Naphthalene derived chromogenic enzyme substrates

Abstract

Conjugates of 2,3-dihydroxynaphthalene and its derivatives with enzyme cleavable groups are chromogenic substrates that form colored compounds when complexed with metal ions, e.g. iron ions, on cleavage by enzymes, and are useful in microbial detection and identification. The cleavage products form purple or red-brown colored complexes, that can easily be observed by the naked eye. Microbes can be grown in the presence of the substrates and the metal salts that provide the metal ion for complexing with the 2,3-dihydroxynaphthalene product. Substituents in the naphthalene ring may affect the solubility of the substrates and also the diffusibility and color of the metal complexes. Some of the substrates yield soluble complexes on cleavage and are of particular value in liquid growth media. Other substrates produce less soluble complexes that are more suitable for use in solid agar media. Some substrates are new compounds, such as those having the general formula II ##STR00001## wherein one of the following applies i) m=0, R.sup.4R.sup.5=Z.sup.1H, Y.sup.1 is selected from the group consisting of D-glucuronyl and D-ribofuranosyl; ii) m=2, each R.sup.6 is Br, R.sup.4R.sup.5H or Br, Z.sup.1H, Y.sup.1 is glycosyl or phosphate; iii) m=1, R.sup.6 is SO.sub.3X, X is H or M.sup.+ wherein M.sup.+ is an alkali metal cation or a non-metal cation, Y.sup.1 is glycosyl and R.sup.4R.sup.5=Z.sup.1H; iv) m=0, R.sup.4NO.sub.2, R.sup.5Z.sup.1H, Y.sup.1=glycosyl. Methods of synthesizing the substrates are described.

Claims

1. A composition comprising a) an enzyme substrate of formula I: ##STR00037## wherein Y is an enzyme cleavable group; Z is H, a metal cation or non-metal cation, acyl or the same enzyme cleavable group as Y; R.sup.2 and R.sup.3 are each independently selected from the group consisting of H, OH, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.24 acyl, halogen and nitro, provided that if Z is H or a salt, then R.sup.2 must not be OH; R.sup.1 is C.sub.1-C.sub.8 alkyl, halogen, OH, NO.sub.2, C.sub.2-C.sub.24 acyl, or SO.sub.3X, where X is H, a metal cation or a non-metal cation; and n is 0-4; b) a metal compound such that a product of enzymatic substrate cleavage is capable of chelating a metal ion of the metal compound, thereby forming a colored compound, wherein the metal compound is an iron salt; and c) microbial growth nutrients.

2. The composition according to claim 1, wherein the microbial growth nutrients include a carbon source, a nitrogen source, amino acids, salts, vitamins and/or cofactors.

3. The composition according to claim 1, wherein the molar ratio of enzyme substrate to metal compound is in the range 0.05 to 50.

4. A composition comprising a) an enzyme substrate of formula I: ##STR00038## wherein Y is an enzyme cleavable group; Z is H, a metal cation or non-metal cation, acyl or the same enzyme cleavable group as Y; R.sup.2 and R.sup.3 are each independently selected from the group consisting of H, OH, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.24 acyl, halogen and nitro, provided that if Z is H or a salt, then R.sup.2 must not be OH; R.sup.1 is C.sub.1-C.sub.8 alkyl, halogen, OH, NO.sub.2, C.sub.2-C.sub.24 acyl, or SO.sub.3X, where X is H, a metal cation or a non-metal cation; and n is 0-4; and b) a metal compound that is an iron salt.

5. The composition according to claim 4, further comprising microbial growth nutrients.

6. The composition according to claim 5, wherein the microbial growth nutrients include a carbon source, a nitrogen source, amino acids, salts, vitamins and/or cofactors.

7. The composition according to claim 4, wherein the molar ratio of enzyme substrate to metal compound is in the range 0.05 to 50.

8. A composition comprising an enzyme substrate of formula II ##STR00039## wherein one of the following applies: i) m=0, R.sup.4R.sup.5Z.sup.1H, Y.sup.1 is selected from the group consisting of D-glucuronyl and D-ribofuranosyl; ii) m=2, each R.sup.6 is Br, R.sup.4R.sup.5H, Z.sup.1H; Y.sup.1 is glycosyl or phosphate; iii) m=1, R.sup.6 is SO.sub.3X, X is H or M.sup.+ wherein M.sup.+ is an alkali metal cation or a non-metal cation, Y.sup.1 is glycosyl or phosphate and R.sup.4R.sup.5Z.sup.1H; iv) m=0, R.sup.4NO.sub.2, R.sup.5Z.sup.1H, Y.sup.1=glycosyl.

9. The composition of claim 8, further comprising a metal compound, a metal ion of which is chelatable by the product of enzyme cleavage of the group Y.sup.1 and, in the case of option iv), also of the group Z.sup.1, from the substrate.

10. The composition according to claim 9, wherein the metal compound is an iron salt.

11. The composition according to claim 9, further comprising microbial growth nutrients.

12. The composition according to claim 11, wherein the microbial growth nutrients include a carbon source, a nitrogen source, amino acids, salts, vitamins and/or cofactors.

13. The composition according to claim 9, wherein the molar ratio of enzyme substrate to metal compound is in the range 0.05 to 50.

Description

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(1) Among the compounds possessing the ortho-dihydroxyaromatic system, DHN and DHN-6-sulfate are well-known metal chelators. They form coloured complexes with a variety of different metals, including first row transition metals such as iron, titanium and vanadium [V. Patrovsky, Coll. Czech. Chem. Commun., 35, 1599-1604, (1970)]. The use of transition metal complexes of DHN, DHN-6-sulfate and some related ortho-dihydroxyaromatic compounds as potential or actual analytical reagents has been reviewed [P. K. Tarafder and R. K. Mondal, Rev. Anal. Chem., 30, 73-81, (2011)]. Until the present invention, DHN and its simple derivatives have not been considered as the core molecule part of chromogenic enzyme substrates. When the DHN substrates of the present invention are incorporated into microbiological growth media containing an iron compound, enzymatic cleavage by microorganisms liberates the free DHN molecule. This reacts spontaneously with the iron compound to generate a highly coloured chelate that allows easy detection of any enzyme-positive reactions by eye (i.e., by the means of incident visible light). The colour given by the reaction depends upon the substitution pattern of the DHN-derivative, and may also be influenced by pH and the mix of oxidation states of the iron, as well as the presence of other components contained in the growth medium. The colour may be described variously as brown, purple or maroon. However, whatever its hue, the colour is completely different to that of most microbiological growth media and is intense enough to allow unambiguous detection of enzyme-positive reactions.

(2) Until the present work, only a very limited number of DHN derivatives having the potential to act as chromogenic enzyme substrates had been made, although DHN itself has been known for over 100 years. It is only much more recently that glycosides of DHN have been synthesised. Ellervik and co-workers described the synthesis of DHN--D-xylopyranoside [M. Jacobsson and U. Ellervik, Tetrahedron Letters, 43, 6549-6552, (2002)] and DHN-di--D-xylopyranoside [R. Johnsson et al, Bioorganic and Medicinal Chemistry, 15, 2868-2877, (2007)] as part of a large set of other compounds to investigate their antiproliferative effects on cancer cells. Sakuma and Yokoe described the preparation of DHN--D-glucopyranoside (4) [K. Sakuma and I. Yokoe, Japanese Patent Publication No. 2004-224763, (2004)]. This compound was advocated as a potential bleaching agent for use in cosmetics [K. Sakuma and I. Yokoe, Japanese Patent Publication No. 2004-224762, (2004)]. Clearly, none of the aforementioned prior art concerning DHN compounds is pertinent to the present application wherein they are employed as chromogenic enzyme substrates for the detection of microorganisms. Nor is the more recent technique of Bhowmik and Maitra [(S. Bhowmik and U. Maitra, Chem. Commun., 48, 4624-4626, (2012)] wherein these authors described the development of a time-delayed luminescence method for detecting enzyme activity using terbium(III) acetate in conjunction with either DHN--D-glucopyranoside (4) or DHN-diesters. This method differs greatly from the present invention in several important respects. Firstly, it is a luminescence method in which the luminescence is triggered by excitation of the sample with ultraviolet light, whereas the present invention is a chromogenic method involving absorbance of light in the visible spectrum. Bhowmik and Maitra reported that their technique (which they state does not involve chelation) works exclusively in a gel medium, and this is a further major difference from the present invention which works very well in a fluid or liquid medium as well as in agar gels. A third important difference is that the necessary gelling agent of Bhowmik and Maitra was the sodium salt of a bile acid, cholic acid. Cholic acid and other bile acids have long been known to inhibit the growth of certain microorganisms [Binder et al, Amer. J. Clin. Nutr., 28, 119-125, (1975); Kurdi et al, J. Bacteriol., 188, 1979-1986, (2006)]. The concentration of sodium cholate used by Bhowmik and Maitra was 15 mM, and there was no suggestion by these authors that microorganisms are able to grow in or on their reaction medium. In this context it should be noted that the growth of some intestinal bacteria is completely suppressed with cholic acid concentrations lower than 15 mM [Floch et al, Amer. J. Clin. Nutr., 25, 1418-1426, (1972)]. Additionally, it should be remarked that the present invention requires the incorporation of metal compounds, e.g. first transition metal series metal compounds, preferentially iron compounds, into the medium. The iron compounds may be either iron(II) or iron(III); or a mixture of both states. In some instances, if other metal ions are substituted for iron, including ions such as Ti(IV) that are known to form coloured complexes with DHN, then little or no visible colour is developed after enzymatic hydrolysis. Similarly, using the lanthanide terbium(III) acetate instead of an iron compound does not furnish a coloured end-product. The metal compound used in the invention must therefore be selected having regard to the colour-forming capability with the cleaved product of the enzymatic reaction.

