WO2004037772A1

WO2004037772A1 - Convenient and scalable synthesis of ethyl n-[(2-boc-amino) ethyl] glycinate and its hydrochloride salt

Info

Publication number: WO2004037772A1
Application number: PCT/CA2003/001586
Authority: WO
Inventors: Robert H.E. Hudson; Russell D. Viirre
Original assignee: The University Of Western Ontario
Priority date: 2002-10-22
Filing date: 2003-10-22
Publication date: 2004-05-06
Also published as: AU2003278025A1

Abstract

The present invention discloses an improved synthesis of ethyl N-[(2-Boc-amino)ethyl]glycinate and its hydrochloride salt. The synthesis is based on the reductive alkylation of Boc-ethylenediamine with ethyl glyoxylate hydrate and furnishes the title compound in near quantitative yield and high purity without chromatography. This compound is suitable, as is, for the synthesis peptide nucleic acid monomers. Further, conversion to the hydrochloride salt provides a stable, non-hygroscopic solid that is a convenient form for handling and storage.

Description

CONVENIENT AND SCALABLE SYNTHESIS OF ETHYL N-[(2-BOC- AMINO)ETHYL]GLYCINATE AND ITS HYDROCHLORIDE SALT

FIELD OF THE INVENTION

The present invention relates to an improved synthesis of ethyl N-[(2-Boc- amino)ethyl]glycinate and its hydrochloride salt and its use for the synthesis of peptide nucleic acid monomers. The improved process for ethyl N-[(2-Boc- amino)ethyl]glycinate would be useful in other compounds derived from reaction of the alpha-amino group with a variety of possible electrophiles.

BACKGROUND OF THE INVENTION

At present, a ubiquitous requirement in the field of peptide nucleic acid (PNA) researchl is the preparation of monomers for subsequent oligomerization. Although the monomers suitable for both t-Boc- and Fmoc- strategies of solid- phase peptide synthesis are commercially available, they are costly and of limited variety.

Such "submonomer" approaches with which the presently reported invention would be compatible have reported. Examples of submonomer synthesis for which a differentially protected 2-aminoethy glycinate is pre-formed or a synthetic intermediate are exemplified by: (a) Sforza, Stefano; Tedeschi,

Tullia; Corradini, Roberto; Ciavardelli, Domenico; Dossena, Arnaldo; Marchelli, Rosangela. Fast, solid-phase synthesis of chiral peptide nucleic acids with a high optical purity by a submonomeric strategy. European Journal of Organic Chemistry 2003, 6, 1056-1063 and references therein, (b) Viirre, Russell D.; Hudson, Robert H. E. Optimization of a Solid-Phase Synthesis of a PNA Monomer; Organic Letters 2001, 3, 3931-3934. (c) Di Giorgio, Christophe; Pairot, Sandrine; Schwergold, Caroline; Patino, Nadia; Condom, Roger; Farese-Di Giorgio, Audrey; Guedj, Roger. Liquid-phase synthesis of polyamide nucleic acids (PNA); Tetrahedron 1999, 55, 1937-1958. (d) Farese, Audrey; Patino, Nadia; Condom, Roger; Dalleu, Sandrine; Guedj, Roger. Liquid phase synthesis of a peptidic nucleic acid dimer. Tetrahedron Letters 1996, 37, 1413-16. (e) Falkiewicz, B.; Kolodziejczyk, A. S.; Liberek, B.; Wisniewski, K. Synthesis of achiral and chiral peptide nucleic acid (PNA) monomers using Mitsunobu reaction; Tetrahedron 2001, 57, 7909-7917. (f) Davis, Peter W.; Swayze, Eric E. Automated solid-phase synthesis of linear nitrogen-linked compounds;

Biotechnology and Bioengineering 2000, 71, 19-27.

Therefore it would be very advantageous to provide a method to improve the processes leading to PNA monomer synthesis. As well, a differentially protected [2-(amino)ethyl]glycinate could have use in a submonomer synthetic approach to PNA.

SUMMARY OF THE INVENTION

The present invention provides an improved synthesis of ethyl N-[(2-Boc- amino)ethyl]glycinate and its hydrochloride salt. The synthesis is based on the reductive alkylation of Boc-ethylenediamine with ethyl glyoxylate hydrate and furnishes the title compound in near quantitative yield and high purity without chromatography. This compound is suitable, as is, for the synthesis peptide nucleic acid monomers. Further, conversion to the hydrochloride salt provides a stable, non-hygroscopic solid which is a convenient form for handling and storage. The present invention provides a method of synthesizing ethyl N-[(2-Boc- amino)ethyl]glycinate, comprising the steps of: adding a suitable dessicant and Boc-ethylenediamine to a solution comprising substantially pure ethyl glyoxylate hydrate dissolved in a suitable solvent at a suitable temperature and stirring the resulting solution for an effective period of time; filtering the solution and isolating a filtrate and isolating from the filtrate ethyl-(2-Boc-amino-ethylimino) acetate (5); and chemically reducing (5) to produce ethyl N-[(2-Boc- amino)ethyl]glycinate.

The present invention also provides a method of synthesizing ethyl N-[(2- Boc-amino)ethyl] glycinate, comprising the steps of: adding a suitable dessicant and Boc-ethylenediamine to a solution comprising substantially pure ethyl glyoxylate hydrate dissolved in a suitable solvent at a suitable temperature and stirring the resulting solution for an effective period of time; filtering the solution and isolating a filtrate and hydrogenating said filtrate in the presence of an effective catalyst; and isolating ethyl N-[(2-Boc-amino)ethyl]glycinate from the hydrogenated filtrate.

In another aspect of the invention there is provided a method of synthesizing ethyl N-[(2-Boc-amino)ethyl]glycinate, comprising the steps of: adding a molecular sieve material and Boc-ethylenediamine to a solution comprising substantially pure ethyl glyoxylate hydrate dissolved in a suitable solvent at a suitable temperature and stirring the resulting solution for an effective period of time; filtering the resulting solution and hydrogenating the filtered solution in the presence of an effective catalyst; and isolating ethyl N-[(2-Boc-amino)ethyl]glycinate from the hydrogenated filtered solution.

