CA1096376A - Process for preparing nitroaromatic glycosides - Google Patents

Abstract

E.I. du Pont de Nemours and Company ABSTRACT

A process for preparing nitroaromatic glycosides is described. The process comprises contacting an acetylated gly-coside of maltotetraose, maltopentaose or maltohexaose with a phenol, nitrating the resulting product to place a NO2 group on the aromatic moiety and deacetylating the nitrated product. The nitroaromated glycosides are useful as standard substrates for the assay of .alpha.-amylase.

Description

~637~i ;~ BACKGROUND OF THE INVENTION

Field of the Invention This invention relates to processes for preparing nitroaromatic glycosides, particularly nitroaromatic derivatives of maltotetraose, maltopentaose, and maltohexaose, useful as standard substrates for the assay of ~-amylase in serum and other biological liquids.
Relation to the Prior Art 1. U. S. Patent 3,879,263, issued April 22, 1975 discloses the determination of the ~-amylase content of bio-logical samples by adding maltotetraose or maltopentaose to the sample at constant temperature and pH. The process allows rapid determination of ~-amylase, and can be used to differ-entiate between saliva ~-amylase and pancreas ~-amylase. The latter produces glucose, whereas the former does not. The glucose produced may be estimated spectrophotometrically, e.g~, by nicotinamide-adenine dinucleotide (reduced form) (NADH) absorption at 340 nm. Because this assay depends upon glucose, a glucose detecting reaction is necessary. Furthermore, if glucose is present in the sample, it must either be removed or compensated for. The compounds of the present invention differ from this in that 4-nitrophenol is released as the substance which can then be related to ~-amylase. This makes the assay independent of the glucose detecting step.

2. A. P. Jansen and P. G. A. B. Wydeveld, Nature, 182, 525 (1958) postulate that ~ nitrophenyl)maltoside could be a substrate for an amylase assay. However, this `~ paper shows that the authors never identified the active agent responsible for their observations. They reported:

..
~

637~ii (1) Incubation of human urine or saliva samples With ~ -(p-nitrophenyl)maltoside at 37 for 16 hr produced 4-nitrophenol, identified spectrophotometrically by mixing the hydrolyzate with 0.02N sodium hydroxide. (2) ~he hydro-lysis was inhibited by protein precipitants such as 10%
trichloroacetic acid and 0.5~ silver nitrate. (3) me hydrolysis was pH-dependent, being most e~fective at pH
5.9-7Ø mey state that this was evidence for "the possible existence of an unidentified carbohydrase~ -(4-Nitrophenyl) maltoside is not believed to be useful for an amylase assay because the cleavage of this compound by ~ -amylase iS extremely slow.
SUMMARY OF THE INVENTION
According to the present invention there is provided a procese for preparing ~ and ~ nitroaromatic glycosides comprising:
(a) contacting an acetylated glycoside of the formula:

CH2Ac CH2Ac CH2Ac AcO ~ ~ L ~ ~ H

OAC OAc n OAC

wherein AC iS an acetyl group, and n is an ; integer of 2, 3 or 4, ` 20 with a phenol selected from the group consisting of OH

~ Y y, an ~

wherein X and Y are individually H, N~2, halogen, alkyl of 1 to 4 carbon atoms, ORt or C02Rt

- 3 ~

6:~76 where R' is an alkyl group of 1 to 6 carbon atoms, with the proviso that only one of X
and Y is NO2, in the presence of a catalyst at a temperature in the range of about 80-120C;
(b) nitrating the product of (a) by contacting said product with:
(i) nitric acid contained in a mixture of acetic acid and sulfuric acid, or (ii) a nitronium compound selected from nitronium tetrafluoroborate, nitronium hexafluoro-phosphate and nitronium, trifluoromethane-sulfonate contained in dichloromethane, - chloroform or 1, 2-dichloroethane; and (c) deacetylating the product of (b) by contacting said product with:
(i) a catalytic amount of an alkali metal lower alkoxide contained in the corresponding ~` alcohol, or (ii) a solution of anhydrous ammonia or HCl in methanol.
DETAILED DESCRIPTION OF THE INVENTION
.~
The nitroaromatic glycosides of the invention are derived from a series of oligomers and polymers of glucose which are ~[1-~ 4] linked. This series of glucosides has the general formula indicated below. The Gn nomenclature is convenient shorthand for n ~[1- ~ 4] linked glucose units.