(3) Substitution products of the DHN core molecule yield both enzyme substrates and endpoints of very different solubilities, and this is a further useful feature of the present invention. Thus, unsubstituted-DHN compounds are fairly water-soluble and the soluble DHN-iron chelate is best suited to liquid growth media. Compounds derived from DHN-6-sulfate are even more water soluble and may be preferred when the solubility of the enzyme substrates in aqueous media is problematic, for instance in the development of assays for esterases such as lipase which require substrates that are derivatives of fatty acids and may have limited water solubility unless derivatised by ionic groups elsewhere in the molecule. In contrast, substrates based on 6,7-dibromo-DHN (as described below) give insoluble precipitates with iron compounds and they are particularly suited to agar plate media, where they are able to visualise single colonies.

(4) Preferred and non-limiting aspects of the invention are described in more detail below, which includes worked examples of microbial test methods using chromogenic substrates, followed by synthetic methods used to make the substrates.

Example 1Testing of DHN--D-Galactopyranoside with a Variety of Microorganisms

(5) DHN is inexpensive and readily available commercially. We found that novel glycosides of it could be prepared by standard means. Thus the protected DHN--D-galactopyranoside (7) was made by coupling acetobromogalactose with DHN in acetone-water in the presence of sodium hydroxide. After work-up, deprotection in methanol containing a catalytic quantity of sodium methoxide furnished the desired DHN--D-galactopyranoside (8) as an off-white solid. Details of the synthesis are given below in syntheses 7 and 8.

(6) This compound was tested on agar plates by multi-point inoculation. The plates were made from Columbia agar (Oxoid, Basingstoke, UK) (100 ml) supplemented with ferric ammonium citrate (FAC) (50 mg). After the agar had been autoclaved (121 C.) and cooled to 50 C., a filter sterilized solution of the substrate (8) (30 mg) dissolved in N-methylpyrrolidone (NMP) (200 L) was added, thus giving a substrate concentration of 300 mg/L. After pouring, the plates were inoculated with a range of microorganisms and incubated at 37 C. for 18 h in air (Table 1). Those organisms positive for -galactosidase could be easily distinguished from those negative for this enzyme by the development of a strong purple-maroon colour in the former.

(7) TABLE-US-00001 TABLE 1 DHN--D- galactopyranoside 8 Organism Colour 1 Escherichia coli NCTC 10418 ++ 2 Klebsiella pneumoniae NCTC 9528 ++ 3 Providencia rettgeri NCTC 7475 4 Enterobacter cloacae NCTC 11936 ++ 5 Serratia marcescens NCTC 10211 + 6 Salmonella typhimurium NCTC 74 7 Pseudomonas aeruginosa NCTC 10662 8 Yersinia enterocolitica NCTC 11176 9 Burkholderia cepacia NCTC 10931 10 Acinetobacter baumannii NCTC 19606 11 Steptococcus pyogenes NCTC 8306 12 Staphylococcus aureus (MRSA) NCTC 11939 13 Staphylococcus aureus NCTC 6571 14 Staphylococcus epidermidis NCTC 11047 15 Listeria monocytogenes NCTC 11994 16 Enterococcus faecium NCTC 7171 Tr. 17 Enterococcus faecalis NCTC 775 18 Bacillus subtilis NCTC 9372 19 Candida albicans ATCC 90028 20 Candida glabrata NCPF 3943

(8) Unless indicated otherwise, the symbols in each table have the following meaning; ++ means strong colour; + means less colour than ++; +/ means less colour than +; Tr means a trace of colour, less than +/; means no colour. The substrate (8) showed no obvious toxicity with any of the Gram-positive or Gram-negative strains tested. Essentially identical results were obtained if other iron salts (e.g. iron(II) gluconate, iron(II) acetate, iron(II) citrate, iron(II) acetylacetonate, and iron(III) acetylacetonate) were substituted in place of FAC. The iron compound can be either iron(II) (i.e. ferrous) or iron(III) (i.e. ferric). Both types of compound work well. The coloured chelate was still formed when the plates were incubated under anaerobic conditions, which is another useful feature of the present invention. It will be appreciated that the media and the reagents will all contain at least traces of iron compounds. However, the invention does not work unless the medium is supplemented with a sufficient amount of an iron compound. Concentrations of 200-600 mg/L of iron compound were found to be satisfactory. In contrast, supplementing the growth media with compounds of other metals gives either no coloured endpoint or an extremely poor one to visualise. In an alternative to the above procedure, the substrate may be added to the agar prior to being autoclaved (121 C. for 20 minutes) with no decrease in sensitivity. The colour generated by DHN in combination with an iron salt is of about the same intensity as that previously demonstrated by the reaction between 1,2-dihydroxybenzene (catechol) and an iron salt at the same concentration [M. Burton, EP1438423, (2007)]. However, the colours are different (the catechol-iron complex is black in solution) and this may be an advantage in designing a particular test medium. DHN--D-galactopyranoside (8) may find application as an alternative to the widely used enzyme substrate ONPG.

Example 2Other DHN Substrates and Cleavage by Microorganisms with a Range of Enzymatic Marker Activities

(9) Several other DHN substrates, intended for some of the most frequently encountered hydrolase activities in diagnostic microbiology, were synthesised by the methods described below. These were the glycosides -D-glucopyranoside (4), -D-glucopyranoside (6), -D-galactopyranoside (10) and N-acetyl--D-glucosaminide (15), the esterase substrate DHN-dicaprylate (16) and the phosphatase substrate, DHN-phosphate disodium salt (17). DHN--D-ribofuranoside (2) was also made. When evaluated in the same manner as DHN--D-galactopyranoside (8), in general these substrates were hydrolysed according to the known enzyme profiles of the test strains (Tables 2 and 3). However, DHN--D-ribofuranoside (2) (Table 2) was of particular interest.

(10) TABLE-US-00002 TABLE 2 DHN--D- DHN--D- DHN--D- DHN-N-acetyl--D- ribofuranoside 2 galactopyranoside 10 glucopyranoside 6 glucosaminide 15 Organism Colour Colour Colour Colour 1 Escherichia coli NCTC 10418 ++ Tr. + 2 Serratia marcescens NCTC 10211 ++ ++ ++ 3 Pseudomonas aeruginosa NCTC 10662 4 Burkholderia cepacia 1222 5 Yersinia enterocolitica NCTC 11176 Tr. + 6 Salmonella typhimurium NCTC 74 ++ 7 Citrobacter freundii NCTC 9750 or 46262 ++ 8 Morganella morganii 462403 (wild) ++ 9 Enterobacter cloacae NCTC 11936 ++ Tr. ++ +/ 10 Providencia rettgeri NCTC 7475 ++ 11 Bacillus subtilis NCTC 9372 ++ 12 Enterococcus faecails NCTC 775 ++ ++ 13 Enterococcus faecium NCTC 7171 Tr. ++ 14 Staphylococcus epidermidis NCTC 11047 + 15 Staphylococcus aureus NCTC 6571 + +/ 16 MRSA NCTC 11939 + +/ 17 Steptococcus pyogenes NCTC 8306 ++ ++ 18 Listeria monocytogenes NCTC 11994 ++ ++ 19 Candida albicans ATCC 90028 20 Candida glabrata NCPF 3943

(11) TABLE-US-00003 TABLE 3 DHN- DHN--D- DHN-phosphate dicaprylate 16 ghicopyranoside 4 disodium salt 17 Organism Colour Colour Colour 1 Escherichia coli NCTC 10418 Tr. +/ 2 Klebsiella pneumoniae NCTC 9528 ++ ++ 3 Providencia rettgeri NCTC 7475 + ++ + 4 Enterobacter cloacae NCTC 11936 + +/ 5 Serratia marcescens NCTC 10211 + ++ ++ 6 Salmonella typhimurium NCTC 74 ++ 7 Pseudomonas aeruginosa NCTC 10662 + 8 Yersinia enterocolitica NCTC 11176 +/ 9 Burkholderia cepacia NCTC 10931 +/ 10 Acinetobacter baumannii NCTC 19606 + 11 Steptococcus pyogenes NCTC 8306 12 Staphylococcus aureus (MRSA) NCTC 11939 + 13 Staphylococcus aureus NCTC 6571 + 14 Staphylococcus epidermidis NCTC 11047 15 Listeria monocytogenes NCTC 11994 + + 16 Enterococcus faecium NCTC 7171 + 17 Enterococcus faecalis NCTC 775 + 18 Bacillus subtilis NCTC 9372 + 19 Candida albicans ATCC 90028 20 Candida glabrata NCPF 3943

(12) When challenged with three strains of staphylococci, only the S. aureus strains NCTC 6571 and MRSA NCTC 11939 were able to hydrolyse it. It was unaffected by S. epidermidis NCTC 11047. This strongly suggests that DHN--D-ribofuranoside (2) has potential utility in the detection of MRSA and is able to differentiate this organism from other species of staphylococci that can cause interference in its positive identification. Other chromogenic -D-ribofuranosides have already been evaluated for this purpose [M. Burton, EP1438424, (2006)]. The unsubstituted DHN-glycosides tested in the present invention were all prepared by means that have been reported in the literature for other mono- or di-phenolic aglycones. The various glycosyl donors employed were all readily prepared intermediates such as acetohalosugars or trichloroacetimidates. One disadvantage of DHN enzyme substrates in agar plate media is that the coloured iron-complex diffuses, as occurs also with substrates made from 1,2-diydroxybenzene or esculetin. The diffusion is no greater than that which occurs with esculin (the -D-glucopyranoside of esculetin). As esculin is used commercially in agar plate media, substrates derived from DHN may also find application in agar plate media notwithstanding their capacity to diffuse. However, it would seem that they are much more advantageously employed in liquid broth media or in agar tube media.