A hydrochloride salt of ethyl N-[(2-Boc-amino)ethyl]glycinate (1ΗC1), may be produced by the steps of: adding ethereal HO to a cooled solution containing dissolved ethyl N-[(2-

Boc-amino)ethyl] glycinate and after sufficient agitation filtering and isolating therefrom the hydrochloride salt of Ethyl N-[(2-Boc-amino)ethyl]glycinate (1-HCl).

The present invention provides a method of synthesizing peptide nucleic acid (PNA) monomers from hydrochloride salts of ethyl N-[(2-Boc- amino)ethyl]glycmate (1ΗCI) comprising the reaction steps of:

6a ^{2a: 48}%

The present invention provides a method of synthesizing peptide nucleic acid (PNA) monomers from hydrochloride salts of ethyl N-[(2-Boc- amino)ethyl]glycinate (1ΗC1) comprising the reaction steps of:

6b 2b: 67% wherein PMB is p rø-methoxybenzyl.

The present invention provides a method of synthesizing peptide nucleic acid (PNA) monomers from hydrochloride salts of ethyl N-[(2-Boc- amino)ethyl]glycinate (1-HCI) comprising the reaction steps of:

6c 2c: 61 %

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed a reliable, convenient, and scalable route to (ethyl N-[2-Boc-aminoethyl]glycinate, 1 which is disclosed herein (wherein Boc = t-butyloxycarbonyl). It is shown that compound 1 may be used for PNA monomer synthesis without explicit purification and is a key intermediate in the synthesis of all standard PNA monomers (2 and Scheme 1), compatible with Merrifield graded acidolysis oligopeptide synthesis. Scheme 1

BocHN

1 2

Definitions: DCC: N,N'-dicyclohexylcarbodiimide, HOBt: N- hydroxybenzotriazole, -OH: hydroxide source, H+: acid source.

The 'backbone' polymer of PNA is comprised of 2-aminoethylglycine repeat units. This structure is commonly prepared by the reaction of Boc- ethylenediamine (3) with a haloacetic acid derivative, most often ethyl bromoacetate although other haloacetates such as ethyl chloroacetate, methyl chloroacetate, methyl bromoacetate or similar compounds are suitable. Even when great care is taken, this method invariably produces a mixture of the desired product and varying amounts of the undesired dialkylated amine, often containing unreacted 3 as well. This procedure is therefore inefficient in the use of 3, and inconvenient to scale up since chromatography is generally used to purify the crude product.

Herein, the inventors report a new procedure that efficiently converts 3 to 1 without the need for explicit purification and is therefore easily scalable. The procedure, in its preferred embodiment, (Scheme 2) is based on the formation of the imine from 3 and ethyl glyoxylate hydrate, itself obtained by oxidative cleavage of diethyl tartrate, followed by reduction of the imine to afford the desired compound 1 without the possibility of overalkylation. Scheme 2

4

0 3A MS, CH₂CI₂ N_\

BocHN^^ ^NH2 + 4 *^■ BocHN ^^ ^^ OEt

3 5

Definitions: MS: molecular sieves

A key factor in the success of this scheme is the method by which ethyl glyoxylate is prepared. A variety of methods for the oxidative cleavage of diethyl tartrate were surveyed. In general, it was found that ethyl glyoxylate was not possible to prepare as the free aldehyde, as the major product. For instance, 1H nrnr (nuclear magnetic resonance) analysis of the crude aldehyde prepared by the reaction of periodic acid with diethyl tartrate in reagent grade diethyl ether⁶ showed almost no sign of the desired aldehyde and contained a considerable amount of the diethyl acetal of ethyl glyoxylate, presumably due to the presence of ethanol as stabilizer in the ether. The spectrum also showed signals ascribed to unidentifiable oligomeric or polymeric material from self condensation of the ethyl glyoxylate. When the cleavage was repeated in distilled ether, acetal formation was suppressed, but no increase in the amount of free aldehyde content was observed. Use of dichloromethane as a solvent slowed the reaction and did not improve the quality of the product. The crude aldehyde from any of the above procedures was not competent in imine formation. Given the high reactivity of the free aldehyde,⁷ and the observed low reactivity of the polymeric or ethyl acetal form of this aldehyde, it was decided to intentionally prepare the hydrated form, based on the hypothesis that the reactivity would be improved, relative to the polymeric form of the aldehyde. This was done by the reaction of diethyl tartrate with sodium periodate in 5 : 1 dichloromethane/water although many other organic solvents compatible with the oxidative conditions and containing water may be used, including for example ethers (nonlimiting examples include: alcohol-free diethyl ether, tetrahydrofuran, 1,4-dioxane), acetates (e.g. ethyl acetate), acetonitrile, chlorinated solvents (nonlimiting examples: chloroform, 1,2- dichloroethane), aliphatic solvents (nonlimiting examples: preferably low boiling point aliphatics such as pentane, hexanes, cyclohexane, decalin) and aromatic solvents (nonlimiting examples include: benzene, toluene, xylenes) and alcohols with a low tendency to form acetals with ethyl glyoxylate (t-butanol, phenol, sec- butanol, isopropanol). The use of aqueous sodium periodate supported on silica gel⁹ was also attempted, however this reaction was slower and more cumbersome, especially on scale-up, since the desired aldehyde hydrate was slightly adsorbed to the silica gel, requiring extensive rinsing with dichloromethane or reduced product recovery. Rinsing with reagent grade ether resulted in acetal formation, while the use of more polar solvents such as ethyl acetate or acetonitrile resulted in the leaching of iodine species into solution.¹⁰ Other oxidants could substitute for sodium periodate, such as other periodate-containing compounds (periodic acid, potassium periodate) or non-periodate-based oxidants such lead tetraacetate in combination with any of the solvents listed, vide supra.

Once pure ethyl glyoxylate hydrate (4) was in hand, the remainder of the synthesis was easily carried out. Treatment of mono-Boc-ethylenediamine (3) with 1.1 mole equivalents of 4 in dichloromethane preferably in the presence of a suitable dessicant to remove water, preferably over 3 A MS (molecular sieves serving as the dessicant) at 0°C quantitatively afforded the corresponding imine (5) within an hour. Preferably, the pulverized molecular sieve material is activated by removal of absorbed water at an effective temperature for an effective period of time.

