WO2009138985A2

WO2009138985A2 - Coupling agents for the synthesis of polypeptides and polynucleotides

Info

Publication number: WO2009138985A2
Application number: PCT/IL2009/000486
Authority: WO
Inventors: Fernando Albericio; Ayman El-Faham; Yoav Luxembourg; Ariel Ewenson
Original assignee: Luxembourg Bio Technologies Ltd.
Priority date: 2008-05-15
Filing date: 2009-05-14
Publication date: 2009-11-19
Also published as: WO2009138985A3

Abstract

Novel coupling agents containing a unique leaving group moiety, which can be used beneficially and safely as coupling agents in various chemical polypeptide and/or polynucleotide syntheses, and are particularly useful as yield enhancing and racemization suppressing coupling agents for use in peptide syntheses, are disclosed. Also disclosed are processes of preparing such coupling agents and their use in the preparation of polypeptides and/or polynucleotides.

Description

COUPLINGAGENTS FORTHE SYNTHESIS OF POLYPEPTIDES AND

POLYNUCLEOTn⁾ES

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to coupling agents and, more particularly, but not exclusively, to the preparation and use of novel coupling agents that can be beneficially utilized in the syntheses of substances such as peptides and oligonucleotides.

A key step in the peptide production process is the controlled formation of a peptide bond (an amide bond formed between a carboxylic acid group and an amine group) between two amino acids (the so-called "coupling" reaction). In peptide syntheses, formation of a peptide bond typically requires proper management of protecting groups, and the activation of the carboxylic acid, or a carboxy group in general, which usually involves the use of a peptide coupling agent [for a comprehensive review on peptide coupling agents see, F. Albericio, S.A. Kates, Solid- Phase Synthesis: A Practical Guide, S.A. Kates, F. Albericio Eds; Marcel Dekker, New York, NY, 2000, ρρ.273-328 and F. Albericio, R. Chinchilla, DJ. Dodsworth, C. Najera, 2001, Org. Prep. Proc. Int., 33, 202].

Phosphate groups can also be activated in a way similar to the activation of carboxyl groups for coupling to amino groups in peptide synthesis. The activation of phosphate groups is an essential step in the synthesis of various nucleic acids and oligonucleotides used to build DNA and RNA, as well as molecules which mimic the chemical structure and thus the activity of the latter, which can be effected by a diverse group of activating or coupling reagents. Such activating reagents and reaction conditions which can be used for activation of phosphate groups are described in the art of peptide synthesis (see for example, L. Carpino (1997) Methods in Enzymology, 289: 104 and WO 2006/063717) as discussed herein. Although the synthesis of medium-large peptides for basic research is a well established procedure, the combination of the 20 naturally occurring amino acids and a growing number of unnatural amino acids makes each peptide synthesis unique at the industrial level, oftentimes requiring closer attention to each amino acid coupling. Some of the rules for coupling agents validated in the research scale can be applied at industrial level, but the results are still hardly predictable. Similar circumstances exist also in the field of polynucleotides and nucleic acids synthesis.

The two main classes of coupling techniques involve (a) those that require in situ activation of the carboxylic acid and (b) those that depend on an activated amino- acid species that has previously been prepared, isolated, purified, and characterized. The first type is by far the most convenient for the stepwise elongation of a peptide chain and is the more commonly used in convergent processes, where protected peptides are used instead of protected amino acids.

As mentioned herein, the role of the coupling agent is the activation of the carboxyl group of one amino acid which facilitates its coupling with the amino group of another amino acid. The process of activation is probably the one aspect of peptide synthesis which has been most extensively developed in recent years. An essential feature of all coupling methods is that, in addition to improving the yield of the peptide- bond formation, the configurational integrity of the carboxylic component must be maintained as well, namely no racemization should occur at any of the amino acid chiral centers.

This duality in coupling agent requirements, i.e. high peptide-bond formation yield and absence of amino acid racemization, is often difficult to achieve, since usually the most effective peptide-bond formation methods involve conversion of the acid to an intermediate bearing a good leaving group. Such leaving groups tend to increase the acidity of the α-proton of the activated amino acid, enhancing deprotonation and formation of an oxazolone, both of which lead to loss of the stereo-configuration.

Racemization is a side-reaction that occurs during the preparation of a peptide. In a production scale, the formation of small amounts of epimers can be difficult to detect and more importantly, it makes the removal of these impurities very challenging in any scale and particularly in large industrial scale processes. This constitutes one of the most serious drawbacks for the implementation of peptides as API's.

The currently most-widely used coupling reagents include carbodiimides on the one hand, and phosphonium and iminium salts on the other. It is noteworthy that coupling agents that are useful in peptide synthesis 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, peptoids, oligocarbamates, oligoamides, β-lactams, esters, polyenamides, benzodiazepines, diketopiperazines, and hydantoins.

Carbodiimides are presently the most available and low-cost coupling agents amongst the presently known reagents, and include dicyclohexylcarbodiimide (DCC), l-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDC) and N,N'- diisopropylcarbodiimide (DIC). The primary reactive species, O-acylisourea, is one of the most reactive species for peptide coupling. Shortcomings associated with the use of carbodiimides as coupling agents therefore mostly stem from the high and relatively uncontrollable reactivity thereof and include, for example, racemization, side reactions and low yields due to the formation of the poorly active N-acyl urea. Furthermore, while low dielectric constant solvents such as CHCI₃ or CH₂Cl₂ are optimal for carbodiimides, the use of these solvents with higher dielectric constant such as DMF, which favor the formation of the N-acyl urea, precludes their use alone. Furthermore, dicyclohexylcarbodiimide is also incompatible with Fmoc/t-Bu solid-phase chemistry, because the urea derivative formed in such syntheses is typically not soluble in common solvents. Such urea derivatives are also difficult to remove in solution chemistry.

At the beginning of the 70 's, 1-hydroxybenzotriazole (HOBt) was proposed as an additive to DCC. The addition of HOBt was aimed at reducing the racemization associated with DCC coupling. The relative success of this additive signaled the beginning of a period during which other benzotriazole derivatives such as 1-hydroxy- 6-chlorobenzotriazole (6-Cl-HOBt) and l-hydroxy-7-azabenzotriazole (HOAt) were developed and successfully used. During later years the addition of benzotriazole derivatives as additives to DCC and other carbodiimides became almost mandatory to safeguard the peptide bond formation by carbodiimide activation from low yields, undesired side reactions and loss of chirality.

In the last decade, the use of phosphonium and iminium/uronium salts, referred to herein in short as "onium salts", of hydroxybenzotriazole derivatives as peptide coupling agents, was introduced. Although these reagents have been rapidly adopted for research purposes, only a few of them have been found compatible with current industrial requirements and synthetic strategies and were adopted by the industry. The species that reacts with onium salts is the carboxylate of the amino/organic acid. Therefore, performing the coupling reaction in the presence of at least one equivalent of a base is essential while using these reagents. The most reactive iminium salt coupling agent at present is l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo-[4,5- bjpyridinium hexafluorophosphate 3-oxide (HATU).

The misconception regarding the structure of these and other coupling agents was settled by Carpino, L.A. et al. [Angew. Chem. Int. Ed. 2002, 41, No. 3, p. 441], and it is now accepted that the leaving group in these agents is linked to the iminium moiety via the triazole nitrogen and not via the oxygen, which remains in its N-oxide form. The chemical structures of HATU and analogs thereof are presented in Scheme 1 below.

Scheme 1

X Y Z

HATU H N PF₆

HBTU H CH PF₆

TBTU H CH BF₄

HCTU Cl CH PF₆

TCTU Cl CH BF₄

Iminium salts, such as HATU, TBTU, HBTU, HCTU, or TCTU, which are possibly the most powerful coupling agents known, are formed by a leaving group and a carbocation skeleton. However, the presently known peptide coupling agents are typically limited by their low desired reactivity, side products formed thereby and/or high cost. U.S. Patent No. 6,825,347 and WO 94/07910 teach uronium and iminium salts and their use in effecting the acylation step in amide formation, especially during peptide synthesis. U.S. Patent No. 5,166,394 teaches the coupling agent O- [(cyano(ethoxycarbonyl methylidene)amino]-l,l,3,3-tetramethyluronium tetrafluoroborate (TOTU). The coupling agents taught in these publications have a leaving group attached to an uronium and iminium moiety, which is characterized by having N-alkyl or P-alkyl substituted nitrogen or phosphor atoms. These coupling agents, while being innovative, still provide coupling efficiencies similar to previously known coupling agents. Phosphonium-based coupling agents are gathering grounds in the industry, and include tris(ρyrrolidino)-phosphonium hexafluorophosphate (PyCOP), 7- azabenzotriazol-l-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate

(PyAOP), and benzotriazol-l-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP). However, these are only marginally more effective than their carbocation/iminium counterparts and thus are oftentimes forbiddingly yet unjustifiably more expensive coupling agents. Recently, 6-chloro-l- hydroxybenzorriazol-l-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyClock), one of the most effective phosphonium-based coupling agents, was introduced by the present assignee (see, for example, WO 2007/020620 and U.S. Patent No. 7,304,163).

SUMMARY OF THE INVENTION

Jn a search for a novel coupling agent, the present inventors have designed, prepared and successfully practiced a novel coupling agent which is more effective than commonly used coupling agents, and is simple and safe to produce, store, transport and use in modern peptide and polynucleotide synthesis techniques.

Thus, according to one aspect of some embodiments of the present invention there is provided a coupling agent having a general formula I:

Formula I

wherein D is a cationic moiety; A is an inorganic anion; Z₄ and Z₅ are each independently selected from the group consisting of F, Cl, Br, CORa, COORa, CONRa, CN, CF₃ or NO₂; and Ra is alkyl.

According to. some embodiments of the invention described below, the cationic moiety has a general formula selected from the group consisting of:

Formula III Formula IV Formula V

wherein: n is an integer from 1 to 4; and each of R₁, R₂, R₃, R₄, R₅ and R₆ is independently selected from the group consisting of an alkyl having 1-4 carbon atoms and an alkyl having 1-4 carbon atoms interrupted, substituted and/or terminated by at least one heteroatom, or, alternatively, R₁ and R₂, and/or R₃ and R₄, and/or R₅ and R₆ are each independently joined to form a heteroalicyclic moiety; with the provise than when the cationic moiety has the general formula III, Z₄ is CN, Z₅ is COORa and Ra is ethyl, at least one of Ri, R₂, R₃ or R₄ is an alkyl having 2-4 carbon atoms, or, alternatively, at least one of Ri and R₂, and/or R₃ and R₄ is joined to form a heteroalicyclic moiety. According to some embodiments, Z₄ is COORa; Ra is ethyl; and Z₅ is CN. Alternatively, Z₄ and Z₅ are each independently COORa or CN; and Ra is methyl.

According to another aspect of some embodiments of the present invention there is provided a coupling agent having a general formula II:

Formula II

wherein D is a cationic moiety; L is a leaving group; and A is an inorganic anion; and whereas the leaving group is selected from the group consisting of a pyridin-2(lH)- one-1-oxy, a quinazolin-4(3H)-one-3-oxy, a lH-benzo[d]imidazol-l-oxy and an indolinone-1-oxy.

According to some embodiments of the invention described below, the cationic moiety of the coupling agent has a general formula selected from the group consisting of:

Formula III Formula IV Formula V

wherein: n is an integer from 1 to 4; and each of R₁, R₂, R₃, R₄, R₅ and R₆ is independently selected from the group consisting of an alkyl having 1-4 carbon atoms and an alkyl having 1-4 carbon atoms interrupted, substituted and/or terminated by at least one heteroatom, or, alternatively, Ri and R₂, and/or R₃ and R₄, and/or R₅ and R₆ are each independently joined to form a heteroalicyclic moiety. According to. some embodiments, the heteroatom is selected from the group consisting of O, S, N₅ P and B.

According to some embodiments, the heteroalicyclic moiety is selected from the group consisting of a pyrrolidine, piperidine, morpholine, a thiomorpholine, an imidazolidine, an azaphosphinane, an azaphospholidine, an azaborinane, an azaborolidine, an azaphosphinane, an azaphospholidine and a piperazine.

According to_. some embodiments, the inorganic anion is selected from the group consisting of halide, hexahalophosphate, hexahaloantimonate, tetrahaloborate, trihalomethanesulfonate and bis(trihalomethylsulfonyl)irnide. According to some embodiments, the inorganic anion is hexafluorophosphate.

According to some embodiments, the pyridin-2(lH)-one-l-oxy has the general Formula VII:

Formula VII

According to some embodiments, the quinazolin-4(3H)-one-3-oxy has the general Formula VIII:

Formula VIII

wherein:

Z₆ and Z₇ are each independently selected from the group consisting of H, F, Cl, Br, CN, CF₃, NO₂, aryl or alkyl. According to some embodiments, Z₆ is CH3; and Z₇ is Cl. According to some embodiments, the lH-benzo[d]imidazol-l-oxy has the general Formula IX:

Formula IX

wherein: Z₁ is CH or N; Zg and Z₉ are each independently selected from the group consisting of H, F, Cl,

Br, CN, CF₃, NO₂, aryl or alkyl.

According to some embodiments, Zi is CH; Zs is phenyl; and Z₉ is H or Cl. Alternatively, Zi is N; Z₈ is methyl; and Z₉ is H.

According to some embodiments, the indolinone-1-oxy has the general Formula X:

Formula X.

wherein:

Zio is selected from the group consisting of H, F, Cl, Br, CN, CF₃, NO₂, aryl or alkyl. According to another aspect of some embodiments of the present invention, there is provided a coupling agent having a formula selected from the group consisting of: l,l,3,3-tetramethyl-2-(2-oxopyridin-l(2H)-yl)isouronium hexafluoro phosphate (HTOP) having the formula:

0-[(dicyanomemylidene)amino]-l,l,3,3-tetramethyluronium hexafluoro phosphate (HTODC) having the formula:

0-[(diethoxycarbonylmethylidene)amino]-l,l,3,3-tetramethyluronium hexafluorophosphate (HTODeC) having the formula:

According to another aspect of some embodiments of the present invention, there is provided a method of synthesizing a polynucleotide, the method is effected by coupling a plurality of nucleotides sequentially, one with another, in the presence of the coupling agent presented herein, to thereby obtain a polynucleotide containing the plurality of nucleotides.

According to another aspect of some embodiments of the present invention, there is provided a method of synthesizing a peptide, the method is effected by coupling a plurality of amino acids sequentially, one with another, in the presence of the coupling agent presented herein, to thereby obtain a peptide containing the plurality of amino acids.

According to some embodiments, the method is effected by a solid phase synthesis.

According to some embodiments, the method is effected by a solution phase synthesis.

According to yet another aspect of some embodiments of the present invention, there is provided a crude composition of peptides, the peptides being synthesized in a C- terminus to N-terminus direction from a plurality of amino acids, the composition consisting essentially of a peptide having a desired amino acid sequence and a plurality of peptides having undesired amino acid sequences and being impurities to the peptide having the desired amino acid sequence, wherein a concentration of the peptide having the desired amino acid sequence in the composition is at least 5 % above a concentration of an identical peptide having the desired amino acid sequence, in a composition of peptides being synthesized in the C-terminus to N-terminus direction using the coupling agent of claim 1 as a coupling agent, otherwise prepared under identical conditions.