Example 3DHN--D-Glucuronide Substrates and their Testing on Various Microorganisms

(13) Tube or liquid media containing the fluorogenic compound MUG (4-methylumbelliferyl -D-glucuronide) are extensively used to detect E. coli. Often these systems also contain the substrate ONPG for the detection of -D-galactosidase activity and therefore total coliforms. The media Colitag (CPI International, Santa Rosa, USA) and ColiLert (Idexx Laboratories, Westbrook, USA) both utilise MUG plus ONPG for detecting E. coli and total coliforms. The disadvantage of MUG is that a UV source is required to visualise the fluorescence associated with E. coli. It would be an advantage to have a chromogenic glucuronide that can detect E. coli with incident visible light. Accordingly, the novel compound DHN--D-glucuronide (12) was prepared in both its cyclohexylammonium salt form (12a) (hereinafter referred to as the CHA salt) and its sodium salt form (12b). In the preliminary evaluation against twenty different microorganisms on agar plates containing FAC, both salt forms of the DHN--D-glucuronide (12a and 12b) were hydrolysed equally well by E. coli (as judged by the strong colour produced with each) (Table 4). Just as significantly, E. coli was the only species able to effect hydrolysis; the other 19 strains were all -D-glucuronidase negative.

(14) TABLE-US-00004 TABLE 4 DHN--D-glucuronide DHN--D-glucuronide CHA salt 12a sodium salt 12b Organism Colour Colour 1 Escherichia coli NCTC 10418 ++ ++ 2 Serratia marcescens NCTC 10211 3 Pseudomonas aeruginosa NCTC 10662 4 Burkholderia cepacia 1222 5 Yersinia enterocolitica NCTC 11176 6 Salmonella typhimurium NCTC 74 7 Citrobacter freundii NCTC 9750 or 46262 8 Morganella morganii 462403 (wild) 9 Enterobacter cloacae NCTC 11936 10 Providencia rettgeri NCTC 7475 11 Bacillus subtilis NCTC 9372 12 Enterococcus faecails NCTC 775 13 Enterococcus faecium NCTC 7171 14 Staphylococcus epidermidis NCTC 11047 15 Staphylococcus aureus NCTC 6571 16 MRSA NCTC 11939 17 Steptococcus pyogenes NCTC 8306 18 Listeria monocytogenes NCTC 11994 19 Candida albicans ATCC 90028 20 Candida glabrata NCPF 3943

Example 4Sensitivity of DHN--D-Glucuronide to Various E. coli Isolates Compared to Standard Chromogenic Indoxyl Glucuronide Substrates

(15) In order to obtain a fuller picture of the sensitivity of DHN--D-glucuronide (12a) it was screened with 100 different clinical isolates of E. coli in a liquid medium containing FAC. The isolates were chosen at random from the Microbiology Department, Freeman Hospital, Newcastle Upon Tyne, UK. The effectiveness of DHN--D-glucuronide (12a) was compared with three other media which all contained indoxyl--D-glucuronides. One was a commercial medium, CPS ID 3 (bioMrieux SA, Lyon, France). CPS ID 3 contains complementary chromogenic substrates; Rose--D-glucuronide (6-chloro-3-indolyl -D-glucuronide) of undisclosed salt form [at 250 mg/L] for the detection of -D-glucuronidase activity (producing red or pink colonies) and X--D-glucoside (5-bromo-4-chloro-3-indolyl -D-glucopyranoside) [50 mg/L] for the detection of -D-glucosidase (producing green colonies) [M. Casse et al, U.S. Pat. No. 8,216,802 (2012)]. This medium was employed as a control. Among the -D-glucuronidase producing strains of E. coli there is a large variation in the quantity of the enzyme produced and it is almost certain that the CPS ID 3 media has been rigorously optimised to allow good growth of all the target organisms and maximum expression of the target enzymes. Therefore, two indoxyl glucuronides, X--D-glucuronide CHA salt and Rose--D-glucuronide CHA salt were also tested in a simple agar medium to allow a direct comparison of the sensitivity of these indoxyl glucuronides when used in a medium that has not been optimised for the growth of the target organism. The comparison of the results for Rose--glucuronide and the CPS ID 3 medium was of particular significance as the commercial medium also uses Rose--glucuronide for the detection of E. coli. For consistency, the three glucuronides were all chosen as their CHA salts. As already stated, is not anticipated that the salt form is critical to their performance. Currently, X--D-glucuronide is often used as either the CHA salt or the sodium salt. Brenner and colleagues [K. P. Brenner et al, Appl. Environ. Microbiol., 59, 3534-3544, (1993)] found no difference in the performance of the CHA and sodium salt forms with their application using indoxyl--D-glucuronide, neither in respect of colour development nor in the recovery of E. coli.

(16) The two indoxyl glucuronides used produce insoluble endpoints following hydrolysis, as does the CPS ID 3 medium. It was therefore necessary to test these two substrates on agar plates. In contrast, DHN--D-glucuronide (12) gives a much more soluble endpoint best suited to liquid media and was therefore tested in a broth medium. The broth was prepared using proteose peptone (2 g), NaCl (1 g) and FAC (100 mg) in DI water (180 mL). This mixture was autoclaved and cooled to room temperature before being dispensed into bijoux (1001.8 mL). DHN--D-glucuronide CHA salt (12a) (60 mg) was dissolved in water (20 mL) and filtered to sterilize before being aseptically dispensed into successive bijoux (0.2 mL) containing the broth solution. The broth/substrate solutions were then inoculated with bacterial suspensions made up to 0.5 McFarland standard (2 L per bijoux). X--D-glucuronide CHA salt (Glycosynth Ltd, Warrington, UK) (10 mg) was dissolved in NMP (200 L). Rose--D-glucuronide CHA salt (Glycosynth Ltd, Warrington, UK) (20 mg) was dissolved in NMP (200 L). These solutions were then added to Columbia agar (Oxoid, Basingstoke, UK) (100 mL) and inoculated with bacterial suspensions made up to 0.5 McFarland standard (1 L). The strains of E. coli used are listed in table 5. The plates and broths were incubated at 37 C. for 18 hours in air. The green colonies seen on the CPS ID 3 media were indicative of -D-glucosidase activity.

(17) TABLE-US-00005 TABLE 5 X--D-glucuronide Rose--D-glucuronide DHN--D-glucuronide Ref Organism Reference CPS ID 3 agar CHA salt CHA salt CHA salt 12a broth 1 E. coli 260471B +/Red 2 E. coli 260464G Red Green Red Purple 3 E. coli 260481J Red Green Red Purple 4 E. coli 260480M Red Green Red Purple 5 E. coli 260521S Red Green Red Purple 6 E. coli 260578D +/Red 7 E. coli 260537E Red 8 E. coli 260522Z Red Green Red Purple 9 E. coli 260541R +/Red Green Red Purple 10 E. coli 260538H Red 11 E. coli 260539Y Red Green Red Purple 12 E. coli 260545G Red Green Red Purple 13 E. coli 260459W Red Green Red Purple 14 E. coli 260458Y Red Green Red Purple 15 E. coli 260441G Red Green Red Purple 16 E. coli 260440S Red Green Red Purple 17 E. coli 260508Y Red Green Red Purple 18 E. coli 260504N Red Green Red Purple 19 E. coli 260503Z Red Green Red Purple 20 E. coli 260554D Red Green Red Purple 21 E. coli 260502G Red Green Red Purple 22 E. coli 260532S Red Green Red Purple 23 E. coli 260533G Red Green Red Purple 24 E. coli 260536Q Red Green Red Purple 25 E. coli 260548Q Red Green Red Purple 26 E. coli 260549E Red Green Red Purple 27 E. coli 260553X 28 E. coli 260547N Red Green Red Purple 29 E. coli 260563B Red Green Red Purple 30 E. coli 260564R Red Green Red Purple 31 E. coli 260555L Red Green Red Purple 32 E. coli 260511X Red Green Red Purple 33 E. coli 260515Z Red Red Purple 34 E. coli 260514G Red 35 E. coli 260505Q Red 36 E. coli 260510R Red Green Red Purple 37 E. coli 260364H Tr. Red 38 E. coli 260406Y Red Green Red Purple 39 E. coli 260492J Red Green Red Purple 40 E. coli 260486L Red Green Red Purple 41 E. coli 260485D Red Green Red Purple 42 E. coli 260479Q Red Green Red Purple 42 E. coli 260506E Red Green Red Purple 44 E. coli 260478N Tr. Red 45 E. coli 260463D Red Green Red Purple 46 E. coli 260396Z Red Green Red Purple 47 E. coli 260370C Red Green Red Purple 48 E. coli 260262P Red Green Red Purple 49 E. coli 260280Z Red Green Red Purple 50 E. coli 260375E Red Green Red Purple 51 E. coli 260400N 52 E. coli 260404W Red Green Red Purple 53 E. coli 260401Q Red Green Red Purple 54 E. coli 260402H Red Green Red Purple 55 E. coli 260411Z Red Green Red Purple 56 E. coli 260407F Red Green Red Purple 57 E. coli 260408C Red Green Red Purple 58 E. coli 260509W Red Green Red Purple 59 E. coli 260433H Red Green Red Purple 60 E. coli 260432E Red Green Red Purple 61 E. coli 260431N Red Green Red Purple 62 E. coli 260428T Red Green Red Purple 63 E. coli 260426F Red Green Red Purple 64 E. coli 260425A Red Green Red Purple 65 E. coli 260483R 66 E. coli 260494B Red Green Red Purple 67 E. coli 260495R Purple 68 E. coli 260439C Red Green Red Purple 69 E. coli 260438F Red Green Red Purple 70 E. coli 260437A Red Tr. Red 71 E. coli 260412Q Red Green Red Purple 72 E. coli 260497D Red Green Red Purple 73 E. coli 260416W Red Purple 74 E. coli 260417P Red Green Red Purple 75 E. coli 260422Y Red Green Red Purple 76 E. coli 260406A Red 77 E. coli 260405P Red Green Red Purple 78 E. coli 260399E Red Green Red Purple 79 E. coli 260435W Red Green Red Purple 80 E. coli 260434Y Red Green Red Purple 81 E. coli 2603121P Tr. Red 82 E. coli 260310H Red Green Red Purple 83 E. coli 260436P Red Green Red Purple 84 E. coli 260398Q Red Green Red Purple 85 E. coli 260345F Red Green Red Purple 86 E. coli 260390B 87 E. coli 260414H Red Green Red Purple 88 E. coli 260415Y Red Green Red Purple 89 E. coli 260313A Tr. Red 90 E. coli 260316T 91 E. coli 260424P Red Green Red Purple 92 E. coli 260348K Red Green Red Purple 97 E. coli 260354W Red Green Red Purple 98 E. coli 260333P 99 E. coli 260330Y Red Green Red Purple 100 E. coli 260327V Red Green Red Purple 101 E. cloacae 260329R Tr. Green 102 E. cloacae NCTC 11936 Tr. Green 103 E. faecium NCTC 7171 Green 104 E. faecalis NCTC 775 Green 105 E. coli O157 non- toxigenic Red, Green, Black or Purple mean strong colour; +/means less colour than strong; Tr means a trace of colour, less than +/; means no colour.

