

[0293] Empirical formula C 17H28F6N4O2P2

[0294] CCDC number

[0295] Formula weight 496.37

[0296] Crystal system Orthorhombic

[0297] Space group Pbca

[0298] a / A 13.6740(4)

[0299] b / A 24.9821(7)

[0300] c / A 26.5484(8)

[0301] a / ° 90

[0302] p / ° 90

[0303] y / ° 90

[0304] Volume / A39069.1(5)

[0305] Z 16

[0306] Pcalcg / Cm31.454

[0307] μ / mm-10.262

[0308] F(000) 4128.0

[0309]

[0310] Crystal size / mm30.32 x 0.24 x 0.2120 range for data collection / 03.068 to 51.998

[0311] Index ranges -16 < h < 16, -30 < k < 30, -32 < 1 < 32 Reflections collected 266290

[0312] Independent reflections 8906 [Rint = 0.0590, Rsigma = 0.0159] Data / restraints / parameters 8906 / 13 / 641

[0313] Goodness-of-fit on F21.027

[0314] Final R indexes [I>=2σ (I)] R₁ = 0.0641, wR₂ = 0.1762

[0315] Final R indexes [all data] R₁ = 0.0894, wR₂ = 0.2020

[0316]

[0317] Largest diff. peak / hole / e A-30.53 / -0.35

[0318] The crystal structure of PyHOPO consists of phosphonium cations and PF₆⁻ anions as an exemplary crystal structure. Each cationic species constitutes of a HOPO moiety that is bonded to the phosphorus atom via the oxygen atom of the oxyamine functional group as shown in Scheme 2 below, which shows the molecule diagram of PyHOPO with ellipsoids drawn at 40 % thermal probability, whereas the disordered components and hydrogen atoms have been omitted for clarity. Moreover, the phosphorus atom adopts a distorted tetrahedral geometry with bond angles of 99.8(1)-115.1(1)°.

[0319] Scheme 2

[0320]

[0321] As can be seen in Scheme 2, the HOPO moiety exhibits a near orthogonal orientation with respect to the P — O bond given P2 — 03 — N8 — C30 and Pl — 01 — N4 — C17 torsion angles of 91.3(3) and -98.7(3), respectively. The other intramolecular bond parameters appear to be comparable with those of closely related structures in literature.EXAMPLE 3

[0322] Peptide Synthesis using PyHOPO

[0323] General:

[0324] All peptides were assembled manually in plastic syringes fitted with a porous polypropylene disk. The Fmoc / fBu strategy was used for synthesis using PyHOPO / PyBOP / PyOxim in DMF as a coupling cocktail. Fmoc removal was carried out using 20 % piperidine in DMF (PIP / DMF). All the peptides were synthesized using the protocol presented in Scheme 3 below, showing SPPS of H-Tyr-Ser-Ser-Phe-Leu-NFh using PyHOPO and PyBOP as coupling reagent.

[0325] SPPS of H-Tyr-Ser-Ser-Phe-Leu-NH2:

[0326] To understand the applicability of PyHOPO as coupling reagent, pentapeptide (H-Tyr-Ser-Scr-Phc-Lcu-NHi) was synthesized and compared with the peptide synthesized using PyBOP (see, Scheme 3). HPLC analysis of the two synthesis (results not shown) demonstrated that the synthesis of pentapeptide was efficient and quite comparable in both cases of coupling with either PyBOP or PyHOPO, although the purity of the peptide synthesized with PyBOP was superior to the one synthesized with PyHOPO as expected.

[0327] Scheme 3

[0328] i. Fmoc-Leu-OH:

[0329] PyHOPOorPyBOP

[0330] : DIEA (3:3:3), 1h i. Swelling 10 mins

[0331] H ii Washing DMFx3 ii Washing DMFx3 H Fmoc-Leu— N— A - H2N— A -* - Fmoc— N— A

[0332] ill Fmoc removal (20%plp pmoc removal (20%PIP

[0333] in DMF; 1+7 min) in DMF; 1+7 mm)

[0334] iv. Washing DMFx3 iv. Washing DMFx3

[0335] v Repeat Previous steps

[0336] ’I TFA: TIS: H2O

[0337] (95-25’25)

[0338] H-Tyr(tBu)-Ser(Trt)-Ser(T

[0339]