According to some embodiments, the crude composition is in a form selected from the group consisting of a powdered composition, a lyophilized composition, a composition bound to a solid support, a solubilized composition and a dissolved composition.

According to some embodiments, at least one of the amino acids is selected from the group consisting of an amino acid having a Secondary alpha amine, an amino acid having a tertiary alpha amine, an amino acid having a substituted alpha carbon atom, an amino acid having a substituted alpha amine, an amino acid having an amino- containing side chain, and any combination thereof. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a protein" or "at least one protein" may include a plurality of proteins, including mixtures thereof.

Throughout this disclosure, various aspects of this invention can 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.

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 throughout, the term "comprising" means that other steps and ingredients that do not affect the final result can be added. This term encompasses the terms "consisting of and "consisting essentially of.

The term "method" or "process" refers 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, pharmacological, biological, biochemical and medical arts.

Herein throughout, the following abbreviations are used:

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

Cl-HOBt for 6-chloro-l-hydroxybenzotriazole;

DCC for N.N'-dicyclohexylcarbodiimide, dicyclohexylcarbodiimide; DIBOC for di-t-butyl dicarbonate;

DIEA for N,N-Diisopropylethylamine;

DIC, DIP or DIPCDI for N,N'-diisoproρylcarbodiimide or 1,3- Diisopropylcarbodiimide; DMAP for 4-Dimethylaminopyridine ;

DMF for N,N-dimethyformamide;

EDC for l-ethyl-3-(3'-dimethylaminopropyl)carbodiimide;

EDCHCl for l-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride;

Fmoc for 9-fhiorenylmethoxycarbonyl; HATU for 2-(7-Aza-lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate or l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo-[4,5- bjpyridinium hexafluorophosphate 3-oxide;

HBTU for 2-(lH-Benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate or l-[bis(dimethylamino)methylene]-lH-benzotriazolium hexafluorophosphate 3-oxide;

HCTU, N-[(lH-6-chlorobenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate

HMPyOP for l-(morpholino(2-oxopyridin-l(2H)- yloxy)methylene)pyrrolidinium hexafluorophosphate HDMOCC for l-[(l-(cyano-2-ethoxy-2-oxoethylideneamir₁ooxy)- dimethylamino-morpholinomethylene)] methanaminiurn hexafluorophosphate

HDMODC for l-[(l-(dicyanomethyleneaminooxy)-dimethylamino- morpholinomethylene)] methanaminium hexafluorophosphate

HDMODeC for l-[(l,3-diethyoxy-l,3-dioxopropan-2-ylideneaminooxy)- dimethylamino-morpholinomethylene)] methanaminium hexafluorophosphate

HDMOPC for N-[(cyano(pyridine-2-yl)methyleneaminooxy)(dimethylamino) methylene)-N-morρholinomethanaminium hexafluorophosphate

HOAt for l-hydroxy-7-azabenzotriazole;

HOBt for 1-hydroxybenzotriazole; HPTU for N-[(dimethylamino)-lH-l,2,3-triazolo-[4.5-beta]ρyddino-l- ylmethylene]-N-methylmethanaminium hexafluorophosphate; PyAOP for 7-azabenzotriazol-l-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate;

PyBOP for benzotriazol-l-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate; TBTU for N-[(lH-benzotriazol-l-yl)(dimethylamino)methylene]-N- methylmethanaminium tetrafluoroborate N-oxide or 2-(lH-Benzotriazole-l-yl)-l,l,3,3- tetramethyluronium tetrafluoroborate;

TCTU₅ N-[(lH-6-chlorobenzotriazol-l-yl)(dimethylamino)methylene]-N- methylmethanaminium tetrafluoroborate N-oxide; TFA for trifluoroacetic acid;

TFFH for Fluoro-N,N,N",N"-tetramethylformamidinium hexafluorophosphate; TCP for 2,4,6-trimethylpyridine (collidine); and TNBSA for trinitrobenzenesulfonic acid.

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.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to coupling agents and, more particularly, but not exclusively, to the preparation and use of novel coupling agents that can be beneficially utilized in the syntheses of substances such as peptides and oligonucleotides. The present invention is further of a process of preparing such coupling agents and of peptides prepared by utilizing same.

The principles and operation of the present invention may be better understood with reference to the accompanying descriptions. 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 invention is capable of other embodiments or of being practiced or carried out in various ways.

Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

As discussed in detail hereinabove, peptide-cόupling agents are the most effective tools in the hands of the pharmaceutical peptide industry. Modern peptide- coupling agents are based on their ability to form a transient activate carboxyl entity at the carboxyl end of the emerging peptide while avoiding its racemization due to the effect of the leaving group part thereof. While the research in the field of coupling agent development in the recent years have focused on work towards the identification of reactive and effective coupling agents in general and particularly research which was directed at the cationic moiety of such coupling agents, less attention has been paid to the nature of the leaving group moieties, and moreover to the safety issues arising from the use of potentially hazardous leaving group moieties.

As discussed hereinabove, peptide coupling agents are at large also effective in the coupling of nucleotides as well as derivatives and analogues thereof. It is thus noted herein that in the context of the present invention, any reference to the coupling capacity of peptide coupling agents also encompasses their capacity to activate phosphate groups in the coupling of nucleotides, as well as derivatives and analogues thereof.

As further discussed hereinabove, coupling agents that are used for peptide synthesis are typically also effective as coupling agents in oligonucleotide syntheses. Presently known oligonucleotide coupling reagents, such as l,l-dioxide-3H-l,2- benzodithio-3-one, (Beaucage reagent), 2-cyanoethyltetraisopropylphosphorodiamidite (CAS No. 102691-36-1), 4,5-dicyanoimidazole (DCI), 4,4'-dimethoxytrityl chloride (DMT-Cl), di-tert-butyl N,N-diethylphosphoramidite (CAS No. 117924-33-1), di-tert- butyl N,N-diisopropylphosphoramidite CAS No. 137348-86-8), 4-methoxytrityl chloride CAS No. 14470-28-1) and phenylacetyl disulfide (PADS), are used typically to activate the end phosphate group of one nucleotide so as to react with a sugar moiety of another nucleotide. Thus, similar to the activation of a carboxyl group in peptide synthesis, the phosphate can be activated using the same type of peptide coupling agents.

Recent reports [Wehourstedt et al., J. Hazard. Mat. A126 (2005) 1] have confirmed that HOBt derivatives exhibit explosive properties under certain conditions, presumably due to the explosive properties of 1-hydroxy-benzotriazole and of benzotriazole derivatives in general. Similarly, coupling agents based on other triaza- containing groups (e.g., triazine, triazole) have been associated with explosiveness. Such studies raised the awareness regarding substances which contain a triaza moiety as being potentially explosive.

While searching for a coupling agent that will be as safe for use as it is effective, the present inventors have uncovered and successfully practiced the use of several families of chemical moieties which can be beneficially used as leaving group moieties in coupling agents, while preserving the high desired reactivity, namely a highly directed capacity to activate carboxylic groups in amino acids and phosphate groups in nucleotides, while being devoid of triaza-containing groups.

Hence, according to one aspect of the present invention, there is provided a coupling agent having the general formula I:

Formula I

wherein:

D is a cationic moiety; A is an inorganic anion; and the leaving group is a ketoxime, as defined herein, wherein Z₄ and Z₅ are each independently selected from the group consisting of F, Cl, Br, CORa, COORa, CONRa, CN, CF₃ or NO₂; and Ra is alkyl, as defined herein.

According to another aspect of the present invention, there is provided a coupling agent having the general formula II:

D Formula II

wherein:

D is a cationic moiety; L is a leaving group; A is an inorganic anion; and whereas the leaving group is any one of pyridin-2(lH)-one-l-oxy, quinazolin-4(3H)- one-3-oxy, lH-benzo[d]imidazol-l-oxy and indolinone-1-oxy, as defined herein.

As used herein, the phrase "moiety" describes a part of a chemical entity or compound, which typically has certain functionality or distinguishing features.

The phrase "cationic moiety" refers to the part of the coupling agent which is positively charged and endures the nucleophilic attach of the carboxylic or phosphate group during the coupling process, while the leaving group is released.

According to embodiments of the present invention, the cationic moiety is having the general formula selected from the group consisting of:

Formula III Formula IV Formula V

wherein: n is an integer from 1 to 4; and each of R₁, R2, R₃, R₄, R₅ and R₆ can each be independently an alkyl having 1-4 carbon atoms and an alkyl having 1-4 carbon atoms interrupted, substituted and/or terminated by one or more heteroatoms, or, alternatively, R₁ and R₂, and/or R3 and R₄, and/or R₅ and R₆ are each independently joined to form a heteroalicyclic moiety.

Coupling agents having the general formula I wherein the cationic moiety has the general formula III, R₁-R₄ are each a methyl group; and the leaving group, as this phrase is defined hereinbelow, is a ketoxime as shown in formula I wherein Z₄ is CN,

Z5 is COORa and Ra is ethyl, have been previously described and are therefore excluded from the scope of this aspect of the present invention. However, coupling agents having the general formula I wherein the cationic moiety has the general formula III, Z₄ is CN, Z₅ is COORa and Ra is ethyl, and further wherein at least one of R₁-R₄ is an alkyl having 2 or more carbon atoms, or, alternatively, at least one of R₁ and R₂, and/or R₃ and R₄ is joined to form a heteroalicyclic moiety, have not been described hitherto and are encompassed by the present embodiments.

As used herein, the term "alkyl" describes an aliphatic hydrocarbon including straight chain and branched chain groups. For example, the alkyl group has 1 to 20 carbon atoms, 1-10 carbon atoms, or, for example 1-4 carbon atoms. Whenever a numerical range; e.g., "1-10", 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 10 carbon atoms. The alkyl can be substituted or unsubstituted. When substituted, the substituent can be, for example, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a halide, a hydroxy, an alkoxy and a hydroxyalkyl as these terms are defined hereinbelow. The term "alkyl", as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl. The term "heteroatom", as used herein, refers to a non-carbon and non-hydrogen atom, which can act as a proton acceptor. A heteroatom can therefore be, for a non- limiting example, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B) and selenium (Se). For example, the heteroatom is O, S, N, P or B, and alternatively, the heteroatom is O, S or N. The phrase "proton acceptor", as used herein, refers to a heteroatom which can be protonated at a pH range which is common to coupling reactions, and change its form, from -A- to the form -AH⁺-, wherein -A- is the heteroatom before being protonated and -AH⁺- is its protonated form. In general, a proton accepting heteroatom possesses at least one lone pair of valence electrons, which can attract a proton (H⁺) and hence are known as a proton acceptor.

The phrase "an alkyl interrupted, substituted and/or terminated by at least one heteroatom", as used herein, describes an alkyl, as defined herein, which contains a heteroatom in one or more positions within the alkyl hydrocarbon chain, at the end of the alkyl hydrocarbon chain and/or as a substituent of one of the carbon atoms in the alkyl. Examples of an alkyl interrupted by heteroatom(s) include, without limitation, ethers such as methoxymethyl, thioethers such as 3-thiobutane, amines such as diethylamine and phosphines such as triethylphosphine; examples of an alkyl substituted by the heteroatom(s) include, without limitation, alcohols (alkyls substituted with a hydroxyl group) such as isopropanol, amines such as 2-aminobutane, and thiols (alkyls substituted with a thiohydroxyl group) such as butane-3-thiol; an alkyl terminated by heteroatoms is a particular case in which the alkyl hydrocarbon backbone is substituted by a heteroatom at a terminal carbon.

According to some embodiments, in at least one of the pairs of JV-substituents, i.e., the pair of Ri and R₂, the pair R₃ and R₄, and the pair R₅ and R₆, the substituents are joined to form a heteroalicyclic moiety. Hence, the nitrogen atom of the doubly- substituted amine and the substituents attached thereto form a heteroalicyclic ring containing at least two heteroatoms.

The heteroalicyclic moiety therefore contains at least the nitrogen atom bearing the Ri and R₂, and/or R₃ and R₄, and/or R5 and R₆ substituents and further contains another heteroatom, as defined herein. Exemplary heteroatoms include, but are not limited to, oxygen, sulfur or nitrogen. Examples of heteroalicyclic moieties include, without limitation, pyrrolidines, piperidines, morpholines, thiomorpholines, oxazolidines, imidazolidines, 1,4- azaphosphinane (e.g. 4-methyl-l,4-azaphosphinane), azaphospholidines, azaborinanes (e.g. 4-methyl-l,4-azaborinane), azaborolidines (e.g. 2-methyl-l,2-azaborolidine) and piperazines (e.g., 4-methylpiperazine). For example, Ri and R₂ can each be ethyl, joined together via an oxygen atom to form, together with the nitrogen they are attached to, a morpholine moiety, as illustrated in Scheme 2 below.

In cases where such Ri and R₂ are joined together via a sulfur atom, a thiomorpholine moiety is formed. In cases where such Ri and R₂ are joined together via a nitrogen atom, a piperazine moiety is formed. Similarly, when Ri is an ethyl and R₂ is a methyl joined together via a nitrogen atom, an imidazolidine is formed together with the nitrogen they are attached to. When Ri is B-methylboroethane and R₂ is ethyl joined together via a boron atom, a 4-methyl- 1,4-azaborinane is formed together with the nitrogen they are attached to, and so forth.

These embodiments can be applied also to R₃ and R₄, and R₅ and R₆. As used herein, the term "amine" describes a -NR'R" group where each of R' and R" is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic, aryl or heteroaryl, as these terms are defined herein.

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 hereinabove.

The term "alkynyl", 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.

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 by one or more substituents, as described hereinabove.

The term "heteroaryl" describes a monocyclic or fused ring (Le., 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 by one or more substituents, as described hereinabove. Representative examples are thiadiazole, pyridine, pyrrole, oxazole, indole, purine and the like.

The term "heteroalicyclic", as used herein, 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, alkyl cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and heteroalicyclic. Representative examples are morpholine, piperidine, piperazine, tetrahydrofurane, tetrahydropyrane and the like.

As used herein, the term "phosphine" describes a -PR'R"R'" group where each of R', R" and R"¹ is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic, aryl or heteroaryl, as these terms are defined herein. The terms "hydroxyl" and "thiohydroxyl" refer to a -OH and -SH groups respectively.

The term "acetate" refers to acetic acid anion.

The term "tosylate" refers to toluene-4-sulfonic acid anion.

The term "triflate" refers to trifluoro-methanesulfonic acid anion. The term "sulfonate" refers to a sulfonic acid anion.

The term "azide" refers to an N3^".

The terms "hydroxy" and "thiohydroxy" refer to the OH^' and SH^' anions respectively.

The term "alkoxy" refers to an R' -O^' anion, wherein R' is as defined hereinabove.

The term "cyanate" and "thiocyanate" refer to [O=C=N]^" and [S=C=N]^' anions respectively.

The term "nitro" refers to NO₂ ^".

The term "cyano" refers to [ON]^". Exemplary inorganic anions which are present in the coupling agents of the present embodiments and represented by Formulae I and II include, without limitation, halide, hexahalophosphate, hexahaloantimonate, tetrahaloborate, trihalomethanesulfonate and bis(trihalomethylsulfonyl)imide.