(18) The results of table 5 (lines 1-100) are summarised below (Table 6) for the 100 E. coli strains after 18 h incubation;

(19) TABLE-US-00006 TABLE 6 Negative Positive Substrate/Medium strains strains % Sensitivity CPS ID 3 7 93 93 DHN--D-glucuronide CHA salt 18 82 82 12a broth Rose--D-glucuronide CHA salt 19 81 81 X -D-glucuronide CHA salt 21 79 79

(20) The commercial medium, CPS ID3, was the most sensitive with 93/100 of E. coli strains giving red colonies. The excellent performance of this medium was to be expected, as it most probably contains inducers of -D-glucuronidase activity and/or optimal conditions for the expression of this enzyme. That not all strains were detected by this medium is understandable, as a small percentage of all E. coli strains is negative for -D-glucuronidase. Surprisingly, the next most sensitive medium was DHN--D-glucuronide (12a) [purple solutions] 82/100 strains. The novel substrate showed higher sensitivity than Rose--D-glucuronide [red colonies] (81/100 strains) when it was used in the simple Columbia agar medium. Considering that Rose--D-glucuronide is the same substrate as employed in the CPS ID3 medium, it shows how those skilled in the art can develop a medium to increase the sensitivity of the substrate when challenged with many different strains of microorganisms. X--D-glucuronide [green colonies] gave the lowest sensitivity (79/100 strains) in the simple agar medium, yet this substrate is currently very extensively used in commercial media to detect E. coli. More surprising still, DHN--D-glucuronide (12a) visualised one strain (E. coli 260495R) that was not detected by CPS ID3 or by the other two media containing the indoxyl glucuronides. In addition to the 100 strains of E. coli, all four media were tested with four other stains of Enterobacteriaceae known to be -D-glucuronidase-negative, as well as one -D-glucuronidase-negative strain of E. coli (E. coli 0157 non-toxigenic) (Table 5, lines 101-105). All five of these strains were negative on all the media, thus showing 100% specificity for -D-glucuronidase-producing E. coli over these other organisms.

Example 5Combination of DNH--D-Glucuronide and ONPG to Simulate a Dual Chromogenic Systems for Distinguishing Galactosidase and Glucuronidase Positive/Negative Microorganisms

(21) Because many E. coli produce both -D-glucuronidase and -D-galactosidase, DHN--D-glucuronide (12a) was tested as in the broth medium described above but in the presence of ONPG (at a concentration of 1.5 g/L). This was done to see if the colour produced by the iron complex could mask the yellow of o-nitrophenol. This would be essential to successfully visualise any E. coli in a dual-chromogenic (or possibly multi-chromogenic) system. It was found that E. coli expressing both -D-glucuronidase and -D-galactosidase now gave purple-brown solutions with DHN--D-glucuronide (12a). The colour of these solutions of mixed chromogens could be very readily distinguished with the unaided eye from the yellow colour of those strains, such as E. cloacae, that produced -D-galactosidase only. Similarly, a combination of DHN--D-glucuronide (12a) with ONPG was able to detect the -D-glucuronidase activity of Shigella sonnei, an important pathogen that is generally positive for both -D-glucuronidase and -D-galactosidase. The above results clearly demonstrate the potential of DHN--D-glucuronide (12) to be used in a liquid medium to detect E. coli, either on its own or in combination with other substrates (e.g. with ONPG). Although the strains tested were of clinical origin, it will be appreciated that DHN--D-glucuronide (12) of the present invention may be used to screen samples from food, environmental sources and water. Moreover, those skilled in the art of developing such media could add other ingredients, such as specific enzyme or growth inducers, enzyme or growth inhibitors or other metabolic regulators to ensure the optimum performance of the substrates within the media. Inducers of -D-glucuronidase in an enzymatic method for detecting E. coli have been disclosed [H. Nelis, U.S. Pat. No. 5,861,270, (1999)] and a commercially produced enzyme inducer cocktail has been used in a test for the enumeration of E. coli in drinking water [S. O. Van Poucke and H. J. Nelis, J. Appl. Microbiol., 89, 390-396, (2000)]. In the method of Monget et al for identifying E. coli from biological samples [D. Monget et al, U.S. Pat. No. 8,334,112 (2012)] glucuronate and methyl--glucuronide are cited as the preferred inducers of -glucuronidase. DHN--D-glucuronide-6-methyl ester (13) was also hydrolysed by -D-glucuronidase-positive strains, but the colour was weaker than with (12a) or (12b).

Example 6Nitrated DHN-Substrate

(22) Having established the utility of DHN as a useful core molecule as a base for chromogenic enzyme substrates, we sought to address the issue of diffusion of the core molecule that would limit its application in or on solid plate media, such as agar media. A possible way to reduce the solubility of a core molecule is to increase its molecular weight or size. Nitration of the fully protected DHN--D-ribofuranoside (1) introduced a nitro group at the 1-position of the DHN nucleus. The method is described in detail below. Deprotection afforded 1-nitro-DHN--D-ribofuranoside (19), and this was tested on agar plates with FAC in exactly the same manner as the unsubstituted compound (2). Unfortunately, the 1-nitro-DHN-iron complex still diffused extensively. However, the colour of this chelate was more distinctly red than the DHN-iron complex, so compounds of this type may be preferred over the unsubstituted-DHN derivatives depending on the requirements.

Example 7Halogenated DHN-Substrates

(23) Halogenation of DHN was then explored. Bromination of DHN was carried out by the method of Zincke and Fries [T. Zincke and K. Fries, Annalen, 334, 365, (1904)]. Using a ratio of 4 mol of molecular bromine to 1 mol of DHN in acetic acid as described by these authors and as detailed below gave the anticipated 1,4-dibromo-DHN in good yield. By increasing the amount of bromine to 8 mol, (again as described by Zincke and Fries), the expected 1,4,6,7-tetrabromo-DHN (20) was also obtained in good yield. Both these brominated derivatives were glycosylated as the 3-D-ribofuranosides, after which the substrates were separately incorporated into agar plates containing FAC for microbiological evaluation using a range of organisms previously tried with the unsubstituted DHN-glycosides. The results with both 1,4-dibromo-DHN--D-ribofuranoside and 1,4,6,7-tetrabromo-DHN--D-ribofuranoside were equally disappointing; both gave a very strong background colouration to the whole plate making it impractical to see any positive reactions.

(24) In the publication of Zincke and Fries, the authors reported that treatment of 1,4,6,7-tetrabromo-DHN (20) with tin (II) chloride led to the removal of the bromines in the 1 and 4 positions. Their work was repeated and the expected 6,7-dibromo-DHN (21) was obtained smoothly and in good yield (68%). 6,7-Dibromo-DHN (21) is isomeric with the 1,4-dibromo compound which had proven unsuccessful as an aglycone for the detection of microorganisms. Notwithstanding this latter fact, we proceeded to evaluate the properties of 6,7-dibromo-DHN (17) as an aglycone for artificial chromogenic enzyme substrates. Firstly we found that this molecule was efficiently converted into 6,7-dibromo-DHN--D-ribofuranoside (23). 6,7-Dibromo-DHN--D-ribofuranoside (23) was incorporated into Columbia agar plates containing FAC (500 mg/L) at a substrate concentration of 300 mg/L. The plates were made as described for DHN -D-galactopyranoside (8), except that the substrate was dissolved in DMSO (200 L). After inoculation and incubation for 18 h at 37 C. in air, 6,7-dibromo-DHN--D-ribofuranoside (23) produced largely discrete red-brown or maroon colonies upon hydrolysis by a number of different Gram-negative bacteria (Table 7). There was little or no diffusion of the colour into the surrounding medium and the background colouration of the plates was minimal. The growth of Gram-positive bacteria was mainly supressed by this substrate.