While pure ethyl glyoxylate hydrate (4) is preferred, the inventors contemplate that substantially pure ethyl glyoxylate hydrate may also be used instead to react with mono-Boc-ethylenediamine (3) because it is contemplated that it is possible to successfully apply anhydrous ethyl glyoxylate to this scheme, either by cracking the commercially available polymeric ethyl glyoxylate technical grade material, or by cracking the crude product from any of the other methods of preparing the aldehyde, and using it immediately (or storing very cold until use). Other methods of producing the requisite aldedhyde will be known to those skilled in the art, such as alternative oxidation methods and substrates, for example: ozonolysis of diethyl maleate or diethyl fumarate, sequential dihydroxylation and oxidative cleavage of diethyl maleate or diethyl fumarate, or direct oxidation of diethyl maleate or diethyl fumarate with other agents such as lead tetraacetate. Besides the use of ethyl glyoxylate or ethyl, glyoxylate hydrate obtained as described above, other alkyl glyoxylates not explicitly named may be substituted in the scheme of the present invention. In particular, based on chemical precedent, more sterically demanding alkyl glyoxylates have a lower tendency to form polymers, thus it is expected that the glyoxylate hydrate does not need to be explicitly formed in order to observe high reactivity in some instances. Filtration of this solution, followed by addition of 0.05 equivalents of Pd (10% on activated carbon) and hydrogenation (with molecular hydrogen) afford the PNA backbone monomer (1). Of note, the imine solution (compound 5) may be stored at -20°C or evaporated and stored at -20°C (for example overnight) with little or no effect on the purity of the final product.

When the hydrogenation was carried out in the presence of molecular sieves in an attempt to avoid the filtration step, the quality of the crude product was substantially decreased containing a number of unidentified side products. To demonstrate the scalability of the procedure, 1 has been prepared on 2 g, 10 g and 38 g scales. On each scale, the desired product was isolated pure and in essentially quantitative yield. For scale-up, the amounts of reagents and solvents were scaled equally, and reaction times remained the same. The only difference between the scales was the method of hydrogenation. On the 2 g scale, hydrogenation was complete within 4 hrs. of magnetic stirring under a balloon of H₂. However, on the 10 g scale, these conditions afforded only 77% reduction based on H nrnr analysis of the crude product. By increasing the H₂ pressure to 50 psi and providing vigourous shaking on a Parr hydrogention apparatus, reduction on the 10 g scale was complete within 4 hr. On the 38 g scale, either a suitable hydrogenation apparatus (Parr hydrogenator) or reduction by running a stream stream of H₂ through the solution with a small degree of pressure provided by a mercury bubbler may be carried out. In the latter case, efficient agitation was achieved by the use of vigorous stirring in a Morton-type flask. Under these conditions, reduction was complete within 4 hrs. In general, the major factor in the rate of the hydrogenation appeared to be the efficiency with which H₂ was transferred to the solution, and so effective agitation is important. For this reason, it is recommended (especially on larger scales) that completion of reduction be verified by nmr analysis of a small, filtered, evaporated aliquot of the reaction mixture prior to work-up.

When the procedure is carried out as described, the PNA backbone monomer is obtained in highly pure form. However, the crude product can be further purified by dropwise addition of ethereal HC1 to an ice-cooled ether solution of crude 1. The backbone hydrochloride is a stable white solid, which can be recrystallized from acetone, if necessary. As a solid, 1ΗC1 is more conveniently stored and dispensed than neutral 1, which is usually a viscous oil. We have prepared a few PNA monomers from 1-HCI (Scheme 3), and have observed no disadvantage over the use of the neutral backbone.

Commercially available anhydrous HC1 in 1,4-dioxane or HC1 in tetrahdyrofuran would be suitable source of acid. Other mineral acids such as but not limited to nitric acid, sulfuric acid, phosphoric acid, or tetrafluoroboric acid in solvents such as diethyl ether, tetrahydrofuran, or 1,4-dioxane would be suitable to yield ammonium salts of compound 1. The putative formula of such comounds would may repesectively be: IΗNO₃, IH₂SO₄ (hemisulfate or sulfate), IΗ3PO4, 1*HBF₄. Other acids such as sulfonate-based acids (p-toluene sulphonic acid or similar, triflic acid) and organic acids such the halo-acetic acid derivatives (e.g. trichloroacetic acid, trifluoroacetic acid) in a suitable solvent would be amenable to this process. Solvents for recrystallization oflvHCl, besides acetone, could be ethyl acetate, tetrahydrofuran, 1 ,4-dixoane,or isopropanol, for example. If the identity of the alcohol-derived portion of the glyoxylate changed, then the solvents for precipitation and recrystallization may change according to the solubility properties of the compound. Schemes 3 and 4 illustrates three examples of the preparation of peptide nucleic acid (PNA) monomers using the hydrochloride salt of compound 1 and various nucleobase derivatives. Thus compounds 2a, 2b and 2c are derived from the reactions of 6a, 6b, and 6c, respectively, under the conditions given, and are obtained in the chemical yields as indicated. For example, the conversion of 6a to

2a is done in four successive steps, that follow reported methods and are standard reactions know to those familiar with PNA chemistry. Firstly, the carboxylic acid of 6a is converted to derivative that is reactive towards forming the desired amide bond with 1. This is done, in this instance, by reaction in a separate vessel with N,N'-dicyclohexylcarbodiimide and N-hydroxybenzotriazole (HOBt). After an effective time, the ester between 6a and HOBt will form. Subsequently, 1ΗC1 and an excess of base (triethylamine or similar organic base) to generate 1 in situ, is added to the solution of benzotriazole ester of 6a. After an effective time, the reaction between 1 and the benzotriazole ester of 6a will have taken place, at which time the solvent is removed from the crude product mixture by evaporation in vacuo. The crude reaction mixture is dissolved in a suitable solvent (such as dichloromethane or ethyl acetate) for liquid-liquid extraction (against aqueous solutions), next the organic phase is separated, dried and evaporated to yield a unpurified reaction mixture. The pure ethyl ester of 2a may be obtained by precipitation in diethyl ether or alternatively by other standard methods. The ethyl ester of 2a is then subjected to hydrolytic conditions (aqueous LiOH) to final give pure 2a, after acidification. This same sequence of steps were used for 2b and 2c. Of importance, other conditions may be equally effective in forming the monomers from 1 or 1ΗC1 and suitable carboxylic acids. There are many reagents known to promote amide bond formation either via acyl halides, acyl phosphonium, acyl uronium, active esters or similar intermediates. The combination of DCC/HOBt to achieve amide bond formation is illustrative of a nonlimiting example. Scheme 3