The term "halide", as used herein, refers to the anion of a halo atom, i.e. F^", Cl, Br^" and I^".

The term "halo" refers to F, Cl, Br and I atoms. The term "hexahalophosphate" refers to the inorganic anion PX₆ ^", wherein X is halo. An exemplary hexahalophosphate is hexafluorophosphate or PF₆ ^", which is an exemplary and the most widely used inorganic anion according to the present embodiments. The term "hexahaloantimonate" refers to the inorganic anion SbX₆ ^", wherein X is halo.

The term "tetrahaloborate" refers to the inorganic anion BX₄ ^", wherein X is halo. The term "trihalomethanesulfonate" refers to the inorganic anion CX3SO3^", wherein X is halo. The term "bis(trihalomethylsulfonyl)imide" refers to the inorganic anion

N(SO₂CX₃)₂\ wherein X is halo.

As discussed hereinabove, the type and characteristics of the leaving group, which is a ketoxime in formula I or denoted L in formula II, plays an important role in the effectiveness of the compound as a coupling agent, as well as in its safety by being devoid of a triaza group.

To this effect, the present inventors have developed several series of coupling agents which are based on less hazardous leaving groups, such as ketoxime-, pyridine-, quinazoline-, imidazole- and indolinone-based. Such agents are highly safe in terms of their preparation, storage, transportation and use. As used herein, the phrase "leaving group" describes a labile atom, group or chemical moiety that readily undergoes detachment from an organic molecule during a chemical reaction, while the detachment is facilitated by the relative stability of the leaving atom, group or moiety thereupon. Typically, any group that is the conjugate base of a strong acid can act as a leaving group. According to some embodiments, the leaving group is selected from the group consisting of a ketoxime, a pyridin-2(lH)-one-l-oxy, a quinazolin-4(3H)-one-3-oxy, a lH-benzo[d]imidazol-l-oxy and an indolinone-1-oxy.

Thus, according to some embodiments, the ketoxime, which forms a part of formula I, can be also represented by the general Formula VI: O ^Z5

Formula VI wherein:

Z₄ and Z₅ can each independently be any one of F, Cl, Br, CORa, COORa, CONRa, CN, CF₃ or NO₂; with Ra being an alkyl.

For example, Z₄ is COORa; Ra is ethyl; and Z₅ is CN, resulting in the leaving group:

Alternatively, Z₄ and Z₅ are both COORa with Ra being ethyl, resulting in the leaving group:

Alternatively, Z₄ and Z₅ are both CN, resulting in the leaving group:

According to further features in some embodiments, the -2(lH)-one-l-oxy has the general Formula VII:

Formula VII

According to still further features in some embodiments, the quinazolin~4(3H)- one-3-oxy has the general Formula VIII:

Formula VIII

wherein Z₆ and Z₇ can each independently be any one of H, F, Cl, Br, CN, CF₃, NO₂, phenyl or alkyl.

For example, Z₆ is CH₃; and Z₇ is Cl.

According to still further features in some embodiments, the -benzo[d]imidazol- 1-oxy has the general Formula IX:

Formula DC

wherein Z₈ and Z₉ can each independently be any one of H, F, Cl, Br, CN, CF₃, NO2, aryl or alkyl. For example, Zi is CH, Z₈ is phenyl; and Z₉ is Cl. Alternatively, Z₁ is N, Z₈ is methyl; and Z₉ is H.

According to yet further features in some embodiments, the indolinone-1-oxy has the general Formula X:

Formula X wherein Z10 can be any one of H, F, Cl, Br, CN, CF₃, NO₂, aryl or alkyl.

Processes of preparing some of the iminium/carbocation-type coupling agents is taught, for example, in a PCT Patent Application, filed May 15, 2008 and entitled "Proton Acceptor iminium/Carbocation-Type Coupling Agents", having Attorney's Docket No. 43748, by the present assignee, which is incorporated herein as if fully set forth herewith, and which claims priority from U.S. Provisional Patent Application Nos. 60/924,475 and 60/929,384.

Processes of preparing phosphonium-type coupling agents are taught, for example, in WO 2007/020620 and U.S. Patent No. 7,304,163, also by the present assignee, which is incorporated herein as if fully set forth herewith.

As demonstrated in the Examples section that follows, exemplary coupling agents which are based on an iminuim/carbonium moiety that contains non-hazardous leaving group moieties as presented herein have been prepared.

Exemplary coupling agents according to some embodiments of the invention are listed in Table 1 below.

Table 1

As discussed hereinabove, coupling agents are ranked according to several criteria which are important in peptide synthesis and these are: yield of peptide-bond formation in general and the yield of peptide-bond formation between amino acids which present particular steric coupling hindrance, and the rate of racemization of the activated amino acid on which the coupling agent exerts its activity.

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.

As used herein throughout, the phrase "amino acid residue" describes a residue of an amino acid which forms a part of a peptide, namely, which is coupled to at least one another amino acid residue.

As is well accepted in the art, the term "residue", as used herein, refers to a portion, and typically a major portion of a molecular entity, such as molecule or a part of a molecule such as a group, which has underwent a chemical reaction and is now covalently linked to another molecular entity. For example, the molecular entity can be an amino acid molecule, and the portion of the amino acid which forms a part of a peptide chain after the formation of the peptide chain, is an amino acid residue. An amino acid residue is therefore that part of an amino acid which is present in a peptide sequence upon reaction of, for example, an alpha-amine group thereof with a carboxylic group of an adjacent amino acid in the peptide sequence, to form a peptide amide bond and/or of an alpha-carboxylic acid group thereof with an alpha-amine group of an adjacent amino acid in the peptide sequence, to form a peptide amide bond.

The term "side-chain", as used herein with reference to amino acids, refers to a chemical group which is attached to the α-carbon atom of an amino acid. The side- chain is unique for each type of amino acid and does not take part in forming the peptide bond which connects the amino acids in a polypeptide. For example, the side chain for glycine is hydrogen, for alanine it is methyl, for valine it is isopropyl and for methionine it is methylsulfanyl-ethyl. For the specific side chains of all amino acids reference is made to A. L. Lehninger's text on Biochemistry (see, chapter 4).

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

Tables 2 and 3 below list naturally occurring amino acids (Table 2) and non- conventional, modified and unnatural amino acids (Table 3) which can be used in the context of the present invention.

Table 2

Table 3

The yield of peptide-bond formation can be determined simply by measuring the amount of product (for example an elongated peptide) versus the amount of starting material and/or side-reaction impurities. In cases of coupling of particular sterically- hindered amino acids, such as phenylglycine, or otherwise more difficult-to-couple amino acids such as 2-aminoisobutyric acid, the length and sequence of the resulting peptide will serve as an indication of coupling yield.

During the process of peptide-bond formation which is mediated by a coupling agent, the carboxylic part of the amino acid interacts with the coupling agent to form an activated intermediate, which in turn interacts with the amino part of the next amino acid. During this process a full inversion of the stereo-configuration of the activated amino acid may occur, and more likely a partial inversion may occur which leads to racemization of the chiral center of that particular amino acid.

The rate of racemization, which is one of the most prevailing technological problems in peptide synthesis, is also the most difficult to measure. The term "racemization", as used herein, refers to partial conversion of one enantiomer of a chiral molecule into its other stereoisomeric form, or its "mirror image" inversion.

As used herein, the term "enantiomer" refers to a stereoisomer of a compound, such as an amino acid, that is superposable with respect to its counterpart only by a complete inversion/reflection (mirror image) of each other. Enantiomers are said to have "handedness" since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems.

As presented in the Example section that follows, the coupling agents presented herein can be tested in some of the most challenging peptide synthesis tasks, and can be rated according to their peptide-bond formation yield and rate of racemization.

Hence, according to another aspect of the present invention, there is provided a method of synthesizing a peptide, the method is effected by sequentially coupling a plurality of amino acids, one with another, in the presence of the coupling agent presented herein, to thereby obtain a peptide containing a plurality of amino acids. The quality of a coupling agent is therefore measured according to the quality of the resulting peptide, namely the efficiency of its production (yield of the reaction) and the rate of internal racemization of each of the chiral centers therein (purity of the product). This assessment of quality can be quantified as a stepwise indicator, per each elongation step, or as an overall rate per the complete peptide. Obviously when trying to compare coupling agents it is best to avoid criteria in which the peptide sequence plays a key role in the efficiency of its synthesis, and therefore a stepwise criteria, looking at particular arαino-acid pairs, will serve as an indicative coupling agent quality ranking criteria.

According to some embodiments the yield of coupling per each general coupling step can range from 80 % to 99 %, depending on the coupling agent and other conditions. Of particular importance is the effectiveness of the coupling agent presented herein when used in the syntheses of peptides that incorporate amino acid residues that are otherwise incorporated in relatively low yields and sometimes are particularly difficult to be incorporated.

Exemplary amino acids that are considered difficult to be incorporated in common peptide syntheses include, but are not limited to, amino acids having a secondary alpha amine, amino acids having a tertiary alpha amine, amino acids having a substituted alpha carbon atom and amino acids having an amino-containing side chain.

The incorporation of amino acids that have a secondary or tertiary alpha amine into a synthetically prepared peptide is exceptionally difficult to perform, due to the hindrance of the amine that participates in the coupling reaction.

The incorporation of amino acids having a substituted alpha carbon atom also substantially affects the synthesis yield due to the steric interactions induced by the substituent, which interfere with the coupling reaction. The incorporation of two consecutive such amino acids are even more complicated. As is well known in the art, amino acids that are substituted at the alpha carbon even by a low alkyl such as methyl are difficult to be incorporated in a peptide sequence.

The incorporation of amino acids that have an amino-containing side chain is oftentimes complicated by the unselective reaction of a coupling agent with the side chain amine group. Such an unselective reaction leads to termination of the peptide chain. As mentioned above, the method, according to this aspect of the present invention, is therefore particularly useful for synthesizing peptides that include one or more residues of the amino acids cited above.

Thus, in embodiments of the present invention, at least one of the pluralities of amino acids that are used for synthesizing the peptide is an amino acid having a secondary alpha amine, an amino acid having a tertiary alpha amine, an amino acid having a substituted alpha carbon atom and/or an amino acid having an amino- containing side chain. The peptides prepared by this method therefore include at least one residue of such amino acids. According to some embodiments of the present invention, one or more of the plurality of amino acids in an amino acid having a substituted alpha carbon. In one embodiment the alpha carbon is substituted by an alkyl such as methyl. An exemplary amino acid in this respect is α-amino-isobutyric acid (Aib). In another embodiment, at least two such amino acids are sequentially incorporated in the peptide prepared by the method presented herein, such that the peptide comprises two such amino acids that are coupled one to another. According to another embodiment of the present invention, a peptide containing two or more residues of such an amino acid, coupled one to another can be obtained in a yield greater than 80 %, greater than 85 %, greater than 90 %, greater than 95 %, and even greater than 98 %. In an exemplary embodiment of this aspect of the present invention, one or more of the plurality of amino acids is an amino acid having a substituted alpha carbon which is chiral. As discussed hereinabove, coupling agent mediated peptide synthesis is prone to inversion of the stereo-configuration of the activated amino acid, and more likely a partial inversion may occur which leads to racemization of the chiral center of that particular amino acid, and eventually leads to formation of heterogeneous mixture of peptides when considering stereoisomers thereof. Any amino acid which exhibits a chiral center at the alpha-carbon is sensitive to the coupling reaction with respect to preservation of stereo-configuration. Examples of such amino acids include all D/L- amino acids except glycine, D/L-α-methyl-amino acids such as D-α-methylisoleucine and the likes. According to another embodiment of the present invention, a peptide containing one or more residues of such an amino acid can be obtained in a yield greater than 80 %, greater than 85 %, greater than 90 % and even greater than 95 %. As demonstrated in the Examples section that follows, the coupling agents presented herein were superior over presently known coupling agents with respect to the yield of the challenging stereo-hindered couplings reaction of α-amino-isobutyric acid (Aib), even in the coupling of two consecutive Aib residues. According to embodiments of the present invention, a peptide containing more than 10 residues can be obtained in a yield greater than 40 %, greater than 50 % and even greater than 60 %.

According to some embodiments the rate of racemization per each coupling step can range from about 10 % to about 0.5 %, or from about 5 % to about 0.1 %. As presented in the Examples section that follows, the coupling agents presented herein can be superior over presently known coupling agents with respect to the rate of racemization which occurred upon use of the coupling agents at the chiral center which the coupling agent activates for the coupling reaction. This criterion was estimated for phenylglycine (Phg) after coupling of a proline residue thereto; and for valine after coupling of a proline residue thereto.

In another embodiment of this aspect of the present invention, in each of the sequential attachments of the amino acids, the coupling agent can be used in an amount that ranges from 0.2 mol equivalent to 5 mol equivalents relative to the molar amount of the amino acid, or from about 0.5 mol equivalent to 3 mol equivalents relative to the molar amount of the amino acid, or alternatively a stoichiometric amount of the coupling agent is used.

Using the above described method, which utilized the high reactivity and selectivity of the coupling agent presented herein, peptides compositions which comprise a high concentration of the desired peptide, namely, a peptide having the desired sequence, can be obtained. Such compositions typically have a concentration of a desired peptide which is higher than that obtained using presently known coupling agents.

Thus, according to a further aspect of the present invention there is provided a crude composition of peptides, which is synthesized in a C-terminus to N-terminus direction, and which consists essentially of a peptide having a desired amino acid sequence and a plurality of peptides having undesired amino acid sequences, which are impurities of the peptide that has the desired amino acid sequence. According to this aspect of the present invention, the concentration of the peptide having the desired amino acid sequence in the composition is at least 5 % above a concentration of an identical peptide having the desired amino acid sequence, in a composition of peptides being synthesized in a C-terminus to N-terminus direction using the coupling agents described herein, as compared to a composition otherwise prepared under the same conditions using presently known coupling agents.

As used herein, the phrase "a peptide having a desired sequence" describes a peptide that comprises a desired sequence in terms of the order and the composition of amino acids in the peptide chain and a desired chain length in terms of the number of amino acids in the peptide chain.

The phrase "a peptide having an undesired sequence" describes a peptide that comprises a different sequence, in terms of order and composition of the amino acids in the peptide chain and a different chain length in terms of the number of amino acids in the peptide chain, as compared with a peptide having a desired sequence. The phrase "a crude composition of peptides" describes a crude product of a peptide synthesis (in which the peptides are prepared in a C-terminus to N-terminus direction). As is well known ion the art, such a crude product is consisted of a peptide having a desired sequence, as defined herein, which is contaminated by peptides having an undesired sequence, as defined herein. Depending on the synthetic method used for preparing the peptide and the conditions and procedures employed therewithin, such a crude composition can be in a form of a solid or a solution. Solid compositions can be, for example, powdered or lyophilized. The composition can further be bound to a solid support, onto which the peptide was synthesized. Solutions may include the composition solubilized or dissolved in a liquid media. Such a liquid media is typically an aqueous media, which comprises, in addition to water, various salts, buffers and the like.