OH ~ OH ~ ~ OH 1 ~ OH

HO ~ OH H ~ - o_ ~ ~
OH OH OH OH

~-D-Glucose (Gl) n = 0 maltose (G2) n = 1 maltotriose (G 3 ) n = 2 maltotetraose (G4) n = 3 maltopentaose (Gs) n = 4 maltohexaose (G6) n ~ 2000 amylose For brevity in discussing the invention, trivial names and shorthand abbreviations will be used as shown in Table I.
It is understood that these refer to the systematic names identified in Table I, the rules for which are given in "Naming and Indexing of Chemical Substances for Chemical Abstracts During the Ninth Collective Period (1972-1976)", Chemical Abstracts Service, Columbus, Ohio (1973).

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The glucose oligomer starting materials, i.e., maltotetraose, maltopentaose and maltohexaose can be pre-pared by the procedure described by either R.L. Whistler and coworkers in J. Amer. Chem. Soc., 76, 1671 (1954), 77, 1017, 5761 (1955) or Thomas John Pankratz in United States Patent 4 039 383 which issued 1977 August 2. The preferred nitroaromatic glycosides of G4 and G5 can be made from pure G4 and G5, prepared by chromatography of the hydrolyzate of amylose as described by W. Pigman, "The Carbohydrates", Academic Press, New York, 1957, pages 678-9. Scheme I
illustrates a procedure for preparing the preferred com-pounds of the invention. The details of each step will be described hereinafter.
SCHEME I: SYNTHETIC SCHEME

CH2H CH20Ac ~ Ac20, NaOAc > ~ Ac GnO OH ~ GnO OAc Gn=G3 or G4 Gn=G3(AC)lolor G4(AC)13 C6H50H, ZnC12 \ ~ ~ .
CH20Ac CIH20Ac n OAc ~ NO ~ NITRATION G ~ AcOC6Hs Gn=G3(Ac)lo or G4(Ac)13 Gn=G3(Ac)lo or G4(AC)13 NaOMe MeOH
CH2OH ~ ~
~O\ ~
n ~ ~ NO2 Gn ~ MALTO-N-OSIDE RESID~E~[1-4 Ac = CH3CO
Gn=G3 or G4 G3(Ac)lo=DECAACETYLMALTOTRIOSYL
(Ac)l3 = TRIDECAACETYLMALTo-TETRAOSYL

t ~

i;37~
.
The details of each step of the procedure for preparing the compounds of the invention are as follows:
ACETYLATIO~ REACTION:
me acetylation of glucose oligomers with a mixture of acetic anhydride and anhydrous sodium acetate at elevated temperatures is known (W.J. Whelan and P.J.P. Roberts, J. Chem. Soc., 1928 (1953), W.J. Whelan, J.M. Bailey, and P.J.P. Roberts, J. Chem. SOC A, 1923 (1953), A. Thompson and M.L. Wolfrom, J. Amer. Chem. Soc., ~, 3612 (1954), M.L. Wolfrom, L.W. Georges, A. Thompson, and I.L. Miller, J. Amer. Chem. Soc., 71, 2875 (1949) to give the completely acetylated derivatives with the substituent on the anomeric carbon being mostly in the ~ -configuration. On the other hand replacement of the anhydrous sodium acetate with zinc chloride or acids fa~ors the production of compounds where the sub-stituent on the anomeric carbon is in the ~ -configuration.
In either case a mixture of the ~ and ~ isomers is obtained.
(N. Pigman, loc. cit. p. 140-142). me acetylation is con-ducted in acetic anhudride as the solvent and reactant, the amount of acetic anhydride being from 5 to 50 times the weight of G4 or G5. The preferred amount is 5 to 10 times the weight of G4 or G5 in order to provide sufficient reagent, to keep the reactants in solution, and to permit isolation of the pro-duct when the reaction mixture is poured into water. The amount of anhydrous sndium acetate used may be from 1 to 10 molar equivalents per molar equivalent of G4 or ~5, preferably from 5 to 6 molar equivalents.
The temperature of the reaction can be from 100C
to 140C, the reflux temperature of acetic anhydride, and is - ..~t..

