BACKGROUND OF THE INVENTION At present, a ubiquitous requirement in the field of peptide nucleic acid (PNA) research 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) DiGiorgio, 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 (1'HCl), may be produced by the steps of: adding ethereal HCl 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-HCI).

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-HCl) comprising the reaction steps of : 0 0 ° 1) formation of active ester H3C A, H HAN NAH 2) 1-HCI, neutralization and coupling) IN t L 3) ester hydrolysis -N 4) protonation oxo N OH BocHN~ XOH 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 (1-HC1) comprising the reaction steps of : 0 ! 1) formation of active ester H3C C PMB CH3 PMB 2) 1-HCI, neutralization and coupling N 3) hydrolysis IN 4) protonation N 0 zozo O_ O p OH BocHN~ \J4OH

6b 2b: 67% wherein PMB is para-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-HC1) comprising the reaction steps of : 0 H 1) formation of active ester 1,--HCI, neutralization and coupling 3) hydrolysis N''O N O 4) protonation lb 0 0 0 y OH BocHN OH OH

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. 3 Scheme 1

Base 1) DCC/HOBt Base 0 lao 2) OH-0 Y 3) H N A BocHN OEt OH BocHN OH 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- ethylenediamine4 (3) with a haloacetic acid derivative, most often ethyl bromoacetateS 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

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, lH nmr (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/water8 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. l° 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 3A 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 1H nmr 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 Ha 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, 1 HCl is more conveniently stored and dispensed than neutral 1, which is usually a viscous oil.

We have prepared a few PNA monomers from 1 HCl (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: 1-HNO3, 1H2S04 (hemisulfate or sulfate), 1-H3PO4, l-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 of l HCl, 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, 1 HCl 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 1-HCl 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 (1HCl) : o 0 1) formation of active ester H3C 0 IOH HsC N-H 2) 1-HCI, neutralization and coupling I N X t 3) ester hydrolysis N O 4) protonation L o L, fo'R lN' OH BocHN~ XOH 6a: R'= Me, R"= H 2a : 48% 0 0 1) formation of active ester H3C, PMB CH3 PMB 2) 1-HCI, neutralization and coupling 3 N 3) hydrolysis N/\O 4) protonation t ° 0 0 ouzo N OH BocHN OH

6b 2b: 67% 0 0 1) formation of active ester IOH , H 2) 1 HCI, neutralization and coupling ( 3) hydrolysis N''O N O 4) protonation 9° O N' OH BocHN OH 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 : para-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 1 HCI by use of a suitable base such as NEt3 (triethylamine) or DIEPA (diisopropylethylamine) preferably in the presence of DMAP (4- dimethyaminopyridine). Thus, although l HCI 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, HC1 aqueous solution hydrochloric acid, PMB: para-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 HCl 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 HCl 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

1'HCl, 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. 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-1-ylacetic acidll and 5-iodouracil-1-ylacetic acid12 were prepared according to literature methods. Ethereal HCl was prepared by dropwise addition of a large excess of concentrated HCl to an equal volume of concentrated H2S04, 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 Varian Inova 600 or Varian Mercury 400 spectrometer.

Ethyl N- (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: 8 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. CH2Cl2 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"2nd. Ed. , Wiley-VCH, 1999, New York, NY, USA.

1HCl To an ice-bath cooled solution of 1 (8.71 g, 35.4 mmol) in Et20 (150 mL) was added ethereal HCl (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 Et20, and dried in vacuo, to afford 8.2 g (82%) of 1HCI, an air-stable, non-hygroscopic white solid. M. p. 121-124°C (dec), lH nmr : (400 MHz, D20) 8 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) : 8167. 09,158. 30,81. 83,63. 75, 47.70, 36.87, 27.94, 13.58. HRMS exact mass 247. 1651, calc'd for CllH23N204+ : 247.1652. Anal. calc'd for CllH23ClN204 : 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 1 HCl To an ice-cooled solution of a nucleobase acetic acid derivative (6,1 eq. )<BR> and 1-hydroxybenzotriazole (1.1 eq. ) in a minimum amount of dry DMF was<BR> 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 1HCl (1.1 eq. ), TEA or DIPEA (3.3 eq.), and 4-<BR> (N, N-dimethylamino) pyridine (0.1 eq. ) in a minimum amount of DMF (1 g of

1 HCI 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 2cl2 were in agreement with those previously reported. 2b: M. p. 182°C (decomposition), 1H nmr : (400 MHz, DMSO, 20°C, 2 rotamers) 8 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 N1- CH2 mi), 4.17 (s, 0.7H, oc-CH2 mi), 3.97 (s, 1.3H, oc-CH2 ma), 3.70 (s, 3H, PMB- OCH3), 3. 38 (t, J=6.5 Hz, 1.3H, y-CH2 ma), 3.30 (t, J=6.9 Hz, 0.7H, y-CH2 mi), 3.18-3. 14 (m, 1.3H, 8-CH2 ma), 3.03-2. 99 (m, 0.7H, 8-CH2 mi), 1.80 (s, 3H, thymine CH3), 1.36 (s, 9H, t-Bu). 13C nmr: (100 MHz, DMSO, 20°C, 2 rotamers) 5 170. 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 C1), 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 (a-CH2 mi), 48.99 (thymine N1-CH2 mi), 48.85 (thymine Nl-CH2 ma), 47.51 (oc-CH2 ma), 46.91 (y-CH2 mi), 46.72 (y-CH2 ma), 43.11 (PMB CH2), 38. 04 (8-CH2 ma), 37.58 (8-CH2 mi), 28.22 (C-Mes mi), 28.15 (C-Me (thymine CH3). HRMS exact mass 504.2222, calc'd for C24H32N408 : 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. Acc. Chem. Res. 1999, 32, 624-630.

(2) tBoc-and Fmoc-PNA monomers are available from Applied Biosystems, 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. J. 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, 431 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) Viirre, 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.