In some embodiments of this aspect of the present invention, the concentration of the peptide having the desired amino acid sequence in the composition can be at least 5 %, at least 8 %, at least 10 %, at least 12 %, at least 15 %, at least 18 %, at least 20 %, at least 22 %, at least 25 %, and even at least 30 % above a concentration of an identical peptide having the desired amino acid sequence in a composition of peptides being synthesized in a C-terminus to N-terminus direction using presently known coupling agents otherwise prepared under the same conditions.

Exemplary crude compositions according to this aspect of the present invention are those in which the peptide having the desired amino acid sequence comprises at least one amino acid residue selected from the group consisting of a residue of an amino acid having a secondary alpha amine, a residue of an amino acid having a tertiary alpha amine, a residue of an amino acid having a substituted alpha carbon atom and a residue of an amino acid having an amino-containing side chain, as is defined and detailed hereinabove. Other crude compositions according to this aspect of the present invention are those in which the peptide having the desired amino acid sequence comprises at least two coupled amino acid residues, whereby at least one of the at least two amino acid residues is selected from the group consisting of a residue of an amino acid having a secondary alpha amine and a residue of an amino acid having a tertiary alpha amine. Other crude compositions according to this aspect of the present invention are those in which the peptide having the desired amino acid sequence comprises at least two coupled amino acid residues acids having a substituted alpha carbon atom (e.g., Aib residues).

Any of the presently known techniques for chemically synthesizing a peptide can be used in this context of the present embodiments. These include, for example, standard solid phase techniques including exclusive solid phase synthesis, partial solid phase synthesis methods, and fragment condensation, and classical solution synthesis. Solid phase peptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

The obtained peptides can be further purified, using, for example, preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N. Y.]. Other exemplary purification procedures may include hydroxyapatite, size exclusion and immobilized metal ion adsorption (IMAC) chromatography.

Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-

Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrorn CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of peptides by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Selection of a particular method is determined by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988. The peptides prepared by the method according to this aspect of the present invention are linear, although cyclic forms of the peptide can also be obtained.

Cyclic peptides can either be synthesized in a cyclic form or configured so as to assume a cyclic form under desired conditions (e.g., physiological conditions).

As discussed hereinabove, the coupling agents presented herein can be use in the synthesis of various nucleic acids, oligonucleotides, polynucleotides, analogues and derivatives thereof. The coupling agents activate the phosphate group of nucleotides in a similar way they activate the carboxyl group of amino acids, thereby effecting the coupling of one nucleotide to another.

Thus, according to another aspect of the present invention, there is provided a method of synthesizing a polynucleotide, the method is effected by either a solid phase technique or a solution phase technique, and by sequentially coupling a plurality of nucleotides, one with another, in the presence of the coupling agent presented herein, to thereby obtain a polynucleotide containing a plurality of nucleotides.

As used interchangeably herein, the terms "polynucleotides", "oligonucleotides" and "nucleic acids", and collectively referred to as "polynucleotides", include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. However, in the context of the present invention, since the chemical synthesis of oligonucleotide typically involves single stranded species, the oligonucleotide is essentially a single stranded species at least in the part which is being chemically synthesized using a coupling agent. Hence, nucleotides are monomeric units of a polynucleotide or nucleic acids such as DNA and RNA. As used herein, the term "nucleotide" refers to molecule which comprises a nucleobase-sugar-phosphate combination, meaning a molecule, or an individual unit in a larger nucleic acid molecule, comprising a purine, a pyrimidine or an analogue thereof, a ribose, deoxyribose sugar moiety or an analogue thereof, and a phosphate group or phosphodiester linking group in the case of nucleotides within an oligonucleotide or polynucleotide. Hence, the term "nucleotide" encompasses naturally occurring nucleotides as well as "modified nucleotides" which comprise at least one modification in the linking phosphate-containing group, the purine, the pyrimidine, or the sugar moieties.

The term "nucleotide" includes deoxyribonucleoside triphosphates ("dNTPs") such as dATP (2'-deoxyadenosine 5 '-triphosphate), dCTP (2'-deoxycytidine 5'- triphosphate), dITP (2'-deoxyinosine 5 '-triphosphate), dUTP (2'-deoxyuridine 5'- triphosphate), dGTP (2'-deoxyguanosine 5 '-triphosphate), dTTP (2'-deoxythymidine 5 '-triphosphate), or derivatives thereof. Such derivatives include, for example, α-dATP, 7-deaza-dGTP, 7-deaza-dATP, Aminoallyl-UTP (5-[3-aminoallyl]-uridine-5'- triphosphate), Aminoallyl-dUTP, Aminoallyl-dUTP, Biotin-11-dUTP (biotin-e- ammocaproyl-[5-{3-ammoallyl}-2'-deoxyuridine-5'-triphosphate]), Fluorescein-12- dUTP (fluorescein-6-carboxaminocaproyl-[5-{3-aminoallyl}-2'-deoxyuridine-5^l- triphosphate]), dm⁶ATP (2'-deoxy-N6-methyladenosine 5 -triphosphate), dm⁴CTP (2^!- deoxy-N4-methylcytidine 5 '-triphosphate) or dm⁵CTP (2'-deoxy-5-methylcytidine 5 - triphosphate). The term "nucleotide" as used herein also refers to dideoxyribonucleoside triphosphates ("ddNTPs") and their derivatives, including, but not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. In addition, the term "nucleotide" includes ribonucleoside triphosphates (rNTPs) such as rATP, rCTP, rJTP, rUTP, rGTP, rTTP and their derivatives, which are analogous to the above-described dNTPs and ddNTPs except that the rNTPs comprise ribose instead of deoxyribose or dideoxyribose in their sugar-phosphate backbone. 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.

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.

EXAMPLES

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.

MATERIALS AND METHODS

Solvents were procured from Carlo Erba Reactifs-Sds and used without further purification unless indicated.

N,N-dimethylformamide was bubbled with nitrogen for 2 hours and kept over molecular sieves.

Acetonitrile was distilled before use from anhydrous potassium carbonate and kept over molecular sieves. Diisopropylethylamine was refluxed for 2 hours with nihydrine, and then distilled and kept over molecular sieves.

Potassium hexafluorophosphate, morpholine, triphosgene, thiomorpholine, A- chloro-3-nitrotrifluorobenzene, and 2-chloro-3-notropyridine, N-hydroxysuccinimide, pentafluorophenol were procured from Sigma-Aldrich. Oxalyl chloride, N,N,N-trimethylethylenediamine, N,N-dimethylcarbamoyl chloride, N,N-diethylcarbamoyl chloride, potassium fluoride were procured from Fluka. Potassium fluoride was kept in the oven at 100 ⁰C for 24 hours for drying before use.

HOBt, HOAt, and Rink Amide resin were procured from Biotech GMBH.

6-Cl-HOBt was procured from Luxembourg industries. Other starting materials and reagents were procured from Carlo Erba Reactifs-

Sds, Sigma-Aldrich, Fluka, Biotech GMBH or Luxembourg industries Ltd.

TLC was performed on silica plates (8X4 cm) from Albet using suitable solvent systems and visualization by a Spectroline UV Lamp Model CM-10 (254 run).

Melting points were obtained in open capillary tubes using a Buchi melting point B-540 Apparatus.

Infrared spectra (IR) were recorded on a FT-IR Thermo Nicolet series Fourrier Transformer instrument as KBr pellets. The absorption bands (v_max) were given in wave numbers (cm^"1).

U V- Vis spectra were recorded on a Shimadzu UV-250/PC spectrophotometer,. NMR spectra were recorded on Varian mercury 400 MHz spectrometer at room temperature with chemical shifts reported as ppm relative to internal solvent used.

HPLC analysis was carried out by using a Waters Symmetry Column C18, 5μ, 4.6 X 150 mm with dual wavelength absorbance detector.

Mass spectrometry measurements were carried out on MALDI mass spectrometer with ACH as internal matrix.

X-ray crystallographic analysis was carried out using a Bruker Appex-II CCD diffractometer, using 3363 reflections with a four circle differactometer.

EXAMPLE 1 CHEMICAL SYNTHESES OF VARIOUS UREA DERIVATIVES DESIGNATED

AS URONIUMIIMMONIUM TYPE COUPLINGAGENTS Preparation of urea derivatives - General Procedure A: iV_jiV-Dialkyl-carbamoyl chloride (0.6 mol) is added dropwise to a stirred mixture of a secondary amine compound (0.5 mol) and triethylamine (0.5 mol) in DCM (400 ml) at 0 °C. The mixture is stirred for 3-4 hours at room temperature and is thereafter basified with 10 % NaOH. The organic layer is collected and the aqueous layer is washed with dichloromethane (DCM, 100 ml). The combined DCM phase is washed with H₂O (2 x 100 ml) and with a saturated solution of NaCl (2 x 100 ml), dried over anhydrous MgSO₄, filtered, and the solvent removed under reduced pressure. Typically an oily residue is obtained and purified by vacuum distillation.

Preparation of urea derivatives - General Procedure B: The urea derivative is prepared by using a two phase system consisting of DCM and 10 % NaOH. A secondary amine (0.5 mole) is dissolved in DCM (300 ml) and 10 % NaOH (300 ml). An N,N-dialkyl-carbamoyl chloride (0.6 mol in 200 ml of DCM) is added dropwise for 10 minutes. The reaction mixture is stirred at room temperature for 3 hours and the organic layer is collected. The NaOH layer is washed with DCM (200 ml) and the combined DCM is washed with water (2 x 100 ml), saturated NaOH (2 xlOO ml), dried (MgSO₄), filtered and the solvent is removed under reduced pressure to give the urea derivatives typically as pure colorless oil.

A series of exemplary urea derivatives is prepared according to General Procedure A or B (see Scheme 3 below) using a variety of secondary amines (represented as RiR₂NH in Scheme 3), wherein Ri-R₄ can each be independently an alkyl having 1-4 carbon atoms and an alkyl having 1-4 carbon atoms interrupted, substituted and/or terminated by at least one heteroatom, or, alternatively, Ri and R₂, and/or R3 and R₄ are each independently joined to form a heteroalicyclic moiety.

Scheme 3

Preparation of N,N-dimethylpyrrolidine-l-carboxamide:

NjN-dimethylpyrrolidine-l-carboxamide, a urea derivative, was distillated and collected at 98-100 ⁰C as a colorless oil at 85.5 % yield according to General Procedure A or B using pyrrolidine as a secondary amine. ¹H NMR (CDCl₃): δ = 1.81-2.10 (m, 4H, 2CH₂), 2.81(s, 6H, 2CH₃), 3.15-

3.18(m, 4H, 2CH₂) ppm

Preparation of N JSl-Dimethylmorρholine-4-carboxamide:

N,N-dimethylmorpholine-4-carboxamide, a urea derivative, was distillated and collected at 127-129 ⁰C as a colorless oil at 92.4 % yield (73 grams from 0.5 mole reaction) according to General Procedure A or B using morpholine as a secondary amine. 1H NMR (CDCl₃): δ = 2.84 (s, 6H, 2 CH₃), 3.22-3.2 (m, 4H, 2CH₂), 3.68-3.70

(m, 4H, 2CH₂) ppm;

¹³C NMR (CDCl₃): δ = 38.62, 47.51, 66.89, 164.96 ppm.

Preparation of N_fN-Dimethylthiomorpholine-4_'Carboxamide:

N,N-dimethylthiomorpholine-4-carboxamide, another urea derivative, was obtained as light brown crystals in 89.5 % yield according to General Procedure A or B using thiomorpholine as a secondary amine. m.p.: 61-62⁰C; 1H NMR (CDCl₃): δ = 2.62-2.65 (m, 4H, 2CH₂), 2.81(s, 6H, 2CH₃), 3.48-

3.50(m, 4H, 2CH₂) ppm;

¹³C NMR (CDCl₃): δ = 27.41, 38.77, 49.38, 165.17 ppm.

Preparation of dimorpholinomethanone:

Dimorpholinomethanone, another urea derivative, was synthesized by condensation of morpholine and triphosgene (BTC) in dichloromethane (DCM).

Morpholine (0.4 mol) in DCM (200 ml) was placed in a 250 ml three necked flask fitted with a dropping funnel, and a solution of BTC (0.033 mol), dissolved in DCM (100 ml), was added thereto under nitrogen atmosphere at 0 ⁰C. The reaction mixture was stirred at 0⁰C for 1 hour, and for additional 3-4 hours at room temperature. Water (100 ml) was added to dissolve the white solid, the organic layer was collected and then washed with saturated Na₂CO₃ (50 ml), 1 N HCl (50 ml), saturated NaCl (50 ml), dried (MgSO₄), and filtered, and then the solvent was removed under reduced pressure to give a pure white solid in 86 % yield. m.p. 141-142⁰C;

¹H NMR (CDCl₃): δ = 3.19-3.21 (m, 8H, 4CH₂), 3.59-3.62(m, 8H, 4CH₂) ppm; 1³C NMR (CDCl₃): δ = 47.39, 66.75, 163.94 ppm. Preparation of l,l,3-trimethyl-3-(2-(pyrrolidin-l-yl)ethyl)urea:

l,l,3-trimethyl-3-(2-(pyrrolidin-l-yl)ethyl)urea, another urea derivative, was distillated and collected at 134-135 ⁰C as pale yellow oil in 74.2 % yield (11.6 grams from 78 mmol) according to General Procedure A or B using N-methyl-2-(pyrrolidin-l- yl)ethanamine as a secondary amine.

¹H NMR (CDCl₃): δ = 1.75-1.78 (m, 4H₅ 2 CH₂), 2.52-2.56 (m, 4H, 2 CH₂), 2.65 (t, 2H, CH₂), 2.79 (s, 6H, 2 CH₃), 2.84(s, 3H₅ CH₃), 3.12 (t, 2H₅ CH₂), ppm. ¹³C NMR (CDCl₃): δ = 23.67, 37.18, 38.91, 49.80, 53.94, 54.54, 165.68 ppm. m/z: 199.17.

Preparation of l,l,3-ttimethyl-3-(2-(piperidin-l-yl)ethyl)urea:

l,3-trimethyl-3-(2-(piperidin-l-yl)ethyl)urea, another urea derivative, was distillated and collected at 89-91 ⁰C as colorless oil in 81.7 % yield (34.8 grams from 0.3 mole) according to General Procedure A or B using N-methyl-2~(piperidin-l- yl)ethanamine as a secondary amine.

¹H NMR (CDCl₃): δ = 1.39-1.45 (m, 2H, CH₂), 1.54-1.59 (m, 4H, 2 CH₂), 1.60- 1.65 (br, IH, NH), 2.35-2.41 (m, 4H, 2 CH₂), 2.37-2.44(m, 5H, CH₂, CH₃), 2.66(t, 2H, CH₂) ppm.

Preparation ofl,l,3-trimethyl-3~(2-morpholinoethyl) urea:

l,l,3-trimethyl-3-(2-morpholinoethyl)urea, another urea derivative, was distillated and collected at 152-154 ⁰C as pale yellow oil in 76.8 % yield according to General Procedure A or B using N-methyl-2-morpholinoethanamine as a secondary amine.

¹H NMR (CDCl₃): δ = 2.45 (t, 4H, 2CH₂), 2.54 (t, 2H, CH₂), 2.77 (s, 6H, 2CH₃), 2.82 (s, 3H, CH₃), 3.29(t, 2H, CH₂), 3.66 (t, 4H, 2CH₂) ppm.