The following generalized scheme shows a method of synthesizing peptide nucleic acid (PNA) monomers from hydrochloride salts of ethyl N-[(2-Boc- amino)ethyl]glycinate (1-HCI):

6a: ' = Me, R" = H 2a: 48%

6b 2b: 67%

Η 1) f fnorrmmaαttiinonn n off a arcttiiwvoe o ecsttoerr 2) 1ΗCI, neutralization and coupling 3) hydrolysis 4) protonation

_6c 2c: 61 %

Definitions: DCC: N,N'-dicyclohexylcarbodiimide, HOBt: N- hydroxybenzotriazole, NEt₃: triethylamine, DIEPA: diisopropylethylamine, DMAP: 4-dimethyaminopyridine, LiOH: aqueous solution of lithium hydroxide, HC1 aqueous solution hydrochloric acid, PMB: ^ ra-methoxybenzyl, DMF: dimethylformamide

For example, in step 1 of the above synthesis active ester formation is the formation of a compound that is more reactive towards nucleophilic acyl substitution than the starting carboxylic acid. As indicated in scheme 4, step 1 in the scheme may be effected by the use of carbodiimide reagents such as DCC: (N,N'-dicyclohexylcarbodiimide) in the presence of HOBt (N- hydroxybenzotriazole) in a suitable solvent such as DMF (dimethylformamide). Step 2, in the above scheme, involves the in-stiu neutralization of the l'HCl by use of a suitable base such as NEt₃ (triethylamine) or DIEPA (diisopropylethylamine) preferably in the presence of DMAP (4- dimethyaminopyridine). Thus, although 1ΗC1 is used for the coupling, it must be neutralized to give 1 for the reaction to proceed. Together, steps 1 and 2 effect the condensation of the acid and amine to produce the desired amide bond. With DCC and other carbodiimide-based reagents steps one and two may be combined into a single procedure.

Step 3 in the above scheme may be effected by an aqueous solution of lithium hydroxide to effect the hydrolysis of the ester, yielding the carboxylate which typically remains in solution.

Step 4 in the above scheme is protonation of the carboxylate to facilitate isolation of 2a. Typically, but not exclusively, an aqueous solution hydrochloric acid is used.

The reactions shown in scheme 3 above using specific preferred reagents in each of the steps 1 to 4 are given in scheme 4. Scheme 4

6a: R' = Me, R" = H 2a: 48%

6b 2b: 67%

6c 2c: 61 %

Definitions: DCC: N,N'-dicyclohexylcarbodiimide, HOBt: N- hydroxybenzotriazole, NEt₃: triethylamine, DIEPA: diisopropylethylamine, DMAP: 4-dimethyaminopyridine, LiOH: aqueous solution of lithium hydroxide, HCI aqueous solution hydrochloric acid, PMB: pαr -methoxybenzyl, DMF: dimethylformamide.

With reference to step 1 in the above schemes, alternative reagents to DCC HOBt for effecting the transformation can be used. A wide variety of known 'condensation reagents' represented by, but not limited to, phosphonium- based reagents and uranium-based reagents would be suitable. The use of the term 'active ester' is used to describe generalized reactive acyl compounds such as O- acylureas, acyl phosphonium, acyl halides and esters.

With reference to step 2 in the above scheme, a non-nucleophilic or low nucleophilicity base, typically a trialkylamine (as shown), but not limited to them should be included in the reaction medium. Conversely, 1 could be used in place of 1-HCI, and this would negate the need for a base. This indicates that 1 is the competent species for participation in the condensation reaction. Preferably this reaction occurs in the presence of an acyl-transfer catalyst such as DMAP or N- methylimidazole, whether or not a base is employed. With reference to step 3 in the above schemes, the ester hydrolysis does not rely on the identity of the metal hydroxide (lithium hydroxide is shown), but could be selected from organic bases, metal carbonates or metal hydroxides.

In step 4 of the schemes above the use of HCI, ether and acetone are preferred reagents but the alternatives to these reagents will be known to those skilled in the art. With reference to step 4 in the above scheme, the acid need not be HCI solely, but any proton source that can delivered in a controlled fashion, for instance it could be other mineral acids, organic acids or polymer-supported acids. For example, commercially available anhydrous HCL in 1,4-dioxane or HCI in tetrahdyrofuran would be suitable source of acid. Other mineral acids such as but not limited to nitric acid, sulfuric acid, phosphoric acid, or tetrafluoroboric acid in solvents such as diethyl ether, tetrahydrofuran, or 1,4-dioxane would be suitable to yield ammonium salts of compound 1. Other acids such as sulfonate-based acids (p-toluene sulphonic acid or similar, triflic acid) and organic acids such the halo- acetic acid derivatives (e.g. trichloroacetic acid, trifluoroacetic acid) in a suitable solvent would be amenable to this process. Solvents for recrystallization of l'HCl, besides acetone, could be ethyl acetate, tetrahydrofuran, l,4-dixoane,or isopropanol, for example. If the identity of the alcohol-derived portion of the glyoxylate changed, then the solvents for precipitation and recrystallization may change according to the solubility properties of the compound. In other instances, it is anticipated that the protonation step can be omitted and the carboxylate salt is isolated. Experimental Details

Ethyl glyoxylate hydrate⁸, Boc-ethylenediamine⁴, thymin-1-ylacetic acid³, N3-PMB-thymin-l-ylacetic acid¹¹ and 5-iodouracil-l-ylacetic acid¹² were prepared according to literature methods. Ethereal HCI was prepared by dropwise addition of a large excess of concentrated HCI to an equal volume of concentrated H₂SO₄, and bubbling the gas thus formed through stirred, ice-bath cooled diethyl ether. This reagent was stored at -20°C and titrated prior to use. Molecular sieves were pulverized and activated at 300°C under vacuum for 3 days prior to use. All other reagents and solvents were used as supplied, without further purification. NMR spectra were recorded at 600 MHz or 400 MHz on a Narian Inova 600 or Narian Mercury 400 spectrometer. Ethyl Ν-(2-Boc-aminoethyl) glycinate (1)