'G
.

preferably between 110C and 120C. Below 100C, the acetylation proceeds very slowly and incompletely, and above 140 (e.g., in a pressure vessel), the reaction is very vigorous and gives a dark-colored product.
The reaction time can be from 1 to 6 hours, and is preferably about 2 hours. Prolonged heating at elevated temperatures also gives a dark-colored product. The onset of the reaction is signalled by the reaction mixture becoming a homogeneous solution and by the reaction becoming mildly exothermic.
The completely acetylated product is isolated by pouring the cooled reaction mixture into ice-water from five to twenty times the volume of acetic anhydride taken, stirring the mixture vigorously for a few minutes, and then allowing it to stand at 0-5C for at least 24 hours. The solid product, whose crystallization can be improved and accelerated by seeding, if desired, is filtered, air-dried, and recrystallized from a suitable solvent such as ethanol or methanol.
The identity of this and other intermediate products is established by the usual spectral properties and analyses.
The stereochemistry of the anomeric carbon is readily estab-lished by proton nuclear magnetic resonance spectroscopy (lH nmr), especially at high frequencies such as 220 MHz, and by optical rotation studies, if desired. In the nmr of aldo-pyranose acetates, an anomeric proton in the ~-configuration (Hl~) can be distinguished from an anomeric proton in the ~-configuration (Hl~). In the nmr of aldopyranose acetates, an anomeric proton in the ~-configuration (Hl~) has a chemical shift (~) near 5.75 ppm downfield of internal tetramethylsilane, and the signal appears as a doublet with an axial-axial coupling , , .

3 ~'G

constant (J) of 7-9 Hz. An anomeric proton in the ~-configuration (Hl~) gives a signal 0.2-0.65 ppm downfield of this position, also as a doublet with an axial-equatorial or equatorial-equatorial coupling constant of 3-4 Hz (See, for example, L. M. Jackman, "Applications of NMR Spectroscopy in Organic Chemistry", Pergamon Press (London), (1959), pp 86, 116).
ACETATE DISPLACEMENT REACTION:
The displacement of the anomeric acetate group in a completely acetylated sugar occurs more readily than that of the other acetate groups; this is a useful property for synthetic purposes because it permits preferential reaction at this position. In particular, if tetradecaacetylmalto-tetraoside or heptadecaacetylmaltopentaoside is stirred with ; phenol and anhydrous zinc chloride (ZnC12) at elevated tem-peratures, the anomeric ~-acetoxy group is replaced by an ~-phenoxy group. While at least one molar equivalent of phenol is required per molar equivalent of acetyl compound to satisfy the stoichiometry of the reaction, the reaction can be carried out with from 3 to 20 molar equivalents of phenol, preferably with 4 to 8 molar equivalents to provide sufficient material to form a homogeneous solution. The amount of zinc chloride can be from 0.25 to 5 molar equivalents per molar equivalent of acetyl compound, and is preferably in the range of 0.5 to 1.5 molar equivalents. This reaction can be carried out in the temperature range of from 80C to 120C, with tem-peratures in the range of 100C to 110C being preferred, for the same reasons given above in the first step of the process.
The reaction time can be from 0.25 to 6 hours, with 1 to 3 hours usually being sufficient. Zinc chloride dissolved in a ~~

G~6 mixture of acetic acid: acetic anhydride (e.g., a 95:5 volume mixture) is a useful modification of the above solvent and catalyst system for the introduction of the ~-phenoxy group.
The anhydrous zinc chloride catalyst may be replaced by acids such as _-toluenesulfonic acid and by other anhydrous covalent metal chlorides such as titanium (IV) chloride (TiC14), tin (IV) chloride (SnC14), and iron (III) chloride (FeC13).
The displacement reaction proceeds without solvent because the low melting point of phenol (43C) ensures that when excess phenol is taken to drive the reaction to com-pletion, the mixture remains as a homogeneous solution at the reaction temperature. Similar reactions are also expected to succeed when excess amounts of phenols which are liquid at the reaction temperature are used both as the solvent and reactant, for example, 2-cresol (mp 30C), 3-cresol (mp 11C),