¹³ C NMR (CDCl₃): δ = 37.33, 38.91, 47.36, 54.04, 56.47 ppm. Preparation of chloroiminium derivatives - General Procedure C:

The preparation of chloroiminium derivatives was carried out as depicted in Scheme 4 below:

Scheme 4

Oxalyl chloride (100 mmol) in DCM (100 ml) is added dropwise to a solution of a urea derivative (100 mmol) in dry DCM (200 ml) at room temperature over a period of 5 minutes. The reaction mixture is stirred under reflux for 3 hours, and the solvent is removed under reduced pressure, the residue is washed with anhydrous ether (2 x 100 ml), and then bubbled with N₂ to remove excess of ether. The residue obtained is typically very hygroscopic, and therefore it is dissolved directly in DCM, and a saturated aqueous potassium hexafluorophosphate (100 mmol in 5OmL water, KPF₆) solution is added at room temperature with vigorous stirring for 10-15 minutes. The organic layer is collected, washed once with water (100 ml), dried over anhydrous MgSO₄, filtered and the solvent is removed under reduced pressure to give typically a white solid which can be recrystallized from DCM-ether or acetonitrile-ether to afford typically white crystals.

Preparation of 4-((dimethyamino) chloromethylene)morpholin-4-iminium hexaflurophosphate (DCMH):

Oxalyl chloride (100 mmol) in DCM (100 ml) was added dropwise to a solution of N,iV-Dimethylmoφholine-4-carboxarnide (100 mmol), prepared according to General Procedure A or B, in dry DCM (200 ml) at room temperature over a period of 5 minutes. The reaction mixture was stirred under reflux for 3 hours, the solvent was thereafter removed under reduced pressure and the residue was washed with anhydrous ether (2 x 100 ml), and then bubbled with N₂ to remove excess of the ether. The obtained white solid was dissolved in DCM (500 ml), and a saturated aqueous KPF₆

(18.4 grams in 50 ml H₂O) was added at room temperature with vigorous stirring for

10-15 minutes. The organic layer was collected, washed once with H₂O (50 ml), dried over anhydrous MgSO₄, filtered and the solvent was removed under reduced pressure to give a white solid, which was recrystallized from DCM-ether to give white crystals in 89.6 % yield (28.9 grams). m.p. 94-95 ⁰C;

¹H NMR (CD₃COCD₃): δ = 3.39 (s, 6H, 2CH₃), 3.75(t, 4H, 2CH₂), 3.86 (t, 4H, 2CH₂) ppm;

¹³C NMR (CD₃COCD₃): δ = 44.36, 52.82, 65.99, 162.79 ppm. Preparation of N-(Chloro(pyrrolidin-l-yl)methylene)-N- methylmeihanaminium hexafluorophosphate (DmPyCH):

N-(chloro(pyrrolidin-l-yl)methylene)-N-methylmethanaminium hexafluorophosphate (DmPyCH) was prepared according to General Procedure C and as described for DCMH hereinabove, and obtained as white solid in 89.0 %, yield. m.p.93- 95 ⁰C;

¹H NMR (CD₃COCD₃): δ = 2.00-2.13 (m, 4H, 2CH2), 3.49 (s, 6H, 2CH₃), 3.90- 4.02(m, 4H, 2CH₂) ppm.

Preparation of l-(chloro(morpholino)methylene)pyrrolidinium hexafluorophosphate: l-(chloro(morpholino)methylene)pyrrolidinium hexafluorophosphate was prepared as depicted in Scheme 5 below: Scheme 5

4-Morpholino carbonyl chloride (36 grams, 1.2 equivalents) in 100 ml DCM was added over a period of time 10 minutes to a stirred solution of pyrrolidine (0.2 mole, 17 ml) in 100 ml DCM and 100 ml of 10 % NaOH. The reaction mixture was stirred at room temperature for 3 hours and then the organic layer was collected and then the NaOH layer was washed with 100 ml DCM. The combined DCM was washed with water (2 X 100 ml), dried (MgSO₄), filter and the solvent was removed under vacuum to give colorless oil at a yield of 92.4 % (34 grams) which solidified at room temperature.

The crude urea derivatives further purified by vacuum distillation and collect the fraction at 165-169 °C. 1H NMR (CDCl₃): δ = 1.76-1.81(m, 4H, 2CH₂), 3.24 (t, 4H, 2 CH₂), 3.32-3.35

(m, 4H, 2 CH₂), 3.64 (t, 2H, CH₂) ppm;

¹³C NMR (CDCl₃): δ = 25.72, 46.84, 48.52, 66.97, 162.84 ppm; The chloroiminium salt was prepared according to General Procedure C as described hereinabove. The product was obtained as white solid at a yield of 83.9 %. m.p. 99-100 °C.

¹H NMR (acetone-d₆): δ = 2.10-2.14(m, 4H, 2CH₂), 3.87 (t, 4H, 2 CH₂), 4.00 (t, 4H, 2 CH₂), 4.04-4.06 (m, 4H, 2 CH₂) ppm;

¹³C NMR (CDCl₃): δ = 25.80, 51.75, 55.97, 65.97, 154.85 ppm.

EXAMPLE 2

CHEMICAL SYNTHESES OF VARIOUS COMPOUNDS DESIGNATED AS

LEAVING GROUPS IN COUPLING AGENTS: Preparation ofN-hydroxyindolin-2-one (HOI):

The synthesis is adapted from the art [Kenneth H. Collins, J. Amer. Chem.Soc, 78, 221-224 (1956)] and illustrated in Scheme 6 below. Scheme 6

2-

ydroxyindolin-2-one . (HOI)

2-nitrophenylacetic acid (9 grams, 50 mmol) was dissolved in 50 % H₂SO₄ (40 ml) and then 7 grams of zinc powder was added over a period of time 1 hour. The reaction mixture was stirred for 3hours at 30-35 ⁰C and the insoluble portion was filtered off. The product was dissolved in Na₂CO₃ solution and reprecipitated with HCl, filtered, dried and recrystallized from water to afford pale yellow solid at a yield of 20 % (1.5 grams). m.p. 198-199 ⁰C;

¹H NMR (DMSO-de): δ = 3.97(s, 2H, CH₂), 7.51-7.56(m, 2H, ar) 7.68 (t, IH, ar), 8.04 (d, IH, ar), 12.51 (br, IH, OH) ppm.

Preparation of3-hydroxy-2-meihylquinazolin-4-one (HOMQ): The synthesis is adapted from the art [F. Gutierrez, C. Tedeschi, L. Maron, J-P.

Daudey, R. Poteau, J. Azema, P. Tisnes, and C. Picard, DaIt. Trans. 1334-1347 (2004)] and illustrated in Scheme 7 below.

Scheme 7

BnONH₂.HCI/K₂CO₃ Ac₂O

24 hr, rt 3hr reflux

2-amino-Λ/-(benzyloxy)benzamide lsatoic anhydride

3-(benzyloxy)-2- 3-hydroxy-2-methylquinazolin-4(3H)-one methylquinazolin- (HOMQ)

4(3H)-one K2CO3 (0.93 gram) was slowly added to a solution of o-benzyl hydroxylamine/HCl (2.16 grams, 12.58 mmol) in water (60 ml). This mixture was stirred for 30 minutes and then acetic acid anhydride (2 grams, 12.3 mmol) was added thereto. The reaction mixture was kept at room temperature under stirring for 24 hours and then filtered. The solid was dried under reduced pressure and then recrystallized form dichloromethane-hexane to give light brown solid at a yield of 89.7 % (5.34 grams). m.p. 105-106⁰C; 1H NMR (CDCl₃): δ = 5.02 (s, 2H, CH₂), 6,58-6.62 (m, IH, ar), 6.67-6.69 (m,

IH, ar), 7.1-7.25 (m, 3H, ar), 7.37-7.46 (m, 6H, ar, NH), 8.37-8.46 (brs, IH, NH) ppm.

A mixture of 2-amino-N-benzyloxy benzamide (2 grams, 8.25 mmol) and acetic anhydride (9.12 ml, 8.5 mmol) was heated under reflux for 2 hours. After cooling, water (3.9 ml) and activated carbon were added while it is hot and the mixture was boiled for a further 30 minutes, followed by filtration through celite pad. The celite pad was washed with methanol and the combined filtrates were evaporated under reduced pressure. The residue was filtered through a silica gel using (DCM-MeOH, 9:1), and then the solvent was evaporated under reduced pressure. The crude residue was dissolved in MeOH and treated with (0.02 gram) of Pd/C for 30 minutes, at room temperature, filter through celite and wash with MeOH. The combined MeOH was evaporated under vacuum to afford the product at a yield of 48.1 %. m.p. 161-162⁰C;

¹H NMR (DMSO-d6): δ = 2.27 (s, 3H, CH₃), 7.13 (t, IH, ar), 7.61 (t, IH, ar), 8.13 (d, IH, ar), 8.73 (d, IH, ar), 11.04 (s, IH, OH) ppm. The same compound was prepared using Acetyl chloride-triethylamine in acetic acid as follows. 2-amino-N-hydroxybenzamide (10 mmole) of was suspended in 10 mmol of triethylamine (exothermic dissolution) and then acetic acid (10 ml) was added followed by adding acetyl chloride (1 ml) at room temperature. The reaction mixture was stirred at room temperature for 1 hour and then 20 ml of water were added thereto. The precipitate was collected by filtration, dried to give the pure product at a yield of 78.5 %.

Preparation of 3-hydroxyquinazolin-4-one (HOQ): The synthesis was adapted from the art [Robert H. Clark and E. C. Wagner, J.

Org. Chem., 1, 55-67 (1944)], and started with the synthesis of 2-amino-N- hydroxybenzamide, as illustrated in Scheme 8 below.

Scheme 8

lsatoic anhydride 2-amino-/V-hydroxybenzamide 3-hydroxyquinazolin-4(3H)-one

(HOQ)

Na₂COs (5.5 grams, 0.55 mole, 1 equivalent) was added to a solution of hydroxylamine hydrochloride (6.9 g, 0.1 mole, 1 equivalent) in 250 ml of water. lsatoic anhydride (8.15 grams, 0.05 mole) was added to the solution, which started the reaction spontaneously. The reaction mixture was allowed to stand at room temperature overnight. The solid product was filtered and recrystalized from water at 40 ⁰C, dried briefly in the air and completely under reduced pressure (this compound is sensitive for moist air) to afford the product at a yield of 85.8 %. m.p 86⁰C.

The procedure proceeded with the synthesis of 3-hydroxyquinazolin-4-one (HOQ), effected by two methods as follows.

Method (A): A mixture of 1.52 grams (10 mmol) of the hydroxamic acid and 4 ml of 98 % formic acid was heated under reflux for 15 minutes, after which 10 ml of water was added and the whole mixture was boiled for 15 minutes and cooled to room temperature. The precipitate was collected by filtration and washed with cold water (2 x 5 ml) dried to afford 0.35 grams (22 % yield) of yellowish white solid. m.p. 212-21 A ⁰C (decomposed);

¹H NMR (DMSO-d6): δ = 7.54 (td, IH, ar), 7.71 (dd, IH, ar), 7.82 (td, IH, ar), 8.17 (dd, IH, ar), 8.53 (s, IH, ar), 11.92 (br, IH, OH) ppm.

Method (B): 8 mmol of the hydroxamic acid was dissolved in 20 ml of MeOH contained 8 mmol of NaOH, and the solution was stirred at room temperature for 30 minutes. The solvent was evaporated to dryness and the residual solid was dissolved in 8 ml of 98 % formic acid. The reaction mixture was refluxed for 30 minutes and then cooled to room temperature. Dilute with water (20 ml) to afford a yellowish white solid, filtered, dried and then recrystallized from water to afford light brown solid 0.59 grams (37 % yield). m.p. 270-272⁰C (decomposed);

Preparation of6-Chloro-3-hydroxyquinazolin~4-one (6-Cl-HOQ):

The compound was prepared according to the Method (B) described hereinabove and illustrated in Scheme 9 below.

Scheme 9

6-Chloro isaioic anhydride 2-amino-5-chloro-Λ/-hydroxybenzamide

min

6-chloro-3-hydroxyquinazolin-4(3H)-one (6-CI-HOQ)

The product was obtained in 0.71 grams (36.6 % yield from 10 mmol reaction) as light brown solid. m.p. 268-269⁰C;

¹H NMR (DMSO-d6): δ = 7.74 (d, IH), 7.85 (dd, IH), 8.09 (d, IH), 8.56 (s, IH), 12.02 (s, IH, OH, exchangeable with D₂O). Preparation of6-CMoro-3-hydroxy2-methylquinazolin-4-one (6'Cl-HOMQ): The synthesis of 6-Chloro-3-hydroxy2-methylquinazolin-4-one (6-Cl-HOMQ) is illustrated in Scheme 10 below.

Scheme 10

6-Chloro isiloic anhydride ≥-amino-δ-chloro-W-hydroxybenzamide

6-chloro-3-hydroxy-2-methylquinazolin-4(3W)-one (6-CI-HOMQ)

2-Amino-5-chloro-N-hydroxybenzamide (1.86 grams, 10 mmole) was suspended in triethylamine (1.3 ml, 10 mmole) and then acetic acid (10 ml) was added followed by the addition of acetyl chloride (1 ml) at room temperature. The reaction mixture was stirred at room temperature for 1 hour and then 20 ml of water were added thereto. The precipitate was collected by filtration, washed with cold water, dried to give 1.69 grams of the pure product at a yield of 80.3 %. m.p.183-185 ⁰C;

¹H NMR (DMSO-d6): δ = 2.11 (s, 3H, CH₃.), 7.63 (dd, IH), 7.87(d, IH), 8.45(d,lH), 10.93 (s,lH, OH) ppm.

Preparation ofN-hydroxy-2-phenylbenzimidazole (HOPBI):

The synthesis of N-hydroxy-2-phenylbenzimidazole (HOPBI) is illustrated in

Scheme 11 below:

Scheme 11

2-nitroaniline 1-(benzyloxy)-2-pheny!-1H-benzo[cφrnidazole

2-phenyl-1H-benzo|cφmidazol-1-ol (HOPBI) Method (A): Adapted from the art [N. D. Kokare, R. R. Nagwade, V. P. Rane,

D. B. Shinde, Synthesis, 766-772 (2007)] is effected as follows. NaH (48 mmol, 3.25 grams of 60 % dispersed in mineral oil, washed three times with dry THF before use) suspended in THF dry (30 ml) and 2-nitroaniline (2.68 grams, 20 mmol) was added portion- wise with cooling. After 15 minutes, benzyl bromide (5.8 grams, 50 mmol) was added slowly and the reaction mixture was heated at 80 ⁰C for 4 hours. The reaction mixture was cooled to room temperature, quenched with water (25 ml) and extracted into ethylacetate (2 x 15 ml). The organic layer wad dried (Na₂SO₄) and concentrated under vacuum. The product was isolated by trituration with hexane and filtration to afford an off-white solid, at a yield of 87.2 % (5.1 grams). The NMR showed some traces of starting materials (about 12-15 %), and further recrystallized or purified by column was required.