To an ice-bath cooled solution of ethyl glyoxylate hydrate (4, 5.37 g, 44.7 mmol) in CH₂C1₂ (-90 mL) was added 3A MS (~5 g), followed by dropwise addition of Boc-ethylenediamine (3, 6.50 g, 40.6 mmol) in CH₂C1₂ (~10 mL) over -10 minutes. The mixture was stirred at 0°C for 1 hr. and then filtered through a short pad of Celite. Evaporation of a few drops of this solution, followed by 1H nmr analysis (600 MHz, CDC1₃) revealed complete conversion of 3 to imine 5: δ

7.60 (s, 1H), 4.93 (bs, 1H), 4.22 (q, J=7.2 Hz, 2H), 3.64 (t, J=5.3 Hz, 2H), 3.36 (app q, J=5.3 Hz, 2H), 1.31 (s, 9H), 1.24 (t, J=7.2 Hz, 3H). Palladium (10% on activated carbon, 2.16 g, 2.03 mmol) was added to the filtered solution and the mixture was hydrogenated at 50 psi on a Parr hydrogenation apparatus. After 4 hr, the mixture was filtered through a hard-packed pad of Celite and rinsed with MeOH under a stream of N₂ (Caution! Pd/C is pyrophoric in open air!). The solution was evaporated in vacuo to afford 9.8 g (98%) of the desired product (1), a yellow oil which partially crystallized on standing. The spectral data were in agreement with those previously reported.⁵

While. CH₂C1₂ is a preferred solvent, other suitable solvents for the formation of compound 5 (ethyl-(2-Boc-amino-ethylimino) acetate) would be dichloromethane or other halogenated solvents such as chloroform, 1,2- dichloroethane; acyclic or cyclic aliphatic solvents such as petroleum ether, pentane, hexane, cyclohexane; ethers such as diethyl ether, tetrahydrofuran, dioxane; aromatic solvents such as benzene, toluene, xylenes; and anhydrous alcohols with a low tendancy to form acetals with compound 4 such as t-butanol, isopropanol.

While the preferred method of chemically reducing (5) to produce ethyl N- [(2-Boc-amino)ethyl]glycinate is by hydrogenation using hydrogen dissociated in the presence of a palladium (10% on activated carbon), it will be appreciated by those skilled in the art that other methods of chemically reducing compound (5) may be used. For example, the reducing agent may be either homogeneous or heterogeneous in nature. A hetereogeneous catalyst (in the present example, palladium supported on carbon) in the presence of hydrogen or a substance that produces hydrogen upon decomposition is preferred due to the ease of removal and the possibility of catalyst recycling. Homogeneous chemical reductants such as sodium borohydride or it's derivatives, especially sodium cyanoborohydride, are well known reductants for imines and may also be used. Homogenous transition metal catalysts may also be employed in the presence of hydrogen or a substance that produces hydrogen upon decomposition. These transformations and the necessary reagents and conditions are described by Richard Larock in

"Comprehensive Organic Transformations" 2^nd. Ed., Wiley-NCH, 1999, New York, NY, USA. 1-HCI

To an ice-bath cooled solution of 1 (8.71 g, 35.4 mmol) in Et₂O (150 mL) was added ethereal HCI (1.08 M, 35 mL, 37.8 mmol) dropwise over ~5 minutes.

The mixture was stirred at 0°C for 1 hour, then filtered, rinsed with Et₂O, and dried in vacuo, to afford 8.2 g (82%) of 1ΗC1, an air-stable, non-hygroscopic white solid. M.p. 121-124°C (dec), 1H nmr: (400 MHz, D₂O) δ 4.13 (q, J=7.3 Hz,

2H), 3.85 (s, 2H), 3.27 (t, J=5.3 Hz, 2H), 3.07 (t, J=5.4 Hz, 2H), 1.27 (s, 9H), 1.12 (t, J=7.3 Hz, 3H). ¹³C nmr (100 MHz, D₂O): δ 167.09, 158.30, 81.83, 63.75,

47.70, 36.87, 27.94, 13.58. HRMS exact mass 247.1651, calc'd for C_πH₂₃N₂O₄ ⁺: 247.1652. Anal, calc'd for C_πH₂₃ClN₂O₄: C, 46.72; H, 8.20; Cl, 12.54; N, 9.91; O, 22.63. Found: C, 46.44; H, 8.45; Cl, 12.70; N, 9.70; O, 22.80. General method for the preparation of PNA monomers (2) using THCl To an ice-cooled solution of a nucleobase acetic acid derivative (6, 1 eq.) and 1-hydroxybenzotriazole (1.1 eq.) in a minimum amount of dry DMF was added N,N'-dicyclohexylcarbodiimide (1.1 eq.). The mixture was removed from the ice-bath and stirred for 2 hrs. The mixture was then cooled, and to it was added a solution containing 1-HCI (1.1 eq.), TEA or DIPEA (3.3 eq.), and 4- (N,N-dimethylamino)pyridine (0.1 eq.) in a minimum amount of DMF (1 g of 1ΗC1 is clearly soluble in 6 mL DMF). The mixture was again removed from the ice-bath and stirred overnight. The mixture was then worked up as previously described³ to afford the desired PNA monomers in the yields reported in Scheme 3. The spectral data for 2a³, and 2c¹² were in agreement with those previously reported. 2b: M.p. 182°C (decomposition), 1H nmr: (400 MHz, DMSO, 20°C, 2 rotamers) δ 7.41 (s, 0.65H, thymine C6 ma), 7.36 (s, 0.35H,

thymine C6 mi), 7.22 (d, J=8.7 Hz, 2H, PMB CH), 6.94 (t, J=5.8 Hz, 0.65H, NH ma), 6.83 (d, J=8.7 Hz, 2H, PMB CH), 6.75 (t, J=5.5 Hz, 0.35H, NH mi), 4.91 (s, 2H, PMB CH₂), 4.72 (s, 1.3H, thymine N1-CH₂ ma), 4.54 (s, 0.7H, thymine Nl-