4-cresol (mp 35C), 2-chlorophenol (mp 8C), 3-chlorophenol (mp 29C), 4-chlorophenol (mp 37C), 4-bromophenol (mp 64C), 2-nitrophenol (mp 45C), 2-methoxyphenol (guaiacol, mp 32C), 4-methoxyphenol (mp 53C), 2-methyl-5-isopropylphenol (carva-crol, mp 1C), 2-isopropyl-5-methylphenol (thymol, mp 51C), and methyl salicylate (mp -8C). With higher melting phenols, as well as those specified above, it is also possible to do the reaction in a solvent such as benzene (bp 80C), toluene (bp 110C) or heptane (bp 98C). With higher-melting phenols such as 3-nitrophenol (mp 96C), 4-nitrophenol (mp 114C), 2,4-dinitrophenol (mp 113C), l-naphthol (mp 94C), and 2-naphthol (mp 122C), the solvent method renders the mixture homogeneous and prevents charring of the acetyl derivative.
The solvent may also be one of the above-mentioned catalysts ~ "7~i which is a liquid at the reaction temperature, such as titanium (IV) chloride (bp 136C) or tin (IV) chloride (bp 114C). If desired, the product of the reaction with the phenol can be reacetylated by the procedure of the first step to protect any free hydroxyl groups which may have arisen by deacetylation side reactions during the introduction of the phenoxy group.
NITRATION REACTION:
While the direct use of 4-nitrophenol to prepare 4-nitrophenylglycosides has been described and may be used (T. D. Audichya, T. R. Ingle, and J. L. Bose, Indian J. Chem., 9, 315 (1971), A. P. Jansen and P. G. A. B. Wydeveld, loc.
cit.), the described process shown in Scheme II to ~-(4-nitrophenyl)tridecaacetylmaltotetraoside and ~-(4-nitro-phenyl)hexadecaacetyl maltopentaoside is preferred for ease of operation. The nitration can be accomplished either in a mixture of acetic and sulfuric acids with nitric acid, or in dichloromethane with a nitronium compound such as nitronium tetrafluoroborate (NO2 BF4 ), nitronium hexafluorophosphate 20 (NO2 PF6 ) or nitronium trifluoromethanesulfonate (NO2 CF3SO3 ).
These are all described in L. F. Fieser and M. Fieser, Reagents for Organic Synthesis 5, 477 (1975), Wiley-Interscience, New York. Nitronium tetrafluoroborate is preferred and will be used to describe this aspect of the procedure. In the first procedure, a solution of the phenyl acetylated glycoside in a mixture of sulfuric and acetic acids at 0C to 25C is treated with a 5 to 30-fold molar excess of 70% nitric acid dissolved in acetic acid. The preferred amount of nitric acid is from 10 to 20 molar equivalents per molar equivalent of acetyl derivative. The reaction temperature is between 0C

.'~i.;

6~7~

and 25C (preferably about 0C) to minimize further nitration of the aromatic ring and cleavage of ester and glycosidic linkages. While the reaction time can be from 1 to 10 hours (about 4 hours preferred), the nitration should be allowed to occur as completely as possible without the formation of the above-mentioned further products. The nitration of the aro-matic glycoside acetates produced from the phenols listed above for the displacement reaction follows the usual ortho-para substitution pattern with the para-position being favored unless it is blocked by another group, as in the 4-cresyl derivative. The amount of m _ substitution is negligible.
In the nitronium tetrafluoroborate procedure, the preferred reaction time is from 0.25 to 1 hour and the preferred reaction temperature is about 25C. The molar proportion of nitronium tetrafluoroborate to acetylated glycoside can be between 1 and 20:1, with 10 to 1 as the preferred ratio to ensure complete introduction of 1 nitro group. In addition to the preferred solvent dichloromethane, chloroform and 1,2-dichloroethane can be used. The nitronium tetrafluoroborate procedure is preferred for its ease of operation.
The resulting nitrated product of the first procedure can be isolated from the mixture of nitric, acetic and sul-furic acids by pouring the reaction mixture into water (usually about 5 to 20 times its volume) and either filtering the crude product, or extracting it with chloroform. In the nitronium tetrafluoroborate process, the dichloromethane solution is added to cold saturated sodium chloride solution, dried over sodium sulfate, and evaporated to leave the crude glycoside.

.~.

