Determination of absolute con fi guration of the phosphonic acid moiety of fosfazinomycins †

Fosfazinomycins A and B produced by Streptomyces lavendofoliae share the same phosphonate moiety with one chiral centre of unknown configuration which was determined by synthesising both enantiomers of 2-hydroxy-2-phosphonoacetic acid methyl ester. A chiral cyclic phosphite was reacted with methyl glyoxylate in a Pudovik reaction to give a pair of diastereomeric α-hydroxyphosphonates, which were separated by HPLC. The configurations at C-2 were assigned on the basis of single crystal X-ray structure analysis. Deprotection of these diastereomers furnished the enantiomeric α-hydroxyphosphonic acids, of which the (S)-configured had the same sign of optical rotation as the phosphonic acid moiety of the two fosfazinomycins.


Introduction
Phosphonates and phosphinates are organic compounds characterised by either one or two phosphorus-carbon bonds. Over the past few decades they have been studied extensively and are nowadays used in medicine and agriculture. 1 They have interesting biological properties, 2 which can be attributed to their structural similarity to phosphoric acid esters and carboxylic acids, as well as to the high stability of the incorporated P-C bonds. 3,4 They can be found both free and bound to structural components such as lipids or proteins. As conjugates with macromolecules they either enhance the structural rigidity of the latter or protect them against enzymatic degradation. 5 There is a steadily growing, fascinating group of about a dozen small molecules of natural origin containing a P-C bond, some of which are bioactive. 1,6 Their properties range from antibacterial, antiviral or antibiotic to pesticidal and enzyme inhibitory.
The current work deals with two other members of the group of small bioactive phosphonates, namely fosfazinomycins A (5a) and B (5b) (Scheme 1). They were first isolated in 1983 from the fermentation broth of Streptomyces lavendofoliae and are active against some filamentous fungi. 12 Structure elucidation revealed fosfazinomycin to be a mixture of two components, A and B. They both contain L-arginine as well as a unique phosphonohydrazine moiety. 13,14 The latter somehow relates them to FR-900137, an antibacterial antibiotic particularly active against Escherichia coli. 15 Fosfazinomycin A differs from B by containing L-valine attached to the α-amino group of L-arginine. Furthermore, they share the same α-hydroxyphosphonate moiety with one stereogenic centre. The corresponding free phosphonic acid has been isolated by acid hydrolysis of fosfazinomycins and purification by ion exchange chromatography. Its optical rotation was determined {[α] 20 D +22.5 (c 1.50 in H 2 O)}, but the absolute configuration remained elusive. 16 The purpose of this work was to synthesise both enantiomers of 2-hydroxy-2-phosphonoacetic acid methyl ester (6) of known absolute configuration. Their specific optical rotation will allow assigning the configuration to the natural product. This information might be helpful in unravelling the biosynthesis of fosfazinomycins.

Results and discussion
Synthetic challenges P-C bonds in phosphonates are generally chemically very stable towards cleavage by bases and acids. However, α-hydroxyphosphonates are chemically labile. 17 Their formation from aldehyde and phosphite and cleavage to the same compounds are catalysed by a base. Although chiral α-hydroxyphosphonate 6 looks very simple, we anticipated some obstacles during its synthesis. First, racemisation can interfere if the chiral, nonracemic hydroxyphosphonate is treated with a base. Second, the stereogenic centre here is base-labile as the α-hydrogen is acidified by the ester and the phosphonate group, irrespective of whether it is protected or not. Third, the base can induce an α-hydroxyphosphonate-phosphate rearrangement 18 assisted by the ester group, which itself can be hydrolysed. To avoid these problems, very mild reaction conditions and catalytic removal of protecting group(s) from phosphorus at the end were mandatory.

Original synthetic strategy
Initially, we envisaged to generate racemic dibenzyl α-hydroxyphosphonate (±)-9, possibly separable by HPLC on a chiral stationary phase. Catalytic removal of the protecting groups would give the enantiomeric 2-hydroxy-2-phosphonoacetic acid methyl esters (R)-and (S)-(6) in the final step (Scheme 2). Therefore, methyl glyoxylate (7) was prepared from glyoxylic acid monohydrate and methyl dimethoxyacetate by a literature procedure. 19 It was reacted immediately in a Pudovik reaction 20 with dibenzyl phosphite (8) at −78°C in the presence of Et 3 N as a base catalyst to give the desired racemic α-hydroxyphosphonate (±)-9 in moderate yield (64%). Unfortunately, the two enantiomers could not be separated by HPLC on a Chiralcel OD-H column using various mixtures of iso-propanolhexanes.