(25) TABLE-US-00007 TABLE 7 6,7-Dibromo- 6.7-Dibromo- 6,7-Dibromo- 6,7-Dibromo- DHN--D- DHN--D- DHN--D- DHN--D- glucuronide ribofuranoside 23 galactopyranoside 25 glucopyranoside 27 CHA salt 29 Strains A Colour Colour Colour Colour 1 Escherichia coli NCTC 10418 ++ Tr. ++ 2 Serratia marcescens NCTC 10211 ++ + ++ 3 Pseudomonas aeruginosa NCTC 10662 + 4 Burkholderia cepacia 1222 +/ +/ + 5 Yersinia enterocolitica NCTC 11176 6 Salmonella typhimurium NCTC 74 ++ 7 Citrobacter freundii NCTC 9750 or 46262 ++ + + 8 Morganella morganii 462403 (wild) ++ 9 Enterobacter cloacae NCTC 11936 ++ 10 Providencia rettgeri NCTC 7475 ++ 11 Bacillus subtilis NCTC 9372 + 12 Enterococcus faecails NCTC 775 Tr. Tr. 13 Enterococcus faecium NCTC 7171 +/ +/ 14 Staphylococcus epidermidis NCTC 11047 15 Staphylococcus aureus NCTC 6571 Tr. 16 MRSA NCTC 11939 Tr. 17 Steptococcus pyogenes NCTC 8306 18 Listeria monocytogenes NCTC 11994 +/ +/ 19 Candida albicans ATCC 90028 20 Candida glabrata NCPF 3943

Example 8Further Halogenated DHN-Glycosides

(26) Additional glycosides based on 6,7-dibromo-DHN were also produced to show the generality of this aspect of the invention (Table 7). The synthetic methods are described in detail in the section below. The -D-galactopyranoside (25), the -D-glucopyranoside (27) and the -D-glucuronide CHA salt (29) all gave non-diffuse colonies on agar plates. Gram-negative species grew well but the growth of some Gram-positive organisms was adversely affected by the substrates. These results showed that the 6,7-dibromo-DHN (21) is a suitable chromogenic core molecule for substrates incorporated into agar plates for the detection of Gram-negative organisms. Therefore, from a single inexpensive core molecule nucleus, i.e., DHN, we have succeeded in producing workable chromogenic enzyme substrates that are suitable for inclusion in either liquid or solid microbiological growth media. It will be appreciated by those skilled in the art that media falling between the two extremes of liquid and solid may be chosen depending on the specific application.

Example 9Substrates Derived from DHN-6-Sulfonic Acid

(27) DHN-6-sulfonic acid is also commercially available, most commonly as its sodium salt. As this is an unsymmetrical molecule, mono-glycosylation (or mono-derivatisation) of the aromatic ring hydroxyl groups is capable of yielding two different products. Prior to the present invention, glycosides of DHN-6-sulfonic acid were unknown. We succeeded in coupling acetobromogalactose to DHN-6-sulfonic acid by means of a phase-transfer reaction with tetrabutylammonium bromide as the catalyst. This gave a mixture of the protected di-galactoside and a protected mono-galactoside (30). The mono-galactoside (30) was isolated by column chromatography. Deprotection with sodium methoxide gave a DHN-6-sulfonic acid--D-galactopyranoside sodium salt (31). When tested in Columbia agar plates containing FAC (500 mg/L) at a substrate concentration of 300 mg/L it produced purple to maroon colonies after hydrolysis (Table 8). The colour was slightly different to that obtained by DHN--D-galactopyranoside (8) under the same conditions, but the colour was equally diffuse on agar plates.

(28) TABLE-US-00008 TABLE 8 DHN-6-sulfonic acid--D- DHN-6-sufonic galactopyranoside acid-phosphate sodium salt 31 trisodium salt 32 Organism Colour Colour 1 Escherichia coli NCTC 10418 + 2 Klebsiella pneumoniae NCTC 9528 Tr. 3 Providencia rettgeri NCTC 7475 + Tr. 4 Enterobacter cloacae NCTC 11936 + 5 Serratia marcescens NCTC 10211 +/ 6 Salmonella typhimurium NCTC 74 Tr. 7 Pseudomonas aeruginosa NCTC 10662 8 Yersinia enterocolitica NCTC 11176 9 Burkholderia cepacia NCTC 10931 10 Acinetobacter baumannii NCTC 19606 11 Steptococcus pyogenes NCTC 8306 12 Staphylococcus aureus (MRSA) NCTC 11939 Tr. 13 Staphylococcus aureus NCTC 6571 Tr. 14 Staphylococcus epidermidis NCTC 11047 15 Listeria monocytogenes NCTC 11994 16 Enterococcus faecium NCTC 7171 17 Enterococcus faecalis NCTC 775 18 Bacillus subtilis NCTC 9372 19 Candida albicans ATCC 90028 20 Candida glabrata NCPF 3943

(29) However, the DHN-6-sulfonic acid--D-galactopyranoside sodium salt (31) has the benefit of being very soluble in aqueous media. It readily dissolved in water at ambient temperature (30 mg of substrate dissolved in 1 mL DI water) without the need to add any polar solvents. This is a practical advantage over other types of artificial chromogenic enzyme substrates because the polar solvents normally employed to aid solution, like DMSO, exhibit toxicity to microorganisms. DHN-6-sulfonic acid-phosphate trisodium salt (32) was also prepared and tested on agar plates at a concentration of 300 mg/L with FAC (500 mg/L) (Table 8). As with compound 31, it was very soluble water and its real value is as a substrate in liquid media.

(30) Synthetic Methods

(31) Materials

(32) The glycosyl donors were all prepared by literature procedures. All other reagents and solvents were purchased from Sigma-Aldrich (Gillingham, UK), Alfa Aesar (Heysham, UK) or Univar (Widnes, UK) except where stated differently. Flash column chromatography was performed on silica gel C.sub.60 (Fluorochem, Derbyshire, UK). TLC was carried out using pre-coated silica plates (0.2 mm, UV.sub.254). These were developed using UV fluorescence at 254 nm and 366 nm followed by spraying with H.sub.2SO.sub.4/MeOH and heating. Mixed solvent compositions are reported as volumetric ratios. NMR spectra were recorded on a 270 MHz Joel NMR spectrometer (at 270 MHz for .sup.1H and 68 MHz for .sup.13C) or NMR spectra were recorded on a 400 MHz Joel NMR spectrometer (at 400 MHz for .sup.1H and 100 MHz for .sup.13C). All chemical shifts are quoted in ppm relative to TMS. Optical rotations were measured on an Optical Activity AA10 polarimeter. Melting points were determined with an Electrothermal A19200 apparatus and are uncorrected. All melting points are quoted to the nearest 0.5 C. High Resolution Mass Spectroscopy (HRMS) data were obtained using the EPSRC mass spectrometry service centre (Swansea, UK).

Synthesis 1 (Reference). DHN-2,3,5-tri-O-acetyl--D-ribofuranoside (1)

(33) ##STR00004##

(34) DHN (14.9 g) was stirred in dichloromethane (DCM) (200 mL). BF.sub.3.etherate (3 mL) was added to the mixture followed by 2,3,5-tri-O-acetyl-D-ribofuranosyl-trichloroacetimidate [I. Chiu-Machado et al, J. Carb. Chem., 14, 551, (1995)] (13 g) in DCM (100 mL). After approx. 5 minutes the reaction mixture was poured into sat. aq. NaHCO.sub.3 solution (300 mL) and DCM (200 mL). The DCM layer was separated and washed with sat. aq. NaHCO.sub.3 solution (4500 mL). TLC showed the reaction mixture still contained a large amount of unreacted DHN, therefore it was washed with sat. sodium carbonate (2500 mL). TLC then showed no remaining free DHN. The DCM layer was washed with water (500 mL) before being dried over MgSO.sub.4 and concentrated under reduced pressure to produce an amber foam. The isolated foam was triturated in MeOH (50 mL) and the resultant white solid harvested by filtration to give compound 1 (6.24 g, 54%). m.p. 142-144 C. [].sub.D.sup.22 69 (c 0.99 in acetone). HRMS (ESI) for C.sub.21H.sub.26O.sub.9N [M+NH.sub.4].sup.+: m/z calcd 436.1602; measured: 436.1609.

Synthesis 2. DHN--D-ribofuranoside (2)

(35) ##STR00005##

(36) Compound 1 (2 g) was suspended in MeOH (6 mL), NaOMe solution in MeOH (2.17 M, 2.0 mL) was added and the reaction mixture was left at +4 C. overnight. TLC showed complete deprotection. The solution was neutralised using AcOH (0.5 mL) and the solution concentrated under reduced pressure to give a white foam. The white foam was dissolved in IMS (15 mL) and left at +4 C. overnight to crystallise. Filtration isolated compound 2 as a white solid (542 mg, 39%). m.p. 181-182 C. [].sub.D.sup.22 143 (c 0.55 in acetone/water 1:1 v/v). HRMS (ESI) for C.sub.15H.sub.16O.sub.6Na [M+Na].sup.+: m/z calcd 315.0839; measured: 315.0844.

Synthesis 3 (Reference). DHN-2,3,4,6-tetra-O-acetyl--D-glucopyranoside (3)

(37) ##STR00006##

(38) DHN (80.6 g, 503 mmol) and acetobromoglucose (172.6 g) were stirred in acetone (1.1 L). A solution of NaOH (20 g) in DI water (400 mL) was added in one portion. The clear orange solution was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure until all the acetone had distilled off whereupon a gum formed in the residue. The aqueous solution was decanted off from the cream coloured gum. The gum was dissolved in DCM (1 L) and washed with sat. NaHCO.sub.3 (41 L) and DI water (21 L) before being dried (MgSO.sub.4) and concentrated under reduced pressure to give a yellow oil. The yellow oil was triturated in MeOH (100 mL) to give a solid that was collected by filtration. The obtained solid (63 g) was recrystallised from boiling MeOH (1 L) using charcoal (20 g) to give compound 3 (37.17 g, 18%) as a white fluffy solid. m.p. 155-156 C., [].sub.D.sup.22 24 (c 0.6 in acetone). HRMS (ESI) for C.sub.24H.sub.30O.sub.11N [M+NH.sub.4].sup.+: m/z calcd 481.1453; measured: 481.1448. The .sup.1H-NMR spectral data were consistent with that found in the literature.