3~

DEACETYLATION REACTION:
The selective removal of 0-acetyl groups from an acetylated polyol derivative is preferably accomplished either with a catalytic amount of sodium methoxide (usually 0.01-0.1 molar equivalent) in methanol, or by a solution of anhydrous ammonia in methanol. In addition to the preferred sodium methoxide, other alkali metal lower alkoxides such as potas-sium methoxide, sodium and potassium ethoxide and potassium t-butoxide contained in the corresponding alcohol can be used.
These deacetylation reactions occur readily at temperatures of 0-25C within 12 to 24 hours. The deacetylated product is isolated by evaporation of the alcohol, freed of inorganic ions (if desired) by passage through an acidic ion-exchange column, and recrystailized from a suitable solvent such as methanol or ethanol. An alternative deacetylation procedure uses a 3%
solution of hydrogen chloride in methanol (L. F. and M. Fieser, "Reagents for Organic Syntheses", Wiley, N~Y., 1967, p. 11) at temperatures of 0-25C for periods of 4-24 hours. This is especially useful for dinitro compounds.
An alternative synthesis is to use one of the phenols to displace the halogen from tridecaacetylmaltotriosyl or hexadecaacetylmaltopentaosyl chloride or bromide of the following structure.
_ CH2OAc CH2OAc CH2OAc ~~ ~\1 I~\X, ~ OAc ~ -o J \ OAc ~ -o ~ OAc AcO
AcO AcO n AcO

n = 2 or 3 X = Cl or Br 3~7~

mese halides are prepared either by treatment of the completely acetylated oligosaccharide with anhydrous hydrogen halide, solutionæ of hydrogen halide in mixtures of acetic anhydride and acetic acid, solutions o~ aluminum chloride and phosphorus pentachloride or of titanium tetrachloride in chloroform, or from the oligosaccharide itselr by treatment with acetyl chloride (W. Pigman, loc. cit., p. 150-151).
The halogen atom is either displaced with phenol or a substituted phenol in the presence of a halogen acceptor such as silver (I) oxide (Ag20), silver (I) carbonate (Ag2C03), mercury (II) acetate (Hg(C~ C00)2), or with iron (III) chloride (Koenigs-Knorr reaction), or with the sodium or potassium salt o~ the phenol (W. Pigman, loc. cit., p. 194-198).
The rest of the synthesis is the same as described above.
The progress o~ any o~ the above reactions can be followed by thln-layer chromatography (TLC) on silica gel in a suitable solvent system, and by nmr spectroscopy.
The purity o~ the products of the reactions can be determined by high-per~ormance liquid chromatography (HPLC), by polarimetry, and by ultraviolet and high-~requency (220 MHz) nmr spectroscopy.
It has been found that the nitroaromatic glycosides o~ the invention are useful substrates ~or serum ~-amylase assay. The assay process is illustrated in Scheme II ~or the pre~erred compounds of the invention, ~ -(4-nitrophenyl)malto-tetraoside and ~-(4-nitrophenyl)maltopentaoside. Serum ~ -amylase converts these two compounds to a mixture of G2 or G3 and ~-(4-nltrophenyl)maltoside. The latter is then hydro-lyzed to glucose and 4-nitrophenol by ~ -maltase, treatment -~ r l~G37~
with dilute alkali produces the 4-nitrophenolate anion which is spectroscopically identifiable and distinguishable ~rom any unreacted glycoside, and which can be related to serum -amylase levels.

SCHEME II: SERUM ~ AMYLASE ASSAY

t ~ ~ R
OH OH n OH

~ MAX 290_305 nm R 4 02NC6H4 n=2 ~ -(4-NITROPHENYL)MALTOTETRAOSIDE
n=3 ~ -(4-NITROPHENYL)MALTOPENTAOSIDE

1 ~-AMYIASE

Ho~l ~t ~
OH OH n OH

R 4 02NC6H4 n=O ~-(4-NITROPHENYL)MALTOSIDE

i ~-MALTASE

4 or 5 Gl~ HO ~ No2 ~ OH--0~ N2 4-NITROPHENOLATE ANION ~ MAx410nm .;~.

37~

The nitroaromatic glycosides of the invention have the following advantages in the assay of serum ~-amylase. In U.S. 3,879,263, serum ~-amylase levels are related to the glucose produced from G4 or G5. Consequently, serum glucose must be removed chromatographically from the sample before assay, requiring the expenditure of sample preparation time and extra apparatus. By using compounds of the present inven-tion, serum ~-amylase levels are related to the nitrophenols produced from the nitroaromatic glycosides of G4, G5 or G6 which are independent of serum glucose levels. Not only does this also do away with the chromatography system for removing serum glucose, but it also simplifies the detection system by replacing the hexakinase-ATP-NADP unit with dilute alkali, (ATP-NADP is adenosine triphosphate and nicotinamide-adenine dinucleotide phosphate).
EMBODIMENTS OF THE INVENTION
The following illustrative examples demonstrate ways of carrying out the invention. All parts and percentages are by weight, and all temperatures are Centigrade unless other-wise stated. Proton nuclear magnetic resonance (lH nmr)chemical shifts are in parts per million from internal tetra-methylsilane in chloroform-d (CDC13) unless otherwise stated;
qualitative lH nmr results were obtained at 60 MHz and more accurate measurements were made at 220 MHz. Thin layer chromatograms (TLC) were run on silica gel using 250 ~m plates for analytical work and 2 mm plates for preparative work.
High performance liquid chromatograms (HPLC) were run on a Du Pont 830 instrument for analytical work, and on a Du Pont 841 instrument for preparative work.