Revised strategy and synthesis
Therefore the synthetic strategy had to be changed. We decided to prepare a cyclic phosphite of known absolute configuration, which will yield a pair of diastereomeric cyclic α-hydroxyphosphonates upon reaction with methyl glyoxylate. Flash column chromatography and deprotection would give the free phosphonic acids (R)-and (S)-6.
As it proved to be useful, we tried to prepare diol (R,R)-14 by a literature procedure from (S)-2-chloro-1-phenylethanol via the dilithiated species, which was added to benzaldehyde. Contrary to the literature, which reported only the (R,R)product to be formed in 79% yield, our (R,R)-14 : meso-14 ratio was 1.0 : 0.8 and the yield was low (35%). 25 As it could not be improved, enantioselective reduction of dibenzoylmethane (17) with Ru( p-cymene)[(R,R)-Ts-DPEN] (18) seemed to be an attractive alternative to get this chiral diol (Scheme 5). 26 This asymmetric transfer hydrogenation worked best with a mixture of 4.4 equiv. of HCO 2 H and 2.6 equiv. of Et 3 N as a hydrogen source and the yield of the crystallised product was 71% (ee >99% by chiral HPLC). 27 Having the enantiomerically pure diol (R,R)-14 in hand, it was used to generate the cyclic phosphite (R,R)-15 which was added to methyl glyoxylate (7) at −78°C in a Pudovik reaction 20 catalysed by Et 3 N (Scheme 4). When the reaction mixture was allowed to warm to −50°C in the cooling bath, the two diastereomeric α-hydroxyphosphonates 16a and 16b were formed. Addition of an equimolar amount of CF 3 CO 2 H relative to Et 3 N, extractive workup and flash chromatography gave an inseparable mixture of the two diastereomers (in a 1 : 1 ratio) in low (50-55%) yield. Replacing CF 3 CO 2 H by CH 3 SO 3 H to neutralise the amine caused a lower yield. However, when the transformation was finished (as can be easily determined by NMR spectroscopy) and the reaction mixture was directly applied to the silica gel column for flash chromatography without the addition of an acid, the diastereomeric mixture of 16a and 16b was obtained in good (78%) yield. Unfortunately, the two diastereomers could not be separated by flash column chromatography, but only by preparative HPLC (CH 2 Cl 2 -EtOAc = 1 : 1; t R (16a) 7.8 min, t R (16b) 9.9 min). Crystallisation of both diastereomers from CH 2 Cl 2 produced crystals suitable for single crystal X-ray structure analysis ( Fig. 2 and 3). Diastereomers 16a and 16b were found to have (S)-and (R)-configuration, respectively, at the carbon atom bearing the hydroxyl group.
These α-hydroxyphosphonates were used for the next and final step in the reaction sequence, removal of the diol protecting group from phosphorus by hydrogenolysis in MeOH. The 1,3-diphenylpropane formed was removed by extraction with hexanes. Concentration of the methanolic solutions furnished the α-hydroxyphosphonates (R)-and (S)-6 in sufficient purity for collecting the analytical data. Their specific optical rotations Scheme 5 Highly enantioselective preparation of (R,R)-14.   16 By comparison with the synthetic samples, it was concluded that it has (S)-configuration and has evidently partly racemised during the cleavage process. The finding that the only other known α-hydroxyphosphonate of biological origin, which is 2-amino-1-hydroxyethylphosphonic acid (4), has (R)-configuration will have biosynthetic implications. 11 To underpin the consistency between the structure of the α-hydroxyphosphonic acid 6 isolated from fosfazinomycins and the synthetic samples, and to determine the ee of our samples, (R)-6 was esterified with diazomethane in methanol (Scheme 6).
The crude product obtained by concentration of the reaction mixture under reduced pressure was dimethyl phosphonate (R)-19 (ee 99%, by NMR spectroscopy using (R)-(+)-t-butyl-(phenyl)monothiophosphinic acid as a chiral shift reagent) 28,29 in admixture with a small amount of a compound tentatively assigned the structure of methyl ether (R)-20 (molar ratio of (R)-19 : (R)-20 = 88 : 12). They were separated by flash chromatography. The NMR spectrum of homogeneous (R)-19 was identical to that of an authentic sample of (±)-19, 30 prepared by base-catalysed addition of dimethyl phosphite to methyl glyoxylate in 72% yield, but its ee was only 31%. Partial racemisation evidently occurred during flash chromatography on silica gel by removal of the fairly acidic proton α to phosphorus. To prove the formation of methyl ether (R)-20 during esterification, its racemate was synthesised by etherification of racemic methyl 2-(dimethoxyphosphinyl)-2-hydroxyacetate [(±)-19] with CH 2 N 2 in the presence of HBF 4 ·OEt 2 as a catalyst 31 (Scheme 7). The product yield was poor (29%), but sufficient for collecting the necessary analytical data and proving that (R)-20 was formed as a side product during the esterification of (R)-6.

Experimental
General experimental 1 H, 13  and CDCl 3 (δ C 77.23), CD 3 OD (δ C 49.15) and external H 3 PO 4 (85%). Chemical shifts (δ) are given in ppm and coupling constants (J) in Hz. IR spectra were run using a Bruker VERTEX 70 IR spectrometer as ATR spectra. Optical rotations were measured at 20°C using a Perkin-Elmer 341 polarimeter in a 1 dm cell.
[α] D values are given in 10 −1 deg cm 2 g −1 . Analytical HPLC for the determination of the ee of (R,R)-14 was performed on a Jasco system (PU-980 pump, UV 975 and RI 930) using a Chiralcel OD-H column, Ø 0.46 cm × 25 cm. Preparative HPLC for the separation of 16a and 16b was performed using a Dynamix Model SD-1 equipped with a Model UV-1 absorbance detector using a Nucleosil 50-5 column, Ø 3.2 cm × 25 cm. Melting points were determined using a Leica Galen III Reichert Thermovar instrument and were uncorrected.
TLC was carried out on 0.25 mm thick Merck plates with silica gel 60 F 254 . Spots were visualised by UV and/or dipping the plate into a solution of (NH 4 ) 6 Mo 7 O 24 ·4H 2 O (25.0 g) and Ce(SO 4 ) 2 ·4H 2 O (1.0 g) in 10% aqueous H 2 SO 4 (500 mL), followed by heating with a heat gun. Flash (column) chromatography was performed with Merck silica gel 60 (230-400 mesh).

Paper
Organic & Biomolecular Chemistry