Synthesis 4. DHN--D-glucopyranoside (4)

(39) ##STR00007##

(40) Compound 3 (1.5 g) was suspended in MeOH (4.5 mL) and deprotected by the method used to make compound 2. This afforded compound 4 as a white solid (930 mg, 94%). m.p. >210 C. decomp, [].sub.D.sup.22 100 (c 1.01 in water). HRMS (ESI) for C.sub.16H.sub.22O.sub.7N [M+NH.sub.4].sup.+: m/z calcd 340.1391; measured: 340.1397. The .sup.1H-NMR spectral data were consistent with that found in the literature.

Synthesis 5. (Reference) DHN-2,3,4,6-tetra-O-acetyl--D-glucopyranoside (5)

(41) ##STR00008##

(42) In a 500 mL round bottom flask, a mixture of DHN (21.8 g), HgBr.sub.2 (17.3 g) and HgCN.sub.2 (12.3 g) were stirred together in MeCN (250 mL) with 3 molecular sieves (10 g) for 10 min. Acetobromoglucose (56 g) was added and the mixture stirred at room temperature overnight. TLC then showed no remaining acetobromoglucose. The reaction mixture was filtered through Celite, washing through with DCM (250 mL). The filtrate was washed with sat. NaHCO.sub.3 (4300 mL) and DI water (2300 mL) before being dried (MgSO.sub.4) and concentrated under reduced pressure to give a pale brown foaming oil (73.92 g). The oil was purified by flash chromatography using C.sub.60 silica gel (1 kg), eluting with toluene/acetone 10:1 v/v, collecting fractions of 200 mL. Fractions 6-20 were concentrated under reduced pressure to produce an orange oil (30.71 g). The orange oil was triturated in IMS (50 mL) and the resultant solid (compound (3)) was collected by filtration and discarded. The filtrate from the obtained solid was concentrated under reduced pressure to give a yellow oil which was triturated in IMS (150 mL) and left at +4 C. overnight. The resultant pale yellow solid was collected by filtration (6.45 g). Recrystallisation from IMS (30 mL) using charcoal (2 g) gave compound 5 as an off-white solid (2.77 g). [].sub.D.sup.26 +202 (c 0.42 in CHCl.sub.3). The filtrate from the recrystallization was concentrated under reduced pressure to afford a second crop of compound 5 (1.9 g, 2.8%) [].sub.D.sup.26 +213 (c 0.5 in CHCl.sub.3), m.p. 130-130.5 C. HRMS (ESI) for C.sub.24H.sub.30O.sub.11N [M+NH.sub.4].sup.+: m/z calcd 508.1813; measured: 508.1815.

Synthesis 6. DHN--D-glucopyranoside (6)

(43) ##STR00009##

(44) Compound 5 (1 g) was suspended in MeOH (3 mL) and deprotected by the method used in Synthesis 2 to make compound 2 to give compound 6 as a white fluffy solid (105 mg, 16%). m.p. 164-166 C., [].sub.D.sup.26 +244 (c 0.25 in water). HRMS (ESI) for C.sub.16H.sub.22O.sub.7N [M+NH.sub.4].sup.+: m/z calcd 340.1391; measured: 340.1394.

Synthesis 7. DHN-2,3,4,6-tetra-O-acetyl--D-galactopyranoside (7)

(45) ##STR00010##

(46) This compound was prepared from DHN (80.6 g) and acetobromogalactose (172.6 g) by the method used in Synthesis 3 to make compound 3. This gave compound 7 (31.5 g, 15%) as a white fluffy solid. m.p. 83-84 C., [].sub.D.sup.22 +6 (c 1 in acetone). HRMS (ESI) for C.sub.24H.sub.30O.sub.11N [M+NH.sub.4].sup.+: m/z calcd 508.1813; measured: 508.1805.

Synthesis 8. DHN--D-galactopyranoside (8)

(47) ##STR00011##

(48) Compound 7 (1.5 g) was suspended in MeOH (4.5 mL) and deprotected by the method used to make compound 2 (Synthesis 2) to afford compound 8 (385 mg, 39%) as an off-white solid. m.p. >230 C. decomp, [].sub.D.sup.22 87 (c 0.62 in water). HRMS (ESI) for C.sub.16H.sub.22O.sub.7N [M+NH.sub.4].sup.+: m/z calcd 340.1391; measured: 340.1397.

Synthesis 9 (Reference). DHN-2,3,4,6-tetra-O-acetyl--D-galactopyranoside (9)

(49) ##STR00012##

(50) Compound 9 was prepared from DHN and acetobromogalactose by the method used to make the analogous glucopyranoside 5 (Synthesis 5). HRMS (ESI) for C.sub.24H.sub.30O.sub.11N [M+NH.sub.4].sup.+: m/z calcd 508.1813; measured: 508.1814.

Synthesis 10. DHN--D-galactopyranoside (10)

(51) ##STR00013##

(52) Compound 9 (5 g) was suspended in MeOH (15 mL) and NaOMe solution in MeOH (2.17 M, 1.5 mL) was added. After overnight reaction mixture was concentrated under reduced pressure to give a brown foam. The obtained foam was purified by flash chromatography using C.sub.60 silica gel (220 g), eluting with DCM/MeOH 15:1 v/v, collecting fractions of 50 mL. Fractions 53-72 were combined and concentrated under reduced pressure to give compound 10 as a pink foaming solid (1.2 g, 37.5%). [].sub.D.sup.23 +196 (c 0.23 in water).

Synthesis 11 (Reference). DHN-2,3,4,-tri-O-acetyl--D-glucuronide-6-methyl ester (11)

(53) ##STR00014##

(54) A mixture of DHN (34.4 g) and 1,2,3,4-tetra-O-acetyl--D-glucuronide-6-methyl ester (MTAG) [G. N. Bollenback et al, J. Am. Chem. Soc., 77, 3310, (1955)] (40 g) was heated in an oil bath to 120 C. on a rotary evaporator under reduced pressure until a homogeneous melt was obtained. PTSA (150 mg) in 1:1 v/v AcOH/Ac.sub.2O (1 mL) was added and the mixture stirred at 120 C. on a rotary evaporator under reduced pressure for 1 hour. TLC showed some remaining MTAG, therefore PTSA (150 mg) in 1:1 v/v AcOH/Ac.sub.2O (1 mL) was added and the mixture stirred at 120 C. under reduced pressure for a further 30 min. TLC then showed no remaining MTAG. The dark oil was allowed to cool to room temperature overnight before being dissolved in DCM (300 mL). The solution was washed with sat. NaHCO.sub.3 (450 mL), DI water (500 mL) and brine (500 mL) before being dried (MgSO.sub.4) and concentrated under reduced pressure to give a brown foaming oil (59.1 g). The foam was purified by flash chromatography using C.sub.60 silica gel (1 Kg), eluting with toluene/acetone 10:1 v/v, collecting fractions of 200 mL. Fractions 19-26 were combined and concentrated under reduced pressure to produce a red solid (29.66 g). The red solid was triturated in IMS (150 mL) and left at +4 C. overnight to complete crystallisation. The resultant pale yellow fluffy solid was collected by filtration to give compound 11 (12.6 g, 25%). m.p. 191-192 C., [].sub.D.sup.23 26 (c 0.5 in CHCl.sub.3. HRMS (ESI) for C.sub.23H.sub.28O.sub.11N [M+NH.sub.4].sup.+: m/z calcd 494.1657; measured: 494.1646.

Synthesis 12a. DHN--D-glucuronide CHA Salt (12a)

(55) ##STR00015##

(56) Compound 11 (6.1 g) was dissolved in acetone (75 mL). A solution of NaOH (2.81 g) in DI water (37.5 mL) was added. The mixture was stirred at room temperature for 2 hours. TLC showed no remaining protected material. The solution was passed down an AmberliteIR120 H.sup.+ ion exchange resin column (50 g). The eluent containing the product was basified using cyclohexylamine (5 mL). A white precipitate formed. The mixture was left at +4 C. overnight. The white fluffy solid was collected by filtration, washing with DI water then acetone to give compound 12a as a white fluffy solid (2.8 g, 53%). m.p. 223-224 C., [].sub.D.sup.19 96 (c 0.5 in water). HRMS (ESI) for O.sub.16H.sub.16O.sub.8 [M+H].sup.+: m/z calcd 335.0772; measured: 335.0767.

Synthesis 12b. DHN--D-glucuronide sodium Salt (12b)

(57) ##STR00016##

(58) Compound 11 (1.56 g) was dissolved in acetone (21 mL). A solution of NaOH (0.446 g) in DI water (1 mL) was added. The mixture was stirred at room temperature overnight. TLC showed no remaining protected material. A brown precipitate had formed in the solution. This solid was collected by filtration to give the desired compound 12b (1.16 g, 99%). m.p. 53-55 C., [].sub.D.sup.23 25 (c 0.995 in water).

Synthesis 13. DHN--D-glucuronide-6-methyl ester (13)

(59) ##STR00017##

(60) Compound 11 (1.0 g) was suspended in MeOH (3 mL) and NaOMe solution in MeOH (2.17 M, 0.6 mL) was added. The solid slowly dissolved and a cream precipitate began to form. The mixture was neutralised to pH 6-7 using AcOH (0.2 mL). The solid dissolved giving an orange solution which was concentrated under reduced pressure to give compound 13 as an orange foam (849 mg) which appeared to contain about 15% inorganic salt. [].sub.D.sup.19 100 (c 0.1 in water). HRMS (ESI) for C.sub.17H.sub.22O.sub.8N [M+NH.sub.4].sup.+: m/z calcd 368.1340; measured: 368.1339.