¹H NMR (CDCl₃): δ = 5.10(s, 2H), 7.20-7.40(m, 5H), 7.60-7.80(m,5H), 7.8(d, 2H), 8.20(d, 2H)ppm.

Method (B): Adapted from the art [J. M. Gardiner, C. R. Loyns, C. H. Schwallabe, G. C. Barrett, P. R. Lowe, Tetrahedron, 51, 4101-4110 (1995)] is effected as follows. O-Nitroaniline (14.5 mmol) was dissolved in dry THF (100 ml), and NaH(60 % in oil, 1.45 mmol) added at ambient temperature. Benzyl bromide (14.5 mmol) was added slowly, and the reaction was refluxed for 4 hours. The reaction was cooled to ambient temperature, and further 14.5 mmol of NaH were added slowly, the reaction heated a further 4 hours, and the second portion of benzyl bromide (14.5 mmol) was added. After a further 4 hours, the reaction was again cooled to ambient temperature, a third portion of NaH (14.5 mmol) was added and the reaction heated for further 4 hours (NaH must be washed three times with THF before used). The reaction was cooled and quenched with NaCl solution, extracted with DCM (3 X 100 ml). The organic extracts were combined, washed with saturated NaCl solution (100 ml), dried (Na₂SO₄), filter and the solvent was removed under vacuum. The crude solid product was triturated with hexane three times (3 X 100 ml) to afford a white solid at a yield of 88.4 %.

The crude product (5.3 grams, 0.17 mol) was dissolved in MeOH (50 ml), Pd/C (10 %, 500 mg) was added and the reaction mixture was stirred under H₂ atmosphere at room temperature for 15-30 minutes (TLC, AcOEt, hexane 1:1). The reaction mixture was filtered through high-flow celite and the filtrate was concentrated and purified by column chromatograph (MeOH-CHCl₃, 1:9) to yield the N-hydroxy compound as a white solid at a yield of 75.9 % (2.75 grams). 1H NMR (DMSO-d₆): δ = 7.23-7.46(m, 2H), 7.61-7.83 (m, 5H, Ph), 8.22 (d,

2H), 12.22 (S₅IH, OH) ppm.

Preparation of ό_'Chloro-N-hydroxy^-phenylbenzimidazole (6-Cl-HOPBI) : ό-Chloro-N-hydroxy^-phenylbenzimidazole (6-Cl-HOPBI) was prepared asillustrated in Scheme 12 below.

Scheme 12

4- le

6-chloro-2-phenyl-1H-benzo[oTIimidazoI-1-ol (6-Cl-HOPBI) The product was obtained using Method (B) as light brown crystals to afford 4.22 grams from 20 mmol (65.8 % yield). m.p. 123-124⁰C; 1H NMR (CDCl₃): δ = 5.03 (s, 2H, CH₂), 7.19-7.38 (m, 7H), 7.51 (t, 3H)₅ 7.68

(d, IH), 8.13-8.16 (m, 2H).

The OBn derivative (3.345 grams, 10 mmol) was dissolved in 10 ml MeOH and 10 ml THF. Thereafter Pd/C (10 %, 500 mg) was added and the reaction mixture was stirred under H₂ atmosphere at room temperature for 15-30 minutes (TLC, AcOEt, hexane 1:1). The reaction mixture was filtered through high-flow celite and the filtrate was concentrated and purified washing with ether (2 x 10 ml) to afford the pure product as off white solid at a yield of 73 % (1.78 grams), m.p. 262-264⁰C;

¹H NMR (DMSO-d₆): δ = 7.25 (dd, IH), 7.53-7.57 (m, 4H, Ph), 7.68 (d, IH), 8.20-8.25 (m, 2H), 12.19 (s, IH, OH) ppm.

¹³C NMR (DMSO-d₆): δ = 123.72, 125.90, 130.35, 131.35, 132.36, 133.87, 134.88, 135.58, 136.35, 147.34, 148.17, 159.99, 165.26 ppm.

Preparation of2-methyl-3H-imidazole[4,5-bJpyridine-3-ol (HOMPI) : 2-methyl-3H-imidazole[4,5-b]pyridine-3-ol (HOMPI) was prepared as illustrated in Scheme 13 below.

Scheme 13 p NaN3 [j T Toluene T jT⁺)o

IN Cl 10% aq. EtOH/10% HCI N ^S N 4 hr reflux ^"^N ^^N

2-chloro-3-nitropyridine ^{reflux 4B hr N} [1 ,2,5]oxadiazolo[3,4-

8-nitro-[1 ,2,4]triazolo[1 ,5- ϋjpyridine 1 -oxide a]pyridine

CH₃CH₂NO₂ V ff ^{^}Y^-1V-_PH

Piperidine IT ^^Y ^jWVCHB ^c —^sa/ ' ^•M•e — ^{■■ ■} ■ " Q ^•' ' ^ori3

12 hours, r.t. ^N^^N ^oH - O^H

OH 2-methyl-3H-imidazo[4,5-6]pyridin-3-ol

3-hydroxy-2-methyl-3H- (HOMPI) imidazo[4,5-_?]pyridine 1 -oxide 8-Nitro-[l,2,4]triazole[l,5-a]pyridine was prepared according to a published procedure [Charlotte K. Lowe-Ma, Robin A. Nissan, William S. Wilson, J. Org. Chem., 55, 3755-3761 (1990)] as follows. 2-chloro-3-nitropyridine (4 grams, 25.6 rnrnol) and sodium azide (4 grams, 61.6 mmol) were dissolved in 10 % aqueous ethanol (400 ml) at ambient temperature, and 10 % HCl (40 ml) was added. The solution was then heated under reflux for 48 hours. Evaporation to dryness, addition of water (80 ml) and filtration afforded buff solid residue which recrystallized from ethanol to afford light brown crystals 2.4 g (74.1% yield). m.p. 176-178⁰C; 1H NMR (DMSOd₆): δ = 7.65 (t, IH), 8.88 (d, IH), 9.76 (d,lH) ppm.

[1,2,5] -Ioxadiazoleo[3,4-b]pyridine 1-oxide was prepared as follows. 8-nitro-

[l,2,4]triazole[l,5-a]pyridine (1 gram, 6 mmol) was dissolved in toluene (100 ml) and heated under reflux for 4 hours. The solution was decolorized with charcoal and filtered, evaporation to dryness to produce a pale yellow solid which recrystallized from cyclohexane to afford a yellow crystals at a yield of 62.4 % (0.52 grams). m.p. 54-56 ⁰C;

¹H NMR (acetone-d₆): δ = 7.47 (dd, IH), 8.25 (dd, IH), 8.65 (dd, IH) ppm.

3-Hydroxy-2-methyl-3H-imidazole[4,5,b]pyridine 1-oxide was prepared according to a published procedure [M. Boiani, L. Boiani, A. Denicola, S. Torres de Ortiz, E. Serna, N. Verade Bilbao, L. Sanabria, G. Yaluff, H. Nakayama, A. Rojasde

Arias, C. Vega, M. Rolan, A. Gόmez-Barrie, H. Cerecette, M. Gonzalez, J. Med.

Chem., 49, 3215-3224 (2006)] as follows. [1,2,5] ioxadiazoleo[3,4-b]pyridine 1-oxide

(3 mmol) and nitroethane (3 mmol) were dissolved in 5 ml of THF. Piperidine (3 mmol) was added drop-wise (exothermic reaction). After complete addition the reaction mixture was left to stand at room temperature for 12 hours. Filter for the brown precipitate which recrystallized from DCM-hexane.

Preparation of potassium salt of hydroxycarbonimidoyl dicyanide:

The synthesis was effected according to a published procedure [Mitsuru Kitamura, Shunsuke Chiba, Koichi Narasaka, Bull. Chem. Soc. Jpn, 76, 1063-1070 (2003)] as illustrated in Scheme 14 below.

Scheme 14

NCk^CN NaNO₂ / HAc NC_x XN malononitrile 4hr, O ⁰C ^*~ JL

HO" hydroxycarbonimidoyl dicyanide

(Oil)

KOH/MeOH _> NC^CN

O ⁰C, 15 min \f κo'^N

(KONDC) yellow soild

Sodium nitrite (14.2 grams, 206 mmol) was added slowly at ⁰C over 20-30 minutes to a solution of malononitrile (9.06 grams, 138 mmol) in acetic acid (20 ml) and water (50 ml) and then the mixture was stirred at the same temperature for 45 minutes. After quenching the reaction with 2N HCl (100 ml), the reaction was extracted three times with ether (3 x 100 ml). The extracts were dried over anhydrous Na₂SO₄, and the ether was removed under vacuum to give an oily residue. The oily product was added slowly to a cold solution of KOH (8.0 grams) in MeOH (100 ml), and then the reaction mixture was stirred at ⁰C for 20 minutes. Excess ether was added to afford the pot salt as yellow crystals at a yield of 77.5 % (14.1 grams). Preparation of potassium salt of diethyl 2-(hydroxyimino)malonate:

Potassium salt of diethyl 2-(hydroxyimino)malonate was prepared according to a published procedure [Kenneth N. F. Shaw and Chis Nolan, /. Org. Chem., 22, 1668- 1670 (1957)] as illustrated in Scheme 15 below.

Scheme 15

diethyl 2-(hydroxyimino) malonate

(KONDeC) yellow solid

A solution of 16 grams (0.1 mole) of diethylmalonate in 17.5 ml (0.3 mole) of glacial acetic acid was stirred vigorously at 0-5 ⁰C while addition of a solution of 20.7 grams of NaNO₂ (0.3 mole) in 250 ml of water was added drop-wise during 3-4 hours. The ice bath was removed and the mixture was stirred vigorously for additional 20 hours. The nitrosation was carried out in three nicked flask with appropriate fitting and a small vent to permit escape of nitric oxide. The reaction mixture was extracted with 400 ml and then three 100 ml portions of DCM. The combined DCM extracts were dried over anhydrous Na₂SO₄. The DCM was removed under vacuum and the resulting oily product was dissolved in 400 ml of DCM, stirred with anhydrous K₂CO₃ (32 grams) for 15 minutes and filtered, and the DCM was concentrated until 200 ml. Thereafter ether was added until the solution became cloudy, and then kept in the refrigerator over night to afford off white crystals at a yield of 63.4 % m.p. 116-118⁰C;

¹H NMR (CDCl₃): δ = 1.24-1.29 (q, 6H, 2 CH₃), 4.20-4.29(m, 4H, 2CH₂) ppm. Preparation of 2_'Pyridylhydroxyiminoacetonitrile:

2-Pyridylhydroxyiminoacetonitrile was prepared according to a published procedure [Jan Izdebski, Polish J. Chem., 53, 1049-1057 (1979)] as illustrated in Scheme 16 below.

Scheme 16

2-(pyridin-2-yl)acetonitrile

Λ/-hydroxypicolinimidoyl cyanide

A solution of sodium nitrite (4.5 grams, 0.065 mole in 5 ml water) was added slowly to a solution of 2.2 grams (0.019 mole) of 2-pyridylacetonitrile in 4.5 ml of glacial acetic acid. After 12 hours standing the precipitate was filtered off, washed with water, dried and then recrystallized from ethanol to afford the product at a yield of 65 % m.p 220-222⁰C;

¹H NMR (DMSO-d₆): δ = 7.45-7.52 (IH), 7.87-7.90 (2H), 8.67 (d, IH), 14.12 (OH) ppm.

EXAMPLE 3

CHEMICAL SYNTHESES OF VARIOUS URONIUM/IMMONIUM TYPE

COUPLINGAGENTS Preparation of tetraalkyl uroniumjimmonium type coupling reagents - General

Procedure D:

The chloroiminium salt, prepared as illustrated in

Scheme 4 hereinabove (20 mmol) is added to a solution of the potassium salt of the leaving group hydroxyl-precursor compound (denoted KOL₁ in Scheme 17, 20 mmol) in acetonitrile (50 ml) at 0⁰C. The reaction mixture is stirred at this temperature for 30 minutes and allowed to warm to room temperature while stirring for 6 hours. The resulting precipitate is filtered and washed with acetonitrile, the solvent is concentrated to small volume (1/4) under reduced pressure, and then dry ether is added to afford the product typically as a white solid in pure state.

Scheme 17

L₁OH =

H)-one

(

2-(hydroxyimino)malononitrile 6-chloro-2-methylquinazolin-4(3H)- one-1-hydoxy

COOEt indolin-2-one-l-hydroxy HO-N=^

COOEt diethyl 2- (hydroxyimino)malonate

Preparation of l,l,3,3-tetramethyl-2~(2-oxopyridin-l(2H)-yl)isouronium hexqfluorophosphate (HTOP):

l,l,3,3-Tetramethyl-2-(2-oxopyridin-l(2H)-yl)isouronmm hexafluorophosphate (HTOP) was prepared according to General Procedure D, and obtained as a white solid at a yield of 85.6 % (6.08 grams). tn.p. 204-205⁰C; 1H NMR (acetone-d₆): δ = 3.12 (s, 12H, 4 CH₃), 6.49 (d, IH), 6.74 (dd, IH),

7.63 (td, IH), 8.42 (dd, IH) ppm;

¹³C NMR (acetone-d₆): δ = 40.76, 107.11, 122.05, 135.98, 142.14, 156.66, 162.22 ppm.

Preparation of O-[Cyano(eihoxycarbonyl)methylidene)amino]-l,l,3,3- tetramethyluronium Hexafluorophosphate (HTOCC) :

O-[Cyano(ethoxycarbonyl)methylidene)amino] -1 , 1 ,3 ,3-tetramethyluronium hexafluorophosphate (HTOCC), taught in U.S. Patent No. 5,166,394, was prepared according to General Procedure D, and obtained as a white solid at a yield of 82.1 % (6.32 grams). m.ρ. 135-137 ⁰C (decomposed); ¹H NMR (acetone-d₆): δ = 1.37 (1, 3H, CH₃), 3.37 (s, 12H, 4 CH₃), 4.82 (q, 2H,

CH₂) ppm;

¹³C NMR (acetone-d₆): δ = 13.46, 40.71, 64.56, 106.78, 135.09, 156.11, 161.43 ppm. Preparation of O-[(dicyanomethylidene)amino]-l,l,3,3-tetramethyluroniunι hexafluorophosphate (HTODC):

O-[(Dicyanomethylidene)amino]-l,l,3,3-tetramethyluronium hexafluorophosphate (HTODC) was prepared according to General Procedure D, and obtained as a white solid at a yield of 74.0 % (5.0 grams). m.p. 180-181 ⁰C (decomposed); 1HNMR (acetone-d6): δ = 3.27 (s, 12H, 4 CH3) ppm; 1³C NMR (acetone-d6): δ = 40.80, 105.10, 108.21, 119.65, 160.67 ppm.

Preparation of 0-[(dieihoxycarbonylmethylidene)amino]-l,l,3,3- tetramethyluronium hexafluorophosphate (HTODeC):

O-[(Diethoxycarbonylmethylidene)amino]-l,l,3,3-tetramethyluronium hexafluorophosphate (HTODeC) was prepared according to General Procedure D, and obtained as pale yellow oil at a yield of 84.5 %. ¹HNMR (CDC13): δ = 1.35 (t, 6H, 2 CH3), 3.18 (s, 12H, 4 CH3), 4.39-4.48 (q,

4H₅ 2CH2) ppm.