CH₂ mi), 4.17 (s, 0.7H, α-CH₂ mi), 3.97 (s, 1.3H, α-CH₂ ma), 3.70 (s, 3H, PMB-

OCH₃), 3.38 (t, J=6.5 Hz, 1.3H, γ-CH₂ ma), 3.30 (t, J=6.9 Hz, 0.7H, γ-CH₂ mi),

3.18-3.14 (m, 1.3H, δ-CH₂ ma), 3.03-2.99 (m, 0.7H, δ-CH₂ mi), 1.80 (s, 3H,

thymine CH₃), 1.36 (s, 9H, t-Bu). ¹³C nmr: (100 MHz, DMSO, 20°C, 2 rotamers) δ 170.84 (COOH mi), 170.50 (COOH ma), 167.43 (thymine-Nl-CH₂-C=O mi),

167.00 (thymine-Nl-CH₂-C=O ma), 163.127 (thymine C4), 158.48 (PMB C4), 155.79 (Boc C=O ma), 155.61 (Boc C=O mi), 151.12 (thymine C2), 140.90 (thymine C6), 129.41 (PMB C2 and C6), 129.19 (PMB Cl), 113.66 (PMB C3 and C5), 107.47 (thymine C5), 78.06 (C-Me₃ ma), 77.77 (C-Me₃ mi), 55.05 (OCH₃), 49.20 ( -CH₂ mi), 48.99 (thymine N1-CH₂ mi), 48.85 (thymine N1-CH₂ ma),

47.51 ( -CH₂ ma), 46.91 (γ-CH₂ mi), 46.72 (γ-CH₂ ma), 43.11 (PMB CH₂), 38.04

(δ-CH₂ ma), 37.58 (δ-CH₂ mi), 28.22 (C-Me≥ mi), 28.15 (C-Mes ma), 12.59

(thymine CH₃). HRMS exact mass 504.2222, calc'd for C₂₄H₃₂N₄O₈: 504.2220. In summary, there is disclosed herein a new synthesis of the peptide nucleic acid monomer precursor, which is preferable to reported methods due to its efficiency, reduced labour and especially the elimination of the need for purification by chromatography. Also disclosed is the preparation, purification and use of the hydrochloride salt of ethyl N-[(2-Boc-amino)ethyl] glycinate, a more convenient form of this important compound. It will be appreciated by those skilled in the art that although the compound described, ethyl N-[2-Boc-amino)ethyl]glycinate is a key intermediate for the preparation of PNA monomers compatible with graded acidolysis peptide synthesis, other glycinate derivatives would be suitable. Following a substantially similar process as outlined for ethyl N-[2-Boc-amiho)ethyl]glycinate either the Boc-ethylene diamine or diethyl tartrate or both could be substituted by a plurality of similar substance to yield useful glycinate derivatives. For example, dimethyl tartrate or any dialkyl-tartrate could serve a similar function as diethyl tartrate. The key intermediate that would lead to monomers compatible with Fmoc-based (Fmoc = 9-fluorenylmethyloxycarbonyl) chemistry could be prepared by the condensation of Fmoc-ethylenediamine with t-butylglyoxylate (derived from the oxidative cleave of di-t-butyltartrate), for example. With slight modification, the preparation of PNA monomers compatible with trityl/acyl protecting group strategy could be done with trityl-ethylene diamine and an alkyl lglyoxylate (derived from the oxidative cleavage of dialkyltartrates), where trityl refers to any of the triphenylmethyl- class of protecting groups including monomethoxytrityl

(MMTr).

As used herein, the terms "comprises", "comprising", "includes" and "including" are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in this specification including claims, the terms "comprises", "comprising", "includes" and "including" and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. The foregoing description of the preferred embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated. It is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims and their equivalents.

REFERENCES

(1) For more about the application, properties, and standard syntheses of PNA, see the following review and references therein: Nielsen, P.E. Ace. Chem. Res. 1999, 32, 624-630.

(2) tBoc- and Fmoc-PNA monomers are available from Applied Biosysiems, Foster City, CA, USA, 94404.

(3) Dueholm, K.L.; Egholm, M.; Behrens, C; Christensen, L.; Hansen, H.F.;

Vulpius, T.; Petersen, K.H.; Berg, R.H.; Nielsen, P.E.; Buchardt, O. /. Org. Chem. 1994, 59, 5767-5773.

(4) Kofoed, T; Hansen, H.F.; Orum, H; Koch, T. J. Peptide Sci. 2001, 7, 402- 412.

(5) Meltzer, P.C.; Liang, A.Y.; Matsudaira, P. J. Org. Chem. 1995, 60, 4305-

4308.

(6) Kelly, R.K.; Schmidt, T.E.; Haggerty, J.G. Synthesis 1972, 544-545. (7) Wennerberg, J.; Polla, M.; Frejd, T. J. Org. Chem. 1997, 62, 8735-8740.

(8) Bailey, P.D.; Smith, P.D.; Pederson, F; Clegg, W.; Rosair, G.M.; Teat, S.J. Tetrahedron Lett. 2002, 43, 1067-1070.

(9) Zhong, Y.-L.; Shing, T.K.M. J. Org. Chem. 1997, 62, 2622-2624.

(10) Technical grade ethyl glyoxylate, -50% in toluene is commercially available from Fluka cat. # 50705 and Lancaster Synthesis cat # 19207, but this material "exists partly in the polymerized form" (2001/2002 Fluka laboratory chemicals and analytical reagents catalog). We have not yet attempted to synthesize 1 using this product.

(11) Nϋrre, R.D.; Hudson, R.H.E. Org. Lett. 2001, 3, 3931-3934.

(12) Hudson, R.H.E.; Li, G.; Tse, J. Tetrahedron Lett. 2002, 43, 1381-1386.

