Synthesis of Orthogonally Protected Muramic Acid Building Blocks for Solid Phase Peptide Synthesis

Muramic acid is found in many peptide natural products containing oligo(poly)saccharide moieties. Taking into consideration that the Fmoc methodology is routinely used for solid-phase peptide synthesis, preparation of orthogonally protected muramic acid building blocks for total solid-phase synthesis of these natural products is of particular practical importance. Herein a simple and efficient synthesis of benzyl 2-amino-4,6-O-benzylidene-3-O-[(R)-1-carboxyethyl]-2-deoxy-N-9-fluorenylmethyloxycarbonylα-D-glucopyranoside (6) from N-acetylglucosamine (1) is described. Important improvements over previous synthetic approaches to glucopyranosides 2 (benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside) and 3 (benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside), key building blocks in preparation of 6, include synthesis simplification and efficient isolation and purification. Optically pure (S)-2chloropropionic acid 7 was prepared and introduced to the positon 3-O of sugar moiety to give compound 4 (benzyl 2-acetamido-4,6-O-benzylidene-3-O-[(R)-1-carboxyethyl]-2-deoxy-α-D-glucopyranoside) with the (R)-configuration of the lactyl side-chain in excellent overall yield and optical purity. Deacetylation of amino group gave compound 5 (benzyl 2-amino-4,6-O-benzylidene-3-O-[(R)-1-carboxyethyl]-2-deoxy-αD-glucopyranoside) suitable for incorporation of the Fmoc protecting group to give protected muramic acid derivative 6, a useful building block in peptide synthesis.


INTRODUCTION
2][3][4][5][6][7] As such, SAAs are widely spread in nature, and neuraminic acid and N-acetylmuramic acid constitute important structural elements in many oligo-(poly)saccharides and glycoconjugates. 1 Muramic acid has been shown to be the main component of the peptidoglycan chain, which constitutes a building unit of the bacterial cell walls and spores. 8The discovery that the minimal adjuvant active structure of bacterial peptidoglycans is the muramyl dipeptide N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) has generated a great deal of interest in muramic acid derivatives preparation. 9,10The potential utilisation of a simple and well-defined synthetic adjuvant molecule as MDP, showing maximal stimulation of the immune response but little toxicity and other side effects, has heralded a new era in immunology especially in the field of new vaccine development. 11Interestingly, SAAs in nature are almost exclusively linked through interglycosidic bonds, not through amide bonds.The relevance of the hybrid nature of SAAs is evident by the multitude of reported applications in which selected amino acid residues are replaced by SAA in biologically relevant oligopeptides.Thus the outcome of this replacement can be twofold: 1) The nature of the parent carbohydrate (the furan or pyran ring) in combination with the positioning of the amine and carboxylate may affect a desired secondary structure on the target oligopeptide, 2) the residual functionalities on the furan/pyran core may be used to introduce additional desirable properties to the peptide. 124][15][16][17][18] Herein we describe the synthesis of optically pure orthogonally protected muramic acid were the impurities of the isomuramic acid were avoided by using optically pure (S)-2-chloropropionic acid for introduction of lactyl group to sugar moiety.

Benzyl 2-amino-4,6-O-benzylidene-3-O-[(R)-1-carboxyethyl] -2 -deoxy -N -9 -fluorenylmethyloxycarbonyl-α-Dglucopyranoside (6)
Compound 5 (1 g, 2.3 mmol) was dissolved in DMF (20 mL) and sodium hydrogen carbonate (392 mg, 2 eq.) in water (20 mL) was added.Reaction mixture was kept at 0 °C and cold solution of Fmoc N-hydroxysuccinimide ester (Fmoc-OSu, 865 mg, 1.1 eq.) in DMF (20 mL) was added dropwise.Reaction was stirred in ice-water bath for 2 h.Water (100 mL) was added to the reaction mixture and formed precipitate was filtrate and washed with water.Product was evaporated under vacuum and then purified on flash silica gel column in solvent system C which gave 1. (S)-2-Chloropropionic acid ( 7) 24 L-Alanine (6 g, 0.067 mol) was dissolved in 8 M HCl (84 mL) and cooled to 0 °C and then solution of NaNO2 (7.6 g in 24 mL water) in water was added dropwise with vigorous stirring.Temperature was kept at 5 °C for 2 h, and after that the reaction mixture was stirred at 2 °C for additional 4 h, and overnight at room temperature in inert conditions (under nitrogen).Sodium hydrogen carbonate (6.6 g) was added to the reaction mixture in small portions with vigorous stirring.Reaction was extracted with ether (2 × 50 mL, 1 × 30 mL) and collected ether's extracts were washed with saturated solution of calcium chloride (20 mL) and re-extracted with ether (30 mL).Ether's extracts were dried for 2 h on calcium chloride.Ether was evaporated under reduced pressure and oil residue was distilled in vacuum to give pure (S)-2-chloropropionic acid (5.