Preparation of dimethyl morpholino uroniumlimmonium type coupling reagents - General Procedure E:

The preparation of a series of dimethyl morpholino uronium/immonium type coupling reagents is carried out as illustrated in Scheme 18 below.

Scheme 18

L₁OH =

1 -hydroxypyridin-2(1 H)-one

(h

2-(hydroxyimino)malononitrile 6-chloro-2-methy!quinazolin-4(3H)- one-1 -hydoxy

COOEt iπdolin-2-one-l-hydroxy

HO- diethyl 2- (hydroxyimino)malonate

Preparation of l-(N-methyl-N-morpholinomethylene)-(2-oxopyridin-l(2H)- yloxytymethanaminium hexafluomphosphate (HDMOP);

l-(ΛT-methyl-iV-morpholinomethylene)-(2-oxopyridm-l(2H)- yloxyl)methanaminium hexafluorophosphate (ΗDMOP) was prepared according to General Procedure E, and obtained as a white solid at a yield of 78.1 % (6.2 grams).

¹H NMR (acetone-d₆): δ = 3.21 (s, 6H, 2 CH₃), 3.51 (bra, 4H, 2 CH₂), 3.61-3.64 (m, 4H, 2CH₂), 6.44 (td, IH), 6.71(dd, IH), 7.58(td, IH), 8.13 (dd, IH) ppm; 1³C NMR (acetone-d₆): δ = 49.33, 65.47, 106.98, 122.22, 134.98, 141.73,

156.94, 162.06 ppm.

Preparation of l-[(l-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)~ dimethylamino-morphotinomethylene)] methanaminium hexafluorophosphate (HDMOCC):

l-[(l-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino- morpholinomethylene)] methanaminium hexafluorophosphate (HDMOCC) was prepared according to General Procedure E, and obtained as white crystals at a yield of 88.8 % (7.6 grams). m.p. 143-144⁰C;

¹HNMR (acetone-d₆): δ = 1.38 (t, 3H, CH₃), 3.41 (s, 6H, 2 CH₃), 3.82 (t, 4H, 2 CH₂), 3.89 (t, 4H, 2CH₂), 4.48 (q, 2H₅ CH₂) ppm; ¹³C NMR (acetone-d₆): δ = 13.48, 40.70, 49.94, 64.59, 66.04, 106.76, 135.03,

156.14, 160.61 ppm.

Preparation of 1 -[(l-(dicyanomethyleneaminooxy) -dimethylamino- morpholinomethylene)] methanaminium hexafluorophosphate (HDMODC):

l-[(l-(Dicyanomethyleneaminooxy)-dimethylamino-morpholinomethylene)] methanaminium hexafluorophosphate (HDMODC) was prepared according to General Procedure E, and obtained as white solid at a yield of 74.8 % (5.7 grams). m.p. 118-119⁰C;

¹HNMR (acetone-d₆): δ = 3.42 (s, 6H, 2 CH₃), 3.80-3.88 (m, 8H, 4 CH₂) ppm;

¹³C NMR (acetone-d₆): δ = 40.97, 49.93, 65.92, 105.13, 108.15, 119.84, 159.77 ppm.

Preparation of l-[(l,3-diethyoxy-l,3-dioxopropan-2-ylideneaminooxy)- dimethylamino-morpholinomethylene)] methanaminium hexafluorophosphate (HDMODeC):

l-[(l,3-Diethyoxy-l,3-dioxopropan-2-ylideneaminooxy)-dimethylamino- morpholinomethylene)] methanaminium hexafluorophosphate (HDMODeC) was prepared according to General Procedure E, and obtained as pale yellow oil at a yield of 84.3 %. ¹HNMR (CDCl₃): δ = 135 (t, 6H, 2 CH₃), 3.21(s, 6H, 2 CH₃), 3.35 (t, 2H,

CH2), 3.61 (t, 2H, CH2), 3.78 (t, 2H, CH2), 3.87 (t, 2H, CH2), 4.37-4.50 (q, 4H, 2CH₂) ppm.

Preparation of dimethyl pyrrolidine uroniumlimmonium type coupling reagents - General Procedure F:

The preparation of a series of dimethyl pyrrolidino uronium/immonium type coupling reagents is carried out as illustrated in Scheme 19 below using the same type of leaving group moieties (OLi) as shown in Scheme 17 and Scheme 18 above.

Scheme 19

Preparation of l-((dimethylamino)(2-oxopyridin-l(2H)- yloxy)methylene)pyrrolidinium hexafluorophosphate (HDmPyOP):

l-((Dimethylamino)(2-oxopyridin-l(2H)-yloxy)methylene)pyrrolidinium hexafluorophosphate (HDmPyOP) was prepared according to General Procedure F, and obtained as white soild at a yield of 6.6g (86.7%). m.ρ. 146-147⁰C (decomposed);

¹HNMR (acetone-d₆): δ = 1.98-2.15 (m, 4H, 2CH₂), 3.06 (s, 6H, 2CH₃), 3.71- 3.80(m, 4H, 2CH₂), 6.41 (td, IH), 6.73 (dd, IH), 7.50 (td, IH), 7.94(dd, IH) ppm;

¹³C NMR (acetone-d₆): δ = 25.16, 39.77, 51.25, 106.70, 122.04, 135.40, 156.73, 159.84 ppm. Preparation of l~[(l-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)- dimethylamino-pyrrolodinomethylene)] methanaminium hexafluorophosphate (HDmPyOCC):

l-[(l-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino- pyrrolodinomethylene)] methanaminium hexafluorophosphate (HDmPyOCC) was prepared according to General Procedure F, and obtained as white solid at a yield of 82.7 % (6.8 grams). m.p. 126-127⁰C (decomposed);

¹HNMR (acetone-d₆): δ = 1.37 (t, 3H, CH₃), 2.10, 2.13 (m, 4H, 2CH₂), 3.36 (s, 6H, 2CH₃), 3.95-3.99(m, 4H, 2CH₂), 4.48 (q, 2H, CH₂) ppm;

¹³C NMR (acetone-d₆): δ = 13.47, 25.09, 40.40, 51.58, 64.52, 106.74, 134.82, 156.12, 158.66 ppm.

Preparation of l-[(l-(dicyanomethylideneaminooxy)-dimethylamino- pyrrolodinomethylene)] methanaminium hexafluorophosphate (HDmPyODC):

l-[(l-(Dicyanomethylideneaminooxy)-dimethylamino-pyrrolodino methylene)]methanaminium hexafluorophosphate (HDmPyODC) was prepared according to General Procedure F, and obtained as a light yellow solid at a yield of 74 % (5.4 grams). m.p. 146-147⁰C (decomposed); ¹HNMR (acetone-d₆): δ = 1.97-2.00 (m, 4H, 2CH₂), 3.28 (s, 6H, 2CH₃), 3.96-

4.04(m, 4H, 2CH₂) ppm;

¹³C NMR (acetone-d₆): δ = 25.07, 40.41, 51.77, 105.06, 108.23, 119.30, 157.86 ppm. Preparation of pyrrolidine morpholino uronium/immonium type coupling reagents - General Procedure G:

The preparation of a series of dimethyl pyrrolidine uronium/immonium type coupling reagents is carried out as illustrated in

Scheme 20 below using the same type of leaving group moieties (OLi) as shown in Scheme 18 above.

Scheme 20

Preparation of l-(morpholino(2-oxopyridin-l(2H)- yloxy)methylene)pyrrolidinium hexafluorophosphate (HMPyOP):

l-(Moφholino(2-oxopyridin-l(2H)-yloxy)methylene)pyrrolidinium hexafluorophosphate (HMPyOP) was prepared according to General Procedure G, and obtained as white solid at a yield of 70 % (5.9 grams). m.p. 145-146⁰C (decomposed); 1HNMR (acetone-de): δ = 2.05-2.13 (m, 4H, 2CH₂), 3.54 (t, 4H₃ 2CH₂), 3.65(t,

4H, 2CH₂), 3.94-3.98 (m, 4H, 2CH₂), 6.85 (dd, IH), 7.72 (td, IH), 8.21 (dd, IH) ppm; ¹³C NMR (acetone-d₆): δ = 25.16, 39.77, 51.25, 52.87, 106.70, 122.04, 135.44,

156.76, 159.94 ppm.

Preparation of l-((l-cyano-2-ethoxy-2- oxoethylideneaminooxy) (morpholino) methylene) pyrrolidinium hexafluorophosphate (HMPyOCC):

l-((l-Cyano-2-ethoxy-2-oxoethylideneaminooxy)(morpholino)methylene) pyrrolidinium hexafluorophosphate (HMPyOCC) was prepared according to General Procedure G, and obtained as white solid at a yield of 90.3 % (8.2 grams), mp 171-172⁰C;

¹HNMR (acetone-d₆): δ = 1.26 (t, 3H, CH₃), 1.98-2.02 (m, 4H, 2CH₂), 3.65-3.68 (m, 4H, 2CH₂), 3.74-3.76(m, 4H, 2CH₂), 3.84-3.87 (m, 4H, 2CH₂), 4.37-4.40 (q, 2H, CH₂) ppm; 1³C NMR (acetone-d₆): δ = 13.49, 25.09, 49.20, 51.57, 55.98, 51.76, 64.54,

66.06, 106.69, 134.63, 156.11, 157.88 ppm.

Preparation of l-((dicyanomethylene- aminooxy)(morpholino)methylene)pyrrolidinium hexafluorophosphoate

(HMPyODC):

l-((Dicyanomethyleneaminooxy)(moφholino)methylene)pyrrolidinium hexafluorophosphoate (HMPyODC) was prepared according to General Procedure G, and obtained as light.yellow solid at a yield of 78.6 % (6.4 grams). m.p. 158-159⁰C; ¹HNMR (acetone-d₆): δ = 2.11-2.14 (m, 4H, 2CH₂), 3.79.3.85 (m, 4H, 2CH₂),

3.86-3.88(m, 4H, 2CH₂), 3.98-4.01 (m, 4H, 2CH₂) ppm;

¹³C NMR (acetone-d₆): δ = 25.28, 49.15, 65.95, 105.07, 108.17, 119.48, 157.00 ppm. Preparation of ethyl 2-cyano-2-(diphenoxyphosryloxyimino)acetate:

The preparation of ethyl 2-cyano-2-(diphenoxyphosryloxyimino)acetate was adapted from a published procedure [F. Hoffmann, L. Jager, C. Criehl, Phosphorus, sulfur, and silicon, 178, 299-309 (2003)] and carried out as illustrated in Scheme 21 below.

Scheme 21

TEA (1.3 ml, 10 rnmol) was added under argon atmosphere to a solution of diphenylchlorophosphate (2.69 grams, 10 mmol) and the corresponding additive (10 mmol) in anhydrous THF (50 ml). After 3 hours stirring at room temperature the reaction mixture was filtered off and the solvent was removed under reduced pressure. The resulting oil was washed with n-pentane and dried under vacuum. The product was obtained as oil at a yield of 64.5 % (2.42 grams).

¹HNMR (CDCl₃): δ = 1.41 (t, 3H, CH₃), 4.47 (q, 2H, CH₂), 7.22-7.44(m, 1OH, 2Ph) ppm.

Preparation of 2-pyridylacetonitrile oxime containing coupling reagents - General Procedure H: The preparation of a series of 2-pyridylacetonitrile oxime containing coupling reagents is carried out as illustrated in Scheme 22 below.

Scheme 22

Preparation of N-[(cyano(pyridine-2-yl)methyleneaminooxy) (dimethylamino) methylene) -N -Meihylmeihanaminium hexafluorophosphate (HTOPC):

N-[(Cyano(pyridine-2-yl)methyleneaminooxy)(dimethylamino)methylene)-N- methylmethanaminium hexafluorophosphate (HTOPC) was prepared according to General Procedure H, and obtained as a light reddish brown solid at a yield of 82.7 % (6.2 grams). m.p. 169-171⁰C;

¹HNMR (acetone-d₆): δ 3.30 (s, 12H, 4CH₃), 7.57-7.61(m, IH), 7.94(td, IH), 8.10 (dd, IH), 8.70 (dd, IH) ppm.

¹³C NMR (acetone-d₆): δ 40.48, 107.56, 122.52, 127.87, 138.18, 142.46, 146.67, 150.68, 162.03 ppm. Preparation of N-[(cyano(pyridine-2-yl)methyleneaminooxy) (dimethylamino) methylene) -N-morpholinomethanaminium hexafluorophosphate (HDMOPC) :

N-[(Cyano(pyridine-2-yl)methyleneaminooxy)(dimethylamino) methylene)-N- morpholinomethanaminium hexafluorophosphate (HDMOPC) was prepared according to General Procedure H_s and obtained as a light brown solid at a yield of 89.4 % (7.74 grams). m.p. 154-155⁰C;

¹H NMR (acetone-d₆): δ = 3.34 (s, 12H, 4CH₃), 3.79-3.83 (m, 8H, 4 CH₂)7.58- 7.62(m, IH), 7.96(td, IH), 8.12 (dd, IH), 8.71 (dd, IH) ppm; 1³C NMR (acetone-d₆): δ 40.76, 49.79, 66.13, 107.60, 122.53, 127.92, 138.23,

142.74, 146.63, 150.69, 161.06 ppm.

EXAMPLE 4

CHEMICAL SYNTHESES OF VARIOUS TRIS-AMINO PHOSPHONIUM TYPE COUPLINGAGENTS

Preparation of tris~(aminό)phosphonium hexafluorophosphate — General Procedure:

The general procedure is illustrated in

Scheme 23 below. R₁-R₆ can each be independently an alkyl having 1-4 carbon atoms and an alkyl having 1-4 carbon atoms interrupted, substituted and/or terminated by at least one heteroatom, or, alternatively, Ri and R₂, and/or R₃ and R₄, and/or R₅ and R₆ are each independently joined to form a heteroalicyclic moiety. Scheme 23

tris-(amino)phosphine oxide tri-amino-phosphonium chloride hexaflurophosphate

A solution of tris-(amino)phosphine oxide (1 equivalent) in dichloromethane (17.5 ml) is slowly added during 20 minutes to a cooled (water-ice bath) solution of POCl₃ (1 equivalent) in dichloromethane (17.5 ml), under nitrogen atmosphere, while maintaining the reaction mixture temperature below 30 ⁰C. The reaction mixture is further stirred for 1 hour at room temperature.

The reaction mixture is then cooled to 5 ⁰C for 10 minutes, and a suspension of the KPF₆ (1 equivalent) in water (200 ml) is added while rigorously stirring the reaction mixture maintaining its temperature at 5-10 ⁰C. The reaction mixture is further stirred at room temperature for 30 minutes. Thereafter the phases are separated, and the aqueous phase is washed with 2 x

50 ml dichloromethane. The organic phases are combined and washed with 3 x 100 ml water, dried over MgSO₄, and filtrated. The filtrate is evaporated to dryness under reduced pressure to obtain tris-(amino)phosphonium hexafluorophosphate as a solid (more than 90 % yield). Preparation of tris(amino)phosphonium hexafluorophosphate coupling agents

— General Procedure: The preparation of tris(amino)phosphonium-based coupling agents is illustrated in Scheme 24 below.