Synthesis 14 (Reference). DHN-N-acetyl-3,4,6-tri-O-acetyl--D-glucosaminide (14)

(61) ##STR00018##

(62) In a 250 mL round bottom flask a mixture of acetochloroglucosamine (30 g) and DHN (12.92 g) were stirred in acetone (120 mL). K.sub.2CO.sub.3 (21 g) was added and the mixture was heated in a water bath for 15 minutes. The reaction mixture was poured into boiling water (800 mL) and the mixture was left at room temperature for 1 hour. The resultant solid was collected by filtration and washed with water (3800 mL) to give a brown powder (12.8 g). The obtained powder was dissolved in boiling ethyl acetate (300 mL), charcoal (5 g) was added and the reaction mixture was boiled for 10-15 minutes before being filtered through pre-washed (ethyl acetate) Celite. The clear, amber solution was concentrated under reduced pressure until crystallisation began. The resultant solid was harvested by filtration, washing with a little ethyl acetate to give compound 14 (5.5 g, 13.9%) as an off-white powder. m.p. 229-230 C., [].sub.D.sup.27 42 (c 0.5 in CHCl.sub.3). HRMS (ESI) for C.sub.24H.sub.27O.sub.10NNa [M+Na].sup.+: m/z calcd 512.1527; measured: 512.1526.

Synthesis 15. DHN-N-acetyl--D-glucosaminide (15)

(63) ##STR00019##

(64) Compound 14 from the previous stage (1.0 g) was suspended in MeOH (3 mL) and deprotected by the method used to make compound 2 (Synthesis 2). This gave compound 15 (330 mg, 44%) as a pale orange solid. m.p. 204-205 C., [].sub.D.sup.23 10 (c 1 in MeOH). HRMS (ESI) for C.sub.18H.sub.22O.sub.7N [M+H].sup.+: m/z calcd 364.1391; measured: 364.1388.

Synthesis 16. DHN-dicaprylate (16)

(65) ##STR00020##

(66) DHN (636 mg) was suspended in a stirred solution of octanoic acid (1.58 ml) and DCM (2 ml). Dicyclohexylcarbodiimide (DCCI) (2.2 ml) in DCM (1 ml) was added drop wise over 5 minutes. Following addition of the DCCI mixture, the DHN dissolved giving a pink coloured solution. After a few minutes of stirring, a white solid precipitated out of the solution. The reaction mixture was stirred overnight at room temperature. The white precipitate (urea) was removed by filtration and the filtrate was washed with 0.5 M NaOH solution (610 ml), 1% AcOH solution (110 ml) and finally DI water (210 ml). The organic layer was dried (MgSO.sub.4) before being concentrated under reduced pressure to a pale orange oil. The oil was purified by dissolving in cold hexane (15 ml) with added charcoal (100 mg). After filtration and removal of the solvent under vacuum, compound 16 was obtained as a white waxy oil (970 mg, 59.2%). .sup.1H NMR: (DMSO-d.sub.6) 7.78 (2H, m), 7.63 (2H, s), 7.46 (2H, m), 2.57 (4H, t, J 7.6 Hz), 1.76 (4H, q, J 7.4 Hz), 1.46-1.25 (16H, m), 0.89 (6H, m).

Synthesis 17. DHN-phosphate disodium Salt (17)

(67) ##STR00021##

(68) In a 100 mL round bottom flask MeCN (15.51 g), pyridine (6.96 g) and POCl.sub.3 (13.49 g) were stirred in an ice/acetone bath. DI water (1.00 g, 55.5 mmol) was added dropwise keeping the internal temperature less than 20 C. DHN (3.2 g, 19.97 mmol) was added and the mixture was stirred for 4 hours at 2 C. The reaction mixture was poured onto ice (50 g) and 10M NaOH (50 mL) was added. The mixture was concentrated under reduced pressure to a pink solid. Following trituration in MeOH, a pink solid (salt) formed and was discarded. The filtrate containing the product and unreacted DHN was concentrated under reduced pressure to dryness. The obtained solid was triturated in IMS and a pink solid was collected by filtration. This solid was recrystallized from boiling MeOH (100 mL) containing water (25 mL) using charcoal (1 g). The MeOH was removed under reduced pressure and replaced with IMS to induce crystallisation. The resultant pink solid was collected by filtration to give the desired product (1.23 g, 21.6%). m.p. >300 C. decomp

Synthesis 18. 1-Nitro-DHN-2,3,5-tri-O-acetyl--D-ribofuranoside (18)

(69) ##STR00022##

(70) In a 1 L 3-neck round bottom flask, a mixture of copper (II) nitrate (60 g) and DHN-2,3,5-tri-O-acetyl--D-ribofuranoside 1 (20.9 g) was stirred in DCM (500 mL). After rapid stirring at room temperature for 5 min. a very pale yellow colour developed. PTSA (2.0 g) was added and the solution darkened. The reaction mixture was stirred rapidly at room temperature for an additional 50 min. TLC showed a large amount of remaining starting material. More PTSA (3.0 g) was added and the reaction mixture was stirred rapidly for a further 5 hours. TLC then showed no remaining starting material. The reaction mixture was filtered to remove insoluble copper salts and the filtrate was concentrated under reduced pressure to a deep red foaming solid. The solid was purified by flash chromatography using C.sub.60 silica gel (1 Kg), eluting with toluene/acetone 15:1 v/v, collecting fractions of 200 mL. Fractions 3-7 were combined and concentrated under reduced pressure to a yellow powder (2.61 g). The yellow powder was dissolved in boiling IMS (50 mL) and the yellow solution left at +4 C. overnight to allow crystallisation. Collected by filtration to give compound 18 as a yellow powder (2.2 g, 9.5%). m.p. 151-152 C., [].sub.D.sup.23 88 (c 0.5 in CHCl.sub.3). HRMS (ESI) for C.sub.21H.sub.25O.sub.11N.sub.2 [M+NH.sub.4].sup.+: m/z calcd 481.1453; measured: 481.1448.

Synthesis 19. 1-Nitro-DHN--D-ribofuranoside (19)

(71) ##STR00023##

(72) Compound 18 (500 mg) was suspended in MeOH (1.5 mL) and NaOMe solution in MeOH (2.17 M, 2.5 mL) was added. After overnight reaction the gel-like mixture was concentrated under reduced pressure to a red solid, triturated in IMS (10 mL) and the mixture left at +4 C. overnight. The resultant gel-like product was collected by filtration and dried under vacuum over P.sub.2O.sub.5 to give compound 19 (137.5 mg, 38%) as a yellow solid. m.p. >170 C. decomp. [].sub.D.sup.22 73 (c 0.23 in water). HRMS (ESI) for C.sub.15H.sub.19O.sub.8N.sub.2 [M+NH.sub.4].sup.+: m/z calcd 355.1136; measured: 355.1140.

Synthesis 20 (Reference). 1,4,6,7-Tetrabromo-DHN (20)

(73) ##STR00024##

(74) This was made from DHN (18 g) and bromine (23.2 mL) by the method of Zincke and Fries [loc. cit.]. This produced compound 20 (36.19 g, 67%) as a very pale pink solid. m.p. 240-242 C.

Synthesis 21 (Reference). 6,7-Dibromo-DHN (21)

(75) ##STR00025##

(76) The title compound was made by treating 1,4,6,7-tetrabromo-DHN 20 (8 g) with tin (II) chloride (32 g) according to the conditions described by Zincke and Fries [loc. cit.]. This produced 21 (3.65 g, 68%) as a white powder, m.p. 211-213 C.

Synthesis 22 (Reference). 6,7-Dibromo-DHN-2,3,5-tri-O-acetyl--D-ribofuranoside (22)

(77) ##STR00026##

(78) In a 100 mL round bottom flask, 6,7-dibromo-DHN 21 (3 g), 1,2,3,5-tetra-O-acetyl--D-ribofuranose (3.3 g) and 3 mol. sieves (1 g) were stirred in DCM (30 mL) for 10 min. BF.sub.3.etherate (3 mL) was then added. After 15 min. a thick white precipitate had formed. The reaction mixture was poured into a mixture of DCM (200 mL) and sat. NaHCO.sub.3 (200 mL). The white solid dissolved. The organic layer was separated and washed with sat. NaHCO.sub.3 (2200 mL) and DI water (200 mL), dried (MgSO.sub.4) and concentrated under reduced pressure to give a white solid. The solid was triturated in IMS (50 mL) and the resultant solid collected by filtration to give compound 22 (4.42 g, 81%). m.p. 185-186 C., [].sub.D.sup.23 51 (c 1 in CHCl.sub.3). HRMS (ESI) for C.sub.21H.sub.24O.sub.9NBr.sub.2 [M+NH.sub.4].sup.+: m/z calcd 591.9812; measured: 591.9805.

Synthesis 23. 6,7-Dibromo-DHN--D-ribofuranoside (23)

(79) ##STR00027##

(80) Compound 22 (1.0 g) was suspended in MeOH (3 mL) and deprotected by the method used to make compound 2 (Synthesis 2) to give compound 23 (697 mg, 89%). m.p. decomp. 240 C., [].sub.D.sup.23 90 (c 0.5 in MeOH). HRMS (ESI) for C.sub.15H.sub.18O.sub.6NBr.sub.2 [M+H].sup.+: m/z calcd 465.9495; measured: 465.9495.