Claims

THEREFORE IT IS CLAIMED:

1. A method of synthesizing ethyl N- [(2-B oc-amino)ethy 1] glycinate, comprising the steps of: adding a suitable dessicant and Boc-ethylenediamine to a solution comprising substantially pure ethyl glyoxylate hydrate dissolved in a suitable solvent at a suitable temperature and stirring the resulting solution for an effective period of time; filtering the resulting solution and isolating a filtrate and isolating from the filtrate ethyl-(2-Boc-amino-ethylimino) acetate (5); and chemically reducing (5) to produce ethyl N-[(2-Boc- amino)ethyl]glycinate.

2. The method according to claim 1 wherein said substantially pure ethyl glyoxylate hydrate is synthesized using a process comprising the steps of mixing diethyl tartrate with sodium periodate in an effective solvent comprising water and having suitable oxidative properties; and isolating therefrom substantially pure ethyl glyoxylate hydrate.

3. The method according to claims 1 or 2 wherein said suitable solvent is CH₂C1₂, and wherein said suitable temperature is about 0°C.

4. The method according to claims 1 or 2 wherein said suitable solvent is selected from the group consisting of CH₂C1₂, dichloromethane, halogenated solvents including chloroform, 1,2-dichloroethane; acyclic or cyclic aliphatic solvents including petroleum ether, pentane, hexane, cyclohexane; ethers including diethyl ether, tetrahydrofuran, dioxane; aromatic solvents including benzene, toluene, xylenes; and anhydrous alcohols with a low tendancy to form acetals with compound 4 such as t-butanol, isopropanol.

5. The method according to claims 1 , 2, 3 or 4 wherein the effective solvent comprising water is a 5:1 dichloromethane: water mixture.

6. The method according to claims 1, 2, 3, 4 or 5 wherein the step of chemically reducing (5) to produce ethyl N-[(2-Boc-amino)ethyl]glycinate includes hydrogenating said filtrate in the presence of an effective catalyst; isolating ethyl N-[(2-Boc-amino)ethyl] glycinate from the hydrogenated filtrate, and wherein said catalyst is palladium, and wherein said dessicant is produced by pulverizing molecular sieve material to produce a powder.

7. The method according to claims 1, 2, 3, 4 or 5 wherein the step of chemically reducing (5) to produce ethyl N-[(2-Boc-amino)ethyl] glycinate includes reducing (5) in the presence of a homogeneous chemical reductant, and isolating ethyl N-[(2-Boc-amino)ethyl]glycinate from the resulting filtrate, and wherein the dessicant is produced by pulverizing molecular sieve material to produce a powder.

8. The method according to claim 7 wherein homogeneous chemical reductant is selected from the group consisting of sodium borohydride, derivatives of sodium borohydride, homogenous transition metal catalysts in the presence of hydrogen or a substance that produces hydrogen upon decomposition.

9. The method according to claim 8 wherein homogeneous chemical reductant is sodium cyanoborohydride.

10. The method according to claim 6 wherein said pulverized molecular sieve material has a pore size of about 3 angstrom pore size, and wherein said pulverized molecular sieve material is activated by removal of absorbed water at an effective temperature for an effective period of time.

11. The method according to claims 6 or 10 wherein said step of isolating isolating ethyl N-[(2-Boc-amino)ethyl]glycinate from the hydrogenated filtrate includes evaporating liquid from said hydrogenated filtered solution.

12. The method of claims 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 including a step of synthesizing a hydrochloride salt of ethyl N-[(2-Boc-amino)ethyl]glycinate (1ΗC1), comprising the steps of: adding ethereal HCI to a cooled solution containing dissolved ethyl N-[(2- Boc-amino)ethyl]glycinate and after sufficient agitation filtering and isolating therefrom the hydrochloride salt of Ethyl N-[(2-Boc-amino)ethyl]glycinate (1 HC1).

13. The method of claims 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 including a step of synthesizing a salt of ethyl N-[(2-Boc-amino)ethyl]glycinate, comprising the steps of: adding an acid to a cooled solution containing dissolved ethyl N-[(2-Boc- amino)ethyl] glycinate and after sufficient agitation filtering and isolating therefrom the salt of Ethyl N-[(2-Boc-amino)ethyl]glycinate.

14. The method of claim 13 wherein the acid is selected from the group consisting of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and tetrafluoroboric acid in solvents selected from the group consisting of diethyl ether, tetrahydrofuran, or 1,4-dioxane whereby ammonium salts of compound 1 are produced.

15. The method of claim 13 wherein the acid is selected from the group consisting of sulfonate-based acids and organic acids.

16. The method of claim 15 wherein the sulfonate-based acid is selected from the group consisting of p-toluene sulphonic acid and triflic acid.

17. The method of claim 15 wherein the organic acid is selected from the group consisting of halo-acetic acid derivatives in a suitable solvent.

18. The method of claim 17 wherein the halo-acetic acid derivatives is selected from the group consisting of trichloroacetic acid and trifluoroacetic acid.

19. A method of synthesizing ethyl N-[(2-Boc-amino)ethyl]glycinate, comprising the reaction steps of:

3 5

20. A method of synthesizing ethyl N-[(2-Boc-amino)ethyl]glycinate, comprising the steps of: adding a sieve material and Boc-ethylenediamine to a solution comprising substantially pure ethyl glyoxylate hydrate dissolved in a suitable solvent at a suitable temperature and stirring the resulting solution for an effective period of time; filtering the resulting solution and hydrogenating the filtered solution in the presence of an effective catalyst; and isolating ethyl N-[(2-Boc-amino)ethyl]glycinate from the hydrogenated filtered solution.

21. The method according to claim 20 wherein said substantially pure ethyl glyoxylate hydrate is synthesized using a process comprising the steps of mixing diethyl tartrate with sodium periodate in an effective solvent comprising water and having suitable oxidative properties; and isolating therefrom substantially pure ethyl glyoxylate hydrate.

22. The method according to claim 20 wherein said suitable solvent is CH₂C1₂, and wherein said suitable temperature is about 0°C.

23. The method according to claim 21 wherein said effective solvent comprising water is a 5:1 dichloromethane: water mixture.

24. A method of synthesizing ethyl N-[(2-Boc-amino)ethyl]glycinate, comprising the steps of: adding a suitable dessicant and Boc-ethylenediamine to a solution comprising substantially pure ethyl glyoxylate hydrate dissolved in a suitable solvent at a suitable temperature and stirring the resulting solution for an effective period of time; filtering the solution and isolating a filtrate and hydrogenating said filtrate in the presence of an effective catalyst; and isolating ethyl N-[(2-Boc-amino)ethyl]glycinate from the hydrogenated filtrate.