Scheme 24

L₁OH =

1 -hydroxypyridin-2(1 H)-one

2-(hydroxyimino)malononitrile 6-chloro-2-methylquinazolin-4(3H)- one-1-hydoxy 1-hydroxyindolin-2-one (HOI) COOEt Ho-N=<

COOEt diethyl 2- (hydroxyimino)malonate

A solution of leaving group hydroxyl precursor compound (1 equivalent, denoted LiOH in Scheme 24 above) and triethylamine (1 equivalent) in dichloromethane (70 ml) is added dropwise during 20 minutes to a stirred solution of tris- (amino)phosphonium hexafiuorophosphate (1 equivalent) in acetone (500 ml) while maintaining the reaction mixture temperature below 20 ⁰C. Stirring is continued for 3 hours, during which a precipitate (triethyl ammonium HCI salt) was formed. The precipitate is filtered off and the filtrate is evaporated to dryness under reduced pressure.

The solid residue obtained is dissolved in 700 ml dichloromethane, MgSO₄ (10 grams) is added to solution while stirring during 15 minutes, and the resulting suspension is stirred for an additional hour. The suspension is then filtered, and the filtrate is evaporated to dryness under reduced pressure to give the tris(amino)phosphonium-based coupling agents as a solid (more than 60 % yield).

A fully characterized, highly useful and favorably effective example of a phosphoniurα-based coupling agent can be found in WO 2007/020620 and U.S. Patent No. 7,304,163, which are all incorporated herein by reference as if fully set forth herewith.

EXAMPLE 5 CHARACTERIZATION OF COUPLING AGENT

Effect of oxygen on the solubility of the coupling agents:

The maximal solubility of various known and exemplary coupling agents, according to the present embodiments, is tested in DMF in order to assess their capacity to participate in solution- and solid-support peptide forming reactions. Solution synthesis of the di-peptide Z-Phg'Pro-NH2.'

The aptitude of exemplary coupling agents, according to the present embodiments, to couple between two amino-acids without causing racemization thereof is tested in solution. Solution coupling of benzyloxycarbonyl (Z)-protected phenylglycine ( Z-Phg-OH) with H-PrO-NH₂ to give Z-Phg-Pro-NH₂ in DMF in the presence of a base and a coupling agent is used to rank the novel and known peptide- coupling agents, and the extent of Phg residue racemization is determined and used as a ranking criteria. Solution synthesis of the tri-peptide Z-Phe-Val-Pro-NH2:

The ability of exemplary coupling agents, according to the present embodiments, to elongate a di-peptide with a third residue is tested in a solution phase coupling reaction in DMF of Z-Phe- VaI-OH with H-Pro-NH₂ in the presence of a base which yields the tri-peptide Z-Phe-Val-Pro-NH₂. The extent of the racemization of the valine residue is determined and used as ranking criteria. Synthesis of the penta-peptide H-Tyr-Gly-Gly-Phe-Leu-NH₂: The penta-peptide H-Tyr-Gly-Gly-Phe-Leu-NH₂ is prepared by solution phase synthesis using an exemplary coupling agent, according to the present embodiments, and Boc-protected amino acids (except the last one, Fmoc-Tyr(OtBu)-OH). At the end of the reaction the Fmoc-group is removed by using 30 % diethylamine in acetonitrile for 1 hour and the crude peptide is treated with TFA-DCM (1:1) for two hours at room temperature.

Synthesis of Tyr-Aib-Aib-Phe-Leu-NH₂ in solid phase The peptide Tyr-Aib-Aib-Phe-Leu-NH₂, which contains the challenging coupling one α-aminoisobutyric acid residue to another (Aib-Aib), is synthesized on solid-phase using Fmoc protected amino-acids in the presence of an exemplary coupling agent, according to the present embodiments, and the base DIEA (2 equivalents). The extent of failure to form the Aib-Aib peptide bond, namely the rate of (Tyr-Aib-Aib- Phe-Leu-NEb) penta-peptide versus (Tyr-Aib-Phe-Leu-NHb) tetra-peptide formation during the solid-phase assembly of the penta-peptide is used to assess the effectiveness of the coupling agent.

Synthesis of the 15-mer of the (60-74) fragment of the Acyl Carrier Protein (ACP) :

The peptide H-Glu-Lys-Ile-Thr-Thr-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly- NH₂ (15-mer peptide) is elongated manually on an Fmoc-Rink Amide-AM-resin using

Fmoc-protected amino acids and an exemplary coupling agent, according to the present embodiments. Coupling tunes is 30 minutes, excesses of reagents is 3 equivalents, and the excesses of the base DIEA is 6 equivalents.

EXAMPLE 6

CHARACTERIZATION OF PHOSPHONIUM-TYPE COUPLING AGENT Comparative Reactivity Studies:

The stability of a tris(amino)phosphonium hexafluorophosphate type coupling agent, which may serve as a criteria for its reactivity and efficiency as a peptide coupling agent (less stable means more reactive), can be studied by way of comparison to the widely used commercially available phosphonium-based coupling agent benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP).

Materials and experimental methods:

An exemplary tris(amino)phosphonium hexafluorophosphate coupling agent, according to the present embodiments, is prepared as described herein.

HPLC analysis is carried out using a photodiode array detectors equipped with the a separation module, using HPLC reversed phase symmetry columns C18 4.6 x 150 mm, 5 μm. U.V detection is performed at 220 nm. A linear gradient mobile phase starting with CH₃CN (containing 0.036 % TFA) and ending with H₂O (containing 0.045 % TFA) is used at a flow rate of 1.0 ml/minute.

Stability Assays: All the stability assays are carried out using a 0.05 M solution of the coupling agent in DMF.

Samples are taken at different time points and are evaluated by analytical HPLC. The appearance of the breakdown products of the exemplary coupling agent and 1- hydroxy-benzotriazole (HOBt) for PyBOP served as an indication of the stability thereof.

Additional stability studies are performed in the presence of the tertiary amine N,N-diisopropylethylamine (DIEA), which is used as a typical base in peptide syntheses. Thus, DIEA (2 mol equivalents) is added to each of the tested solutions, and the appearance of the breakdown products described above over time is monitored. Comparative Solid-Phase Peptide Syntheses

The reactivity of an exemplary phosphonium-based compound as a coupling agent in solid-phase peptide syntheses is tested and compared with that of PyBOP. To this end, solid phase syntheses of various peptides, which are considered difficult to prepare by conventional synthetic approaches, are performed. Materials and Experimental Methods:

The following Fmoc-N-protected amino acids are used: tyrosine (Fmoc-L- Tyr(tBu)-OH), N-methylvaline (Fmoc-L-NMe VaI-OH), aminoisobutyric acid (Fmoc- Aib-OH), phenylalanine (Fmoc-L-Phe-OH), leucine (Fmoc-L-Leu-OH) and arginine (Fmoc-L-Arg(Pbf)-OH).

Peptide Syntheses:

Solid phase peptide syntheses are carried out in polypropylene syringes (10 ml) fitted with a polyethylene porous disc, using general Fmoc chemistry, as follows: All procedures are performed at 25 ⁰C. Each cycle included Fmoc deprotection, washing, coupling an amino acid to the resin-bound peptidyl in the presence of the tested coupling agent and washing. Fmoc deprotection is carried out using 20 % piperidine in DMF (1 x 1 minute, 2 x 10 minutes).

Washings between repetitive cycles of deprotection and coupling are carried out using DMF (5 x 1 minutes, 10 ml solvent per 1 gram of resin). 5 molar equivalents of a Fmoc-protected amino acid, 5 molar equivalents of the tested coupling agent and 15 molar equivalents of N,N-diisopropylethylamine (DIEA) are reacted with 1 molar equivalent of a resin in each cycle.

DMF (0.2 ml) is added to a mixture of a first Fmoc-protected amino acid, a coupling agent and DIEA and after 5 minutes the resulting mixture is added to the Rink resin (50 mg, 0.66 mmol/gram). Upon washing, Fmoc deprotection and washing, a mixture containing a second Fmoc protected amino acid and a coupling agent is reacted with the resin, as described above. This procedure is repeated until the peptide synthesis is completed.

Upon completion, the peptide is cleaved from the resin by treatment with neat TFA for 1 hour, to remove residual Fmoc and other protection groups. Methyl-t- butylether is then added to the resulting peptide and the resulting solid precipitate is isolated by means of centrifugation. Precipitation is performed trice and the isolated peptide is then subjected to lyophilization.

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. 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.

Claims

WHAT IS CLAIMED IS:

1. A coupling agent having a general formula I:

Formula I

wherein:

D is a cationic moiety;

A is an inorganic anion;

Z₄ and Z₅ are each independently selected from the group consisting of F, Cl, Br, CORa, COORa, CONRa, CN, CF₃ or NO₂; and

Ra is alkyl.

2. The coupling agent of claim 1, wherein said cationic moiety has a general formula selected from the group consisting of :

Formula III Formula IV Formula V

wherein: n is an integer from 1 to 4; and each of Ri, R₂, R₃, R₄, R₅ and R₅ is independently selected from the group consisting of an alkyl having 1-4 carbon atoms and an alkyl having 1-4 carbon atoms interrupted, substituted and/or terminated by at least one heteroatom, or, alternatively, Ri and R₂, and/or R3 and R₄, and/or R₅ and R₆ are each independently joined to form a heteroalicyclic moiety; provided that when said cationic moiety has said general formula III, Z₄ is CN, Z₅ is COORa and Ra is ethyl, at least one of Ri, R₂, R3 or R₄ is an alkyl having 2-4 carbon atoms, or, alternatively, at least one of Ri and R₂, and/or R₃ and R₄ is joined to form a heteroalicyclic moiety.

3. A coupling agent having a general formula II:

Formula III

wherein:

D is a cationic moiety;

L is a leaving group; and

A is an inorganic anion; whereas: said leaving group is selected from the group consisting of a pyridin-2(lH)-one- 1-oxy, a quinazolin-4(3H)-one-3-oxy, a lH-benzo[d]imidazol-l-oxy and an indolinone- 1-oxy.

4. The coupling agent of claim 3, wherein said cationic moiety has a general formula selected from the group consisting of:

Formula III Formula IV Formula V wherein: n is an integer from 1 to 4; and each of Rj, R₂, R₃, R₄, R₅ and R₆ is independently selected from the group consisting of an alkyl having 1-4 carbon atoms and an alkyl having 1-4 carbon atoms interrupted, substituted and/or terminated by at least one heteroatom, or, alternatively, Ri and R₂, and/or R₃ and R₄, and/or R₅ and R₆ are each independently joined to form a heteroalicyclic moiety.

5. The coupling agent of any of claims 2 or 4, wherein said heteroatom is selected from the group consisting of O, S, N, P and B.

6. The coupling agent of any of claims 2 or 4, wherein said heteroalicyclic moiety is selected from the group consisting of a pyrrolidine, piperidine, morpholine, a thiomorpholine , a imidazolidine , an azaphosphinane, an azaphospholidine, an azaborinane, an azaborolidine, an azaphosphinane, an azaphospholidine and a piperazine.

7. The coupling agent of any of claims 1 or 3, wherein said inorganic anion is selected from the group consisting of halide, hexahalophosphate, hexahaloantimonate, tetrahaloborate, trihalomethanesulfonate and bis(trihalomethylsulfonyl)imide.

8. The coupling agent of claim 7, wherein said inorganic anion is hexafluorophosphate.

9. The coupling agent of any of claims 3-8, wherein said pyridin-2(lH)- one-1-oxy has the general Formula VII:

Formula VII

10. The coupling agent of any of claims 3-8, wherein said quinazolin-4(3H)~ one-3-oxy has the general Formula VIII:

Formula VIII

wherein:

Ze and Z₇ are each independently selected from the group consisting of H, F, Cl, Br, CN, CF₃, NO₂, aryl or alkyl.

11. The coupling agent of any of claims 3-8, wherein said IH- benzo[d]imidazol-l-oxy has the general Formula IX:

Formula IX wherein:

Zi is CH or N;

Zg and Z₉ are each independently selected from the group consisting of H, F, Cl₅ Br, CN, CF₃, NO₂, aryl or alkyl.

12. The coupling agent of any of claims 3-8, wherein said indolinone-1-oxy has the general Formula X:

Formula X wherein:

Zio is selected from the group consisting of H, F, Cl, Br, CN, CF₃, NO₂, aryl or alkyl.

13. The coupling agent of claim 2, wherein: Z₄ is COORa;

Ra is ethyl; and Z₅ is CN.

14. The coupling agent of claim 2, wherein:

Z₄ and Z₅ are each independently COORa or CN; and Ra is methyl.

15. The coupling agent of claim 10, wherein: Z₆ is CH3; and

Z₇ is Cl.

16. The coupling agent of claim 11, wherein

Zi is CH;

Zs is phenyl; and

Z₉ is H or CL

17. The coupling agent of claim 11, wherein Zi is N;

Zg is methyl; and Z₉ is H.

18. A coupling agent selected from the group consisting of: l,l,3,3-tetramethyl-2-(2-oxopyridin-l(2H)-yl)isouronium hexafluorophosphate

(HTOP) having the formula:

O-[(dicyanomethylidene)amino]-l,l,3,3-tetramethyluronium hexafluorophosphate (HTODC) having the formula:

O-[(diethoxycarbonylmethylidene)amino]-l,l,3,3-tetramethyluronium hexafluorophosphate (HTODeC) having the formula:

19. A method of synthesizing a polynucleotide, the method comprising: coupling a plurality of nucleotides sequentially, one with another, in the presence of the coupling agent of any of claims 1-17, to thereby obtain a polynucleotide containing said plurality of nucleotides.

20. A method of synthesizing a peptide, the method comprising: coupling a plurality of amino acids sequentially, one with another, in the presence of the coupling agent of any of claims 1-17, to thereby obtain a peptide containing said plurality of amino acids.

21. The method of any of claims 19 or 20, being effected by a solid phase synthesis.

22. The method of any of claims 19 or 20, being effected by a solution phase synthesis.

23. A crude composition of peptides, said peptides being synthesized in a C- terminus to N-terminus direction from a plurality of amino acids, the composition consisting essentially of a peptide having a desired amino acid sequence and a plurality of peptides having undesired amino acid sequences and being impurities to said peptide having said desired amino acid sequence, wherein a concentration of said peptide having said desired amino acid sequence in said composition is at least 5 % above a concentration of an identical peptide having said desired amino acid sequence, in a composition of peptides being synthesized in said C-terminus to N-terminus direction using the coupling agent of claim 1 as a coupling agent, otherwise prepared under identical conditions.

24. The crude composition of peptides of claim 23, being in a form selected from the group consisting of a powdered composition, a lyophilized composition, a composition bound to a solid support, a solubilized composition and a dissolved composition.

25. The method or composition of any of claims 20-23, wherein at least one of said amino acids is selected from the group consisting of an amino acid having a secondary alpha amine, an amino acid having a tertiary alpha amine, an amino acid having a substituted alpha carbon atom, an amino acid having a substituted alpha amine, an amino acid having an ammo-containing side chain, and any combination thereof.