.~

71~i (A) Preparation of Maltotetraose ~-Tetradecaacetate CH OH CH OH CH OH

NaOAc/Ac2O
HO O- ' O- ~
OH OH OH
_ _ 2 CH OAc CH OAc CH OAc O ~ ~ ~ OAc Ac O- J O- J
OAc OAc OAc A mixture of maltotetraose of analytically confirmed structure (10.0 g, 15.0 mmole), anhydrous sodium acetate (10.0 g, 0.15 mole) and acetic anhydride (50 ml) was stirred at 100 for 2 hours, then it was poured into 300 ml of ice water. After 48 hr at 5, the colorless crystalline mass was filtered and air-dried, yield 20.96 g of crude material.
This was recrystallized from methanol (40 ml), recovery 18.18 g (14.49 mmole, 96%) of crystalline maltotetraose ~-tetra-decaacetate in two crops of 2.98 g and 15.20 g. The first crop material had mp 124-126 and its structure was confirmed by: ~max (CHC13) 1750, 1370, 1230 and 1030 cm ; ~max (EtOH) 210 nm (~ 740); [~]D + 104 (_ 1.03 CHC13); lH nmr (220 MHz), ~ 5.76 (d J = 7) (Hl~), 5.43-5.25 (several groups of multiplets) 27H (OCH, OCH2), and 2.19-2.00 (series of sing-lets) 42H (COCH3);
Anal- Calcd- for C52H7035 C, 49-76; H~ 5-62;
Found: C, 49.08; 49.24; H, 5.76, 5.73 In several further experiments up to twice the above scale, the yield of product was from 72-77% after 1~9G3'7~

recrystallization, and the mp was from 122 to 128.
(B) Preparation of ~ - and ~ -Phenyltridecaacetylmalto-tetraosides CH20Ac OAc - CX20Ac ~ 0~ ~ 0\ ~ 0~ OAc /r~------~
Ack~o ~\~ ~f ~
OAc OAc 2 OAc CH20AC CH20Ac CH20Ac Ac~O ~ ~ ~Ac A mixture of maltotetraose ~ -tetradecaacetate ~rom part (A) (11.0 g, 8.77 mmole ), phenol (8.0 g, 85 mmole) and anhydrous zinc chloride (2.0 g, 14 mmole) was heated gently until it became fluid, and it was then stirred 10 mechanically at 100 for 3 hr. me mixture was diluted with water and benzene and separated. The benzene layer was extracted in turn with 3 x 50 ml of 5~ sodium hydroxide, 2 x 50 ml of saturated aqueous sodium chloride, dried, and evaporated to give 10.09 g (7.8 mmole, 89%) o~ crude phenyl derivatlve as a yellow crystalline solid. This material was purified by preparative thln layer and high performance liquid chromatography as ~ollows:
A total amount of 3 . o6 g of crude phenyl derivative was loaded onto 14 2-mm preparative TLC plates and developed 20 3 times with a mixture of 95:5 benzene:methanol. me Rf 0.13-0.27 band was extracted with chloroform and methanol to give 0.90 g (29~) of material which was recrystallized from ethanol, recovery 0.58 g of a mixture of ~- and ~ -phenyltridecaacetylmaltotetraosides. Analytical HPLC (polar _ 19 3~7~i silicone microshperes) showed product with retention time 8,99 min and a minor impurity (2.4~) at 8.23 min. The structure of the crystalline phenyltride~aacetylmaltotetra-oside was confirmed by ~max (CHC13) 1745, 1595, 1585, 1365, 1225, and 1030 cm 1, ~ max (EtOH) 273 nm (~ 800), 266 (960), 260 (730), 210 (7860),L~7 D5 -~ 132 (c 1.00 CHC13); H nmr (220 MHz), ~ 7.37-7-25 (m) 2H, 7-14-6-95 (m) 3H (C6H5)~ 5-oo-3-86 (m) 28H (OCH, OCH2), and 2.19-1.97 ppm (series of singlets), 39H (COCH3), Anal- Calcd- for C56H7234 C, 52-17; H~ ~-63;