[0340] rt)-Phe-Leu-NH~> ■ ■ ■ ■ >„ H-Tyr-Ser-Ser-Phe-Leu-NH2

[0341] Fmoc-Rink-Amide AM-PS resin (100 mg, 0.69 mmol / g resin loading) was washed with DMF (3 times). Deprotection of the Fmoc group was achieved by the treatment of the resin with 20% PIP / DMF (1 x 1 min; 1 x 7 min) followed by washing DMF (3 times). The protected Fmoc-amino acids (3.0 eq.), PyHOPO or PyBOP (3.0 eq.) and DIEA (3 eq.) were dissolved in DMF and added to the resin. Upon addition and after 1 h of coupling, filtration was done to remove excess coupling cocktail. Washing was then performed with DMF (3 times). Coupling and deprotection were repeated until the peptide was assembled onto the resin. The peptidyl resin was dried, and the peptide was cleaved from the resin by treating it with TFA / TIS / H2O (95:2.5:2.5) for 90 min at rt. After that, the peptide was precipitated using chilled diethyl ether. The precipitate wascentrifuged to afford the desired peptide, as confirmed by HPLC and LCMS for purity and mass, respectively.

[0342] SPPS of H-Gly-X-Phe-N H2 (where X = Ser, His, Phe):

[0343] Followed by these promising results, SPPS was performed to synthesize tripeptides (H-Gly-X-Phe-NH2 where X = Ser, His, Cys). Three coupling reagents PyHOPO, PyBOP and PyOxim were evaluated to understand the effect of racemization. Two approaches for activation were adopted for the synthesis, i.e., via pre-activation or in-situ activation (see, Scheme 4). Similar protocol was adopted as above to synthesize H-Gly-X-Phe-NHi (where X = Ser, His, Phe). Different coupling reagents viz-, PyHOPO, PyBOP and PyOxim were used in parallel for synthesis of tripeptide H-Gly-X-Phc-NHi (where X = Ser, His, Phe). D-Amino acids (Ser, His and Phe) were also used to synthesize the above tripeptides for comparison to calculate percentage of racemization. Scheme 4 presents the synthetic protocol for racemization study.

[0344] Scheme 4

[0345] i. Fmoc-Phe-OH

[0346] ■PyHOPO / PyBOg / PyOxim

[0347] : D1EA (3:3:3), 1 h i. Swelling 10 mins

[0348] ii Washing DMFx3 II Washing DMFx3

[0349] Fmoc-Phe— N

[0350]

[0351]

[0352] Fmoc-N H F o r m v l

[0353]

[0354]

[0355] i m c e o a (20% PIP Hi Fmoc removal (20%

[0356] in DMF; 1+7 min) in DMF; 1+7 min)

[0357] iv. Washing DMFx3 iv. Washing DMFx3

[0358] Repeat Previous step

[0359] TFA: TIS: H2O X=Ser * Preactivation or H-Gly-X-Phe-NH - < (95:2.5:2.5)^H-G|y-X-Phe-NH2His in-situ activation Cys approach

[0360] Racemization was studied initially for the synthesis of H-Gly-Scr-Phc-NHi. In this case the tripeptide was synthesized by PyHOPO as coupling reagent using two approaches. In one case, pre-activation was performed wherein, Fmoc-Phe-OH-PyHOPO-DIEA (1: 1: 1; 3 eq) in DMF were mixed and pre-activated for 3 mins and then the coupling cocktail was added to the resin. In both cases, the coupling was allowed to take place for 1 hour at room temperature. Fmoc was then removed and Fmoc-Ser(Bu)-OH was coupled using both approaches as explained earlier. In this way, tripeptide H-Gly-Scr-Phc-NHi was synthesized. The synthesis was repeated replacing PyHOPO with PyBOP and PyOxim for comparison. In parallel, H-Gly-D-Ser-Phe-NHi was also synthesized to determine the amount of racemization in each case. Table 4 presents the analysis of the HPLC resultsm leading to the determination of percentage of racemization in tripeptides. As can be seen in Table 4, racemization was not found (not detected by the HPLC assay utilized) in any of case for H-Gly-Ser-Phe-NHi.Table 4

[0361] Peptide Coupling

[0362] PyHOPO PyBOP PyOxim (HPLC method) Method

[0363] H-Gly-Ser-Phe-NH2Pre-activation - - - (0-30)* In- Situ - - - H-Gly-His-Phe-NH2Pre-activation - 0.4 2.9