RESULTS AND DISCUSSION
6][27][28][29] The synthesis started from N-acetylglucosamine 1, which was protected by benzylation with benzyl alcohol at the anomeric position. 19The p-toluene sulfonic acid was used as a catalyst in the reaction which was carried out in toluene.The synthesis resulted in a α/ β mixture of benzyl anomer 2, however the pure benzyl α-anomer 2 can be obtained with thermodynamic control of the reaction by high temperature in refluxing toluene. 29The incorporation of a benzylidene protection group in the 4-O and 6-O-hydroxy positions was carried out according to previously published Gross and Jeanloz strategy to afford protected N-acetylglucosamine derivative 3 with a free 3-hydroxy group. 21The method for the efficient and easy synthesis of 4,6-benzylidne-derivative of N-acetylglucosamine, using pyridinium perchlorate as catalyst in the reaction with benzaldehyde dimethylacetal, was recently published. 30By treating 3 with optically pure (S)-2-chloropropionic acid 7, the Williamson stereoselective ether synthesis proceeded in good yield (78 %), to afford 4. 31 The synthesis of muramic acid previously reported in the literature is based on condensation of D-glucosamine derivatives with a 2-halogenopropionic acid derivative, forming an ether link with the hydroxyl group at C-3. 23,31 In most of these approaches, methyl 2 -acetamido -4,6 -O -benzylidene -2 -deoxy -α -Dglucopyranoside or benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside (3) have been used as a starting material.However, alkylation of the unprotected 3-O group of 3 with racemic 2-halogeno-propionic acid derivatives led to the formation of a mixture of epimeric (R) and (S) ethers (muramic and isomuramic acid derivatives), and the described methods require additional separation of the epimeric mixture in order to obtain pure diastereomers. 32Improvement in the synthesis of muramic acid was achieved by the use of enantiopure (S)-2-chloropropionic acid 7 as the alkylation agent in the critical condensation step. 23It was found that when (S)-2-chloropropionic acid was condensed with an alkoxide, it underwent Walden inversion. 33herefore, the condensation of 3 with pure (S)-2-chloropropionic acid led stereospecifically to muramic acid, with (R) configuration of the lactic acid moiety.Although this method avoids chromatographic separation of 4, it was not explored due to the difficult preparation of optically pure reagent. 32Our modified approach for (S)-2-chloropropionic acid synthesis from (S)-alanine 24 resulted in the optically pure compound 7 in 73 % yield.The introduction of lactic acid moiety was achieved in presence of NaH according to the paper published by Sinaÿ et al. for the synthesis of manno derivative of muramic acid with some modification in time, temperature and purification of compound. 34The compound 4 was purified by flash chromatography and the stereoselectivity of the reaction was determined by 1 H NMR.
The benzylidene proton at 5.68 ppm for the muramic acid derivative 4 is indicative of the muramic acid derivative with the (R)-configuration of the lactyl sidechain according to the published data by Babič and Pečar which are for the muramic acid 5.66 ppm and for the isomuramic acid 5.55 ppm. 29The determination of the absolute configuration was especially important in the next step in the reaction of saponification of the compound 4, to deprotect amino group and give compound 5.This reaction conditions enable racemisation.
1 H NMR analysis shows benzylidene proton of compound 5 at 5.68 ppm (Table 1) which in comparison to compound 4 showed that we did not lose the optical purity in compound 5 with the (R) configuration of the lactyl group.The introduction of the Fmoc protecting group, the major group that enables utilisation of muramic acid in SPPS, was obtained with Fmoc-OSu in DMF/water solvent mixture with sodium hydrogen carbonate under low temperature (0 °C) to avoid byproducts formation.The synthesis with Fmoc-Cl as reagent was also examined but the reaction yields were poor.Upon the completion of the reaction the product 6 was precipitated with water, filtrated and purified on silica gel column in solvent system C.The optical purity was confirmed by 1 H NMR analysis which shows single signal for benzylidene proton of compound 6 at 5.69 ppm (Table 1) which is in agreement with chemical shifts of compound 4 (5.70 ppm, Table 1) and compound 5 (5.68 ppm, Table 1), respectively, for (R)-configuration of the lactyl group.Synthesised Fmoc protected muramic acid can be used in SPPS as dipeptide isostere building block.Protecting groups are necessary during the synthesis especially for synthesis of longer peptides to avoid by-products formation and to facilitate product purification.Orthogonal protection of muramic acid allows removal of Fmoc protecting group during peptide synthesis, while benzylidene group and benzyl protecting group can be easily removed after peptide condensation under acidic conditions and catalytic hydrogenolysis.

MS/MS fragmentation of prepared compounds
Electrospray ionization (ESI) as soft-ionization method was used to determine the mass spectrometric properties of prepared compounds.The MS/MS fragmentation of all synthesised compounds from 2-6 showed the same tendency.The elimination of benzyl protecting group from 1-O of compounds 2-6 was confirmed with following detected fragments 204 (2), 292 (3), 364 (4), 322 (5, Figure 2) and 544 (6, Figure 3) Da, for each compound, respectively. 35,36The fragments 258 (4), 216 (5, figure 2) and 366 (6, Figure 3) Da are results of benzylidene protecting group elimination.Further most intense signal in MS/MS spectrum of compound 5 is 182 which response to the lactone structure shown on Figure 2. Fragmentation of compound 6 in positive mode completely differs from that one in negative mode.While positive mode shows the elimination of protecting groups as well as dehydration of molecule, in negative mode the main and only observed fragment was 428 Da which responding to the Fmoc deprotected molecule (Figure 3).

CONCLUSION
In this work we reported simple and efficient synthesis of orthogonally protected muramic acid suitable for Fmoc solid-phase peptide synthesis.The advantages of our approach include simplified synthesis, use of readily available starting materials and high yield (~ 70-90 %) of desired optically pure (R)-isomers of products 4-6.
The described synthesis of orthogonally protected muramic acid building blocks will allow an access to a variety of linear and cyclic peptides with unique structural properties and consequently biological activities.