Found: C, 51.44, 51.94, 51.60;
H, 5.35, 5.60, 5.39 A total amount of 4.25 g of the crude phenyl derivative was also purified by preparative HPLC on a 1 m x 23 mm Spherosil (44-50) column eluted with a mixture of 1:1 pentane:dioxane (containing 1.5~ water) to give 1.07 g (25~
recovery) of colorless crystalline phenyltridecaacetylmalto-tetraoside, mp 82-83, identical by spectral and chromatogra-phic data with the material purified by TLC. Analytical HPLC showed this sample to be 99.7~ pure, (C) Preparation of ~ - and ~ Phenyltridecaacetylmalto-tetraosides (Alternate Method) CH20Ac OAc _ CH20Ac ZnC12/
~ ~ ~ 0 OAc ~-----~ AcOH/
A ~ - _ _ ~ ~ OH

CH20Ac CH20Ac CH20AC

~ ~ ~ O ~ O~Ac ~ O
AcO ~ O - ~ O -Ac _ OAc 2 OAc Maltotetraose /~ -tetradecaacetate (2.75 g, 2.19 - 20 _ - ~., ;

37~;

mmole) and phenol (2.27 g, 24.2 mmole) were mixed in a 3-neck flask under nitrogen. Zinc chloride (0.55 g) dissolved in 2.0 ml of a mixture of 95:5 acetic acid:acetic anhydride was added, and the reaction mixture was slowly warmed. When the mixture became homogeneous (temperature ca. 45), the internal pressure was gradually lowered to 23 mm and the reaction mixture was stirred at 100 for 2.5 hr. The material was transferred to a separatory funnel using warm benzene (200 ml), and the cooled mixture was washed twice with 18~ aqueous sodium chloride, twice with 2.5% sodium hydroxide (50 ml) and twice with saturated sodium chloride (30 ml). The organic layer was dried over sodium sulfate, evaporated, and the residue treated with anhydrous sodium acetate (3.0 g) and acetic anhydride (15 ml). The resulting mixture was heated at 120 for 1.0 hr, cooled, and treated with ice-water (200 ml). This procedure reacetylates any unprotected hydroxyl groups which arise from the phenol procedure. The solid which formed upon standing at 0 was filtered and dried, yield 2.85 g. This material was chromatographed on silica gel (Mallinckrodt SilicAR~ CC-7). Elution with a mixture of 97:3 benzene:methanol gave a total of 2.07 g (71%) of pure phenyl-tridecaacetylmaltotetraoside after recrystallization from ethanol. This material had mp 112-117 and its structure was confirmed by: [~]25 + 135 (c 1.0 CHC13), lH nmr (220 MHz) ~ 7.38-6.96 (m) 5H (C6H5), 5.77 (dd J = 9.5 Hz) (Hl~), and

5.61 (d J = 4 Hz) (Hl~) lH, 5.45-3.82 (series of multiplets) 27H (OCH, OCH2), and 2,24-1.95 ppm (series of singlets) 39H
(COCH3); integration was consistent with a 75:25 mixture of ~:~ phenyltridecaacetylmaltotetraosides.