[0364] (5-20)* In- Situ - 1.0 3.0

[0365] H-Gly-Cys-Phe-NH2Pre-activation - 0.8 - (0-40)*

[0366]

[0367] In- Situ - 0.8 - * HPLC Method %B into A

[0368] Further, tripeptide H-Gly-His-Phe-NFh and H-Gly-Cys-Phc-NHi containing Cys and His (amino acids more prone to racemization) were synthesized using the above two protocol of preactivation and in-situ activation. It is evident that no racemization was observed in case of PyHOPO in both the tripeptides containing His and Cys. Peptide synthesized using PyBOP showed racemization in both cases of Cys and His whereas PyOxim lead to racemization in case of His but not in the case of Cys (see, Table 4).

[0369] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0370] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A compound of the formula2. The compound of claim 1, wherein the compound is defined as ((2-oxopyridin-l(2H)-yl)oxy)tri(pyrrolidin-l-yl)phosphonium (PyHOPO), and characterized by a phosphonium cation comprising a 2-hydroxypyridine- 1 -oxide (HOPO) moiety bonded to a phosphorus atom via an oxygen atom of the oxyamine functional group.

3. The compound of any one of claims 1-2, further comprising a counterion, wherein said counterion is selected from the group consisting of hexahalophosphate, tetrahaloborate, trihalomethanesulfonate and bis(trihalomethylsulfonyl)imide.

4. A crystal structure of the compound of any one of claims 1-3, characterized by a distorted tetrahedral geometry around said phosphorus atom.

5. A process for synthesizing the compound of any preceding claim, comprising: (a) providing a reaction mixture by contacting 2-hydroxypyridine- 1 -oxide (HOPO) with a base in a solvent to form an intermediate; and(b) adding chloro tripyrrolidinophosphonium (PyClOP) to said reaction mixture, to thereby obtain PyHOPO.

6. The process of claim 5, further comprising:(c) allowing the reaction to proceed at room temperature for 4 hours;(d) optionally filtering the reaction mixture to remove unreacted base and solvent; and (e) optionally isolating PyHOPO by precipitation in a mixed solvent system.

7. A method for synthesizing a peptide, comprising utilizing the compound of any one of claims 1-3 as a coupling agent in a coupling reaction for forming at least one amide bond, wherein the compound is used in either a pre- activation or in-situ activation protocol.

8. The method of claim 7, wherein said utilizing is effected during solid-phase peptide synthesis (SPPS).

9. The method of any one of claims 7-8, wherein said coupling reaction results in reduced racemization of chiral peptide building blocks in the peptide compared to a coupling reaction utilizing coupling agents selected from PyBOP and PyOxim.

10. The method of any one of claims 7-9, wherein the peptide comprises at least one residue selected from the group consisting of Ser, His, and Cys.

11. The method of any one of claims 7-10, wherein the coupling reagent is utilized in a molar ratio of 1:1:1 with Fmoc-protected amino acid and diisopropylethylamine (DIEA) in dimethylformamide (DMF).

12. The method of any one of claims 7-11, wherein said coupling reaction is for synthesizing peptides containing His and / or Cys and / or Ser residues, exhibits less than 10 % racemization as measured by any industry-acceptable methodology.

13. A crude peptide composition comprising:a detectable amount of 2-hydroxypyridine- 1 -oxide and / or tri(pyrrolidin-1-yl)phosphane, anda detectable amount of tri-pyrrolidyn phosphorotriamide and / or 1-hydroxypyridin-2(1H)-one;a detectable amount of at least one peptide;a detectable amount of unreacted protected and / or unprotected amino acids; and a detectable amount of a solvent.

14. A method of reducing racemization during amide bond formation in peptide synthesis, comprising utilizing the compound of any one of claims 1-3 as a coupling reagent, wherein the method reduces racemization of amino acids prone to racemization, including His,Cys and Ser by at least 90 % compared to amide bond formation in peptide synthesis utilizing PyBOP or PyOxim under similar conditions.

15. A kit for peptide synthesis comprising:the compound of any one of claims 1-3;a plurality of Fmoc -protected amino acids;a resin for solid-phase peptide synthesis; andinstructions for performing pre-activation and in-situ activation protocols.

Images