Synthesis 24 (Reference). 6,7-Dibromo-DHN-2,3,4,6-tetra-O-acetyl--D-galactopyranoside (24)

(81) ##STR00028##

(82) 6,7-Dibromo-DHN 21 (6 g) and acetobromogalactose (10.23 g) were stirred in acetone (1 mL). NaOH (900 mg) in DI water (20 mL) was added in one portion. The reaction mixture was stirred overnight at room temperature at which point TLC showed no remaining acetobromogalactose. The reaction mixture was poured into DCM (500 mL) and sat. NaHCO.sub.3 (500 mL). The DCM layer was separated and washed with sat. NaHCO.sub.3 (6500 mL) and DI water (500 mL), dried (MgSO.sub.4) and concentrated under reduced pressure to give a brown oil (12.37 g). The obtained oil was purified by flash chromatography using C.sub.60 silica gel (550 g) eluting with toluene/acetone 10:1 v/v and collecting fractions of 200 mL. Fractions 5-17 were combined and concentrated under reduced pressure to a green foaming oil (6.6 g). TLC showed that this oil contained the desired product compound 24 and unreacted 6,7-dibromo-DHN 21. The oil was dissolved in DCM (150 mL) and washed with 1M NaOH (150 mL) and DI water (150 mL) before being dried (MgSO.sub.4) and concentrated under reduced pressure to give compound 24 (4.6 g, 37%) as a pale green foaming solid, [].sub.D.sup.19 +20 (c 0.5 in acetone). HRMS (ESI) for C.sub.24H.sub.28O.sub.11NBr.sub.2 [M+NH.sub.4].sup.+: m/z calcd 664.0024; measured: 664.0023.

Synthesis 25. 6,7-Dibromo-DHN--D-galactopyranoside (25)

(83) ##STR00029##

(84) Compound 24 (4.35 g) was deprotected in a similar manner to that used to make compound 2 (Synthesis 2) except that the solution was neutralised with Amberlite IR120H.sup.+ resin. The powder was purified by recrystallization from MeOH/water 1:1 v/v (300 mL) to give compound 25 (1.89 g, 59%) as a white solid. m.p. 256-257 C., [].sub.D.sup.25 64 (c 0.25 in acetone/water 1:1 v/v). HRMS (ESI) for C.sub.16H.sub.16O.sub.7Br.sub.2 [M+H].sup.+: m/z calcd 476.9190; measured: 476.9203.

Synthesis 26 (Reference). 6,7-Dibromo-DHN-2,3,4,6-tetra-O-acetyl--D-glucopyranoside (26)

(85) ##STR00030##

(86) This was made from 6,7-Dibromo-DHN 21 (6 g) and acetobromoglucose (10.23 g) by essentially the same method used for the analogous galactopyranoside 24 (Synthesis 24). The title compound 26 was obtained as a white solid (3.16 g, 25%). m.p. 166-167 C., [].sub.D.sup.25 28 (c 0.5 in acetone). HRMS (ESI) for C.sub.24H.sub.28O.sub.11NBr.sub.2 [M+NH.sub.4].sup.+: m/z calcd 664.0024; measured: 664.0025.

Synthesis 27. 6,7-Dibromo-DHN--D-glucopyranoside (27)

(87) ##STR00031##

(88) Compound 26 (3 g) was suspended in MeOH (8 mL) and deprotected by the addition of NaOMe solution in MeOH (2.17 M, 3 mL). Work-up gave compound 27 (2.04 g, 92%) as a very pale green solid. m.p. decomp. >290 C., [].sub.D.sup.25 168 (c 0.25 in water). HRMS (ESI) for C.sub.16H.sub.15O.sub.7Br.sub.2 [M+H].sup.+: m/z calcd 476.9190; measured: 476.9186.

Synthesis 28 (Reference). 6,7-Dibromo-DHN-2,3,4-tri-O-acetyl -D-glucuronide-6-methyl ester (28)

(89) ##STR00032##

(90) -D-2,3,4-Tri-O-acetyl--D-glucuronyl-trichloroacetimidate-6-methyl ester (10 g) and 6,7-dibromo-DHN 21 (6 g) were stirred in DCM (100 mL). BF.sub.3.etherate (200 L) was added. The reaction mixture was stirred at room temperature for 30 min. before being poured into a mixture of DCM (500 mL) and sat. NaHCO.sub.3 (500 mL). The DCM layer was separated and washed with sat. NaHCO.sub.3 (4500 mL) and DI water (500 mL), dried (MgSO.sub.4) and concentrated under reduced pressure to give a pink solid. The obtained solid was triturated in IMS (100 mL) and left at +4 C. overnight. The resultant solid was harvested by filtration to give compound 28 (2.89 g, 24%) as a white solid. m.p. 176-177 C., [].sub.D.sup.25 16 (c 0.25 in acetone). HRMS (ESI) for C.sub.23H.sub.26O.sub.11NBr.sub.2 [M+NH.sub.4].sup.+: m/z calcd 649.9867; measured: 649.9868.

Synthesis 29. 6,7-Dibromo-DHN--glucuronide CHA Salt (29)

(91) ##STR00033##

(92) Compound 28 (2.5 g) was deprotected with NaOH (870 mg) in a mixture of DI water (11.5 mL) and acetone (23 mL). Work-up as for compound 12a with pro-rata quantities gave compound 29 as a white solid (2.16 g, 95%). m.p. 221-224 C., [].sub.D.sup.25 159 (c 0.25 in water). HRMS (ESI) for C.sub.16H.sub.13O.sub.8Br.sub.2 [M+H].sup.+: m/z calcd 490.8983; measured: 490.8976.

Synthesis 30 (Reference). DHN-6-sulfonic acid-2,3,4,6-tetra-O-acetyl--D-galactopyranoside sodium Salt (30)

(93) ##STR00034##

(94) DHN-6-sulfonic acid sodium salt (5 g) was dissolved in a solution of NaOH (850 mg) in DI water (150 mL). Acetobromogalactose (7.06 g), tetrabutylammonium bromide (6.75 g) and DCM (8.7 g) were added and the two-phase reaction mixture was stirred overnight at ambient temperature. TLC analysis showed no remaining acetobromogalactose. The reaction mixture was diluted with additional DCM (100 mL) and the organic layer was separated before being washed with DI water (300 mL), dried (MgSO.sub.4), and concentrated under reduced pressure to an oil (15.83 g). The isolated oil was purified by flash chromatography using C.sub.60 silica (600 g), eluting with DCM/MeOH. Fractions 7-20 were combined and concentrated under reduced pressure to an oil which was triturated in ethyl acetate (30 mL). The resultant white solid was collected by filtration to give protected mono-galactoside 30 (4.77 g, 49.68%). m.p. 131-133 C., [].sub.D.sup.23 +5 (c 0.592 in acetone). .sup.1H NMR (DMSO-d.sub.6): 9.73 (1H, s), 7.90 (1H, s), 7.55 (1H, dd), 7.45 (1H, m), 7.40 (1H, s), 7.16 (1H, s), 5.47 (1H, d), 5.32 (1H, broad s), 5.25 (2H, m), 4.43 (1H, m), 4.10 (1H, m) 2.13, 1.99, 1.96, 1.91 (43H, 4s). .sup.1H NMR spectroscopy confirmed the obtained solid was the mono-galactoside, although which of the two possible isomers could not be determined from the NMR data.

Synthesis 31. DHN-6-sulfonic acid--D-galactopyranoside sodium Salt (31)

(95) ##STR00035##

(96) Compound 30 (1 g) was suspended in MeOH (3 mL), 2.17 M NaOMe (0.5 mL) was added. Additional 2.17 M sodium methoxide (3 mL, 3.3 mmol) was added to aid crystallisation of the sodium salt. After complete deprotection (by TLC) the mixture was concentrated under reduced pressure and triturated in IMS (10 mL). The solid was collected by filtration and washed with a little IMS to afford compound 31 (462 mg, 62%). m.p. >2300 C. decomp, [].sub.D.sup.23 73 (c 0.26 in water). .sup.1H NMR (DMSO-d.sub.6): 7.45 (1H, s), 7.27 (1H, d), 7.09 (1H, s), 6.98 (1H, dd), 6.41 (1H, s), 4.55 (1H, d), 3.63 (1H, d), 3.52 (3H, m), 3.31 (2H, m). .sup.1H NMR spectroscopy confirmed the obtained solid was the mono-galactoside, although which of the two possible isomers could not be determined from the NMR data.

Synthesis 32. DHN-6-sulfonic acid-phosphate trisodium Salt (32)

(97) ##STR00036##

(98) In a 100 mL round bottom flask MeCN (15.51 g), pyridine (6.96 g) and POCl.sub.3 (13.49 g) were stirred in an ice/acetone bath. DI water (1.00 g, 55.5 mmol) was added dropwise keeping the internal temperature less than 20 C. DHN-6-sulfonic acid sodium salt (5.24 g) was added and the mixture was stirred for 4 hours at 2 C. The reaction mixture was poured onto ice (100 g) and 10M NaOH (35 mL) was added. The mixture was concentrated under reduced pressure to give an orange solid which was triturated in MeOH (200 mL). The resultant solid (33.4 g) was mainly salt and was discarded. The filtrate was concentrated under reduced pressure to an orange solid (4.44 g). This was dissolved in hot DI water (20 mL), filtered, and IMS (200 mL) was added to the clear brown filtrate. A brown oil separated out of the mixture. After decanting the IMS/water solution from the oil, a pale cream precipitate formed in this solution. It was removed by filtration, and this filtrate was now recombined with the previously isolated brown oil. Concentration of this mixture under reduced pressure gave a brown foam. This foam was heated with MeOH (75 mL) in an ultrasonic bath; a fine hygroscopic brown solid was removed by filtration and the clear brown filtrate was concentrated under reduced pressure to a solid. Trituration of the solid in IMS (30 mL) gave a mixture of isomers of title compounds (32) as an off-white solid (1.17 g, 14.7%). m.p. >400 C.