25. The method according to claim 24 wherein said substantially pure ethyl glyoxylate hydrate is synthesized using a process comprising the steps of mixing diethyl tartrate with sodium periodate in an effective solvent comprising water and having suitable oxidative properties; and isolating therefrom substantially pure ethyl glyoxylate hydrate.

26. The method according to claims 20, 21, 22, 23, 24 or 25 wherein said suitable solvent is CH₂C1 , and wherein said suitable temperature is about 0°C.

27. The method according to claims 20, 21, 22, 23, 24 or 25 wherein said suitable solvent is selected from the group consisting of CH₂C1₂, dichloromethane, halogenated solvents including chloroform, 1,2-dichloroethane; acyclic or cyclic aliphatic solvents including petroleum ether, pentane, hexane, cyclohexane; ethers including diethyl ether, tetrahydrofuran, dioxane; aromatic solvents including benzene, toluene, xylenes; and anhydrous alcohols with a low tendancy to form acetals with compound 4 such as t-butanol, isopropanol.

28. The method according to claims 20, 21, 22, 23, 24 or 25 wherein said effective solvent comprising water is a 5:1 dichloromethane: water mixture.

29. The method according to claims 20, 21, 22, 23, 24, 25, 26, 27 or 28 wherein said catalyst is palladium, and wherein said dessicant is produced by pulverizing molecular sieve material to produce a powder.

30. The method according to claim 29 wherein said pulverized molecular sieve material has a pore size of about 3 angstrom pore size, and wherein said pulverized molecular sieve material is activated by removal of absorbed water at an effective temperature for an effective period of time.

31. The method according to claims 20, 21, 22, 23, 24, 25, 26, 27, 29 or 30 wherein said step of isolating isolating ethyl N-[(2-Boc-amino)ethyl]glycinate from the hydrogenated filtrate includes evaporating liquid from said hydrogenated filtered solution.

32. The method of claims 20, 21, 22, 23, 24, 25, 26, 27, 29, 30 or 31 including the step of synthesizing a hydrochloride salt of ethyl N-[(2-Boc- amino)ethyl]glycinate (1ΗC1), comprising the steps of: adding ethereal HCI to a cooled solution containing dissolved ethyl N-[(2- Boc-amino)ethyl]glycinate and after sufficient agitation filtering and isolating therefrom the hydrochloride salt of Ethyl N-[(2-Boc-amino)ethyl] glycinate (1 HC1).

33. The method of claims 20, 21, 22, 23, 24, 25, 26, 27, 29, 30 or 31 including a step of synthesizing a salt of ethyl N-[(2-Boc-amino)ethyl]glycinate, comprising the steps of: adding an acid to a cooled solution containing dissolved ethyl N-[(2-Boc- amino)ethyl]glycinate and after sufficient agitation filtering and isolating therefrom the salt of Ethyl N-[(2-Boc-amino)ethyl]glycinate.

34. The method of claim 33 wherein the acid is selected from the group consisting of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and tetrafluoroboric acid in solvents selected from the group consisting of diethyl ether, tetrahydrofuran, or 1,4-dioxane whereby ammonium salts of compound 1 are produced.

35. The method of claim 33 wherein the acid is selected from the group consisting of sulfonate-based acids and organic acids.

36. A method of synthesizing peptide nucleic acid (PNA) monomers from hydrochloride salts of ethyl N-[(2-Boc-amino)ethyl]glycinate (1ΗC1) synthesized using the method of claim 12, 13, 14, 15, 16, 17, or 18 comprising the reaction steps of:

6a 2a: 48%

37. A method of synthesizing peptide nucleic acid (PNA) monomers from hydrochloride salts of ethyl N-[(2-Boc-amino)ethyl]glycinate (1-HCl) synthesized using the method of claim 12, 13, 14, 15, 16, 17, or 18 comprising the reaction steps of:

6b 2b: 67% wherein PMB is αr -methoxybenzyl.

38. A method of synthesizing peptide nucleic acid (PNA) monomers from hydrochloride salts of ethyl N-[(2-Boc-amino)ethyl]glycinate (1-HCl) synthesized using the method of claim 12, 13, 14, 15, 16, 17, or 18 comprising the reaction steps of: ling

6c 2c: 61 %

39. The method according to claim 36, 37 or 38 wherein step 1 of formation of an active ester is effected using a reagent DCC (N,N'-dicyclohexylcarbodiimide) in the presence of HOBt (N-hydroxybenzotriazole).

40. The method according to claim 36, 37 or 38 wherein the step 2 of in-situ neutralization of the 1-HCl is effected by use of a suitable base such as NEt₃ (triethylamine) or DIEPA (diisopropylemylamine) .

41. The method according to claim 40 wherein the base is in the presence of DMAP (4-dimethyaminopyridine).

42. The method according to claim 36, 37 or 38 wherein the step 3 of ester hydrolysis is effected by an aqueous solution of lithium hydroxide to effect the hydrolysis of the ester, yielding a carboxylate which remains in solution.

43. The method according to claim 36, 37 or 38 wherein the step 4 of protonation is effected by an aqueous solution of an acid, hydrochloric acid.

44. The method according to claim 43 wherein the solution of an acid is an aqueous solution of an acid.

45. A method of synthesizing peptide nucleic acid (PNA) monomers from ethyl N-[(2-Boc-aπιino)ethyl]glycinate (1) synthesized using the method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 comprising the reaction steps of:

6a ^{2a: 48%}

46. A method of synthesizing peptide nucleic acid (PNA) monomers from ethyl N-[(2-Boc-arnino)ethyl]glycinate (1) synthesized using the method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 comprising the reaction steps of:

6b 2b: 67% wherein PMB is αra-methoxybenzyl.

47. A method of synthesizing peptide nucleic acid (PNA) monomers from ethyl N-[(2-Boc-amino)ethyl]glycinate (1) synthesized using the method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 comprising the reaction steps of:

1 ) formation of active ester 2) ^' coupling 3) hydrolysis 4) protonation

6c 2c: 61 %