(D) Preparation of ~- and ~ -(4-Nitrophenyl)tridecaacetyl-maltotetraoside (Nitric Acid Method) CH20AC CH20Ac CH20hc ~ ~ ~
~ OAc ~ K OAc ~ ~ OAc ~ ~ HN03, H2S04 AcO ~ O J ~ ~ O_ 1 ~ \~____~ HOAc OAc _ OAc 2 OAc _ CH20AC CH20Ac CH20Ac ~O~ ~}N2 OAc OAc 2 OAc A mixture of acetic acid (2.0 ml), acetic anhydride (1.0 ml) and preparative TLC-purlfied ~- and ~-phenyltrideca-acetylmaltotetraoside mixture from Part (B) (0.38 g, 0.29 mmole) was cooled to 0 and treated with a mixture of sulfu-ric acid (1.0 g) and acetic acid (2.0 g), and then with a mixture of 70% nitric acid (0.5 g) and acetic acid (1.0 ml).
The mixture was stirred at 25 for 4 hr, poured into ice water (25 ml), and filtered to give 0.40 g of almost color-less solid. mis sample was approximately 50~0 4-nitrophenyl derivative as indicated by ~max (EtOH) 290 nm (~ 3100).
me reaction was repeated on a larger scale (5 g of crude phenyltrldecaacetylmaltotetraoside) to give 4.35 g (84~) of crude nitro compound which was chromatographed on silica gel (200 g) (Mallinckrodt SilicA ~ Ce-7). me product (2.27 g recovered), eluted with 4~ methanol in benzene was also approximately 50~ pure.
A 2.6 g sample of crude precipitated nitro compound was purified by preparative HPLC separation using a 2 m x 2.3 cm Spherosil (44-50) column eluted with a mixture of 1:1 pentane dioxan (containing 1.5~ water). The fractions were examined by TLC and W, and from the middle cuts, 0.2557 g.

-l~G376 (0.19 mole~ 8%) of pure ~ - and ~5 -(4-nitrophenyl)tridecaacetyl-maltotetraoside was obtained. After recrystallization from ethanol (5 ml) this material had mp 112-114. The structure of the material was confirmed by ~max (CHC13) 1745, 1580, 1520~
1420, 1370 and 1220 cm 1, ~max (EtOH) 290 nm ( 7180); ~d~ 25 + 156 (c 0.525 CHC13); lH nmr (220 ~z), ~8.26 (d J = 8) and 7.26 (d J = 8) 4H (C6H4)~ 5.75 (m) lH (Hl~ and Hl~), 5.52-3.95 (series of multiplets) 27H (OCH, OCH2), and 2.25-2.01 ppm (series of singlets) 39H (COCH3), Anal. Calcd. for C56~1N036: , 5 Found: C, 49.95; H, 5.54 (E) ~ - and ~ -(4-Nitrophenyl)tridecaacetylmaltotetraoside (Nitronium Tetrafluoroborate Method) CHzOAc CH OAc CH OAc Ac ~ ~ ~ ~ C~2C12 OAc OAc 2 OAc CH20Ac CH20Ac CH20Ac A ~ O ~ O ~ ~ 2 OAc OAc 2 OAc A slurry of nitronium tetrafluoroborate (1.91 g, 14. 5 mmole) in dry methylene chloride ( 8 ml) was treated with a solution o~ phenyltridecaacetylmaltotetraoside from part (C) (1.24 g, o.g6 mmole) in dichloromethane (10 ml), and the resulting mixture stirred for 20 min under an argon atmosphere. The mixture was added to ice water (60 ml), separated, and washed with cold saturated aqueous sodium chloride. me organic portion was dried over magnesium sulfate and evaporated under reduced pressure to provide a yellow glassy solid (1:30 g) of ~ - and ~5-(4-nitro-phenul) tridecaacetylmaltotetraoside product. Half of this . ., .3~i product was recrystallized from ethanol to provide a cream-colored solid/ Q.48 (75~), mp 116-119. me structure of the product was confirmed by: lH nmr (220 MHz), 7.75 (AA'BB') 4H (C6H4), 5-75 (d J = 4Hz) (Hl~) and 5.81 - 3.86 (series of multiplets) 28H (Hl~, OCH, OCH2), and 2.25 - 1.95 (series of singlets) 39H (COCH3); ~max (E
C~D5 + 152 (c 0.5 CHC13).
(F) Preparation of ~ - and ~5-(4-Nitrophenyl)maltotetraoside CH20Ac CH20Ac H20Ac K~ ~ 1 1~~ ¦ ~ ~ ~} N0 AcO ~ - ~ I ~
OAc OAc 2 OAc HO~OH ~H ~ )H N~2 A mixture of column-chromatographed 4-(nitrophen~l)-tridecaacetylmaltotetraoside from part (D) (6.76 g, 5.1 mmole), methanol (50 ml) and sodium methoxide (50 mg) was left at 25 overnight. me mixture was filtered to remove a small amount of yellow solid, and the filtrate was evaporated to leave 3.17 g (4.02 mmole, 79%) of yellow solid. Three grams of this sample were purified by preparative TLC on 11 plates by development in a mixture of 3:3.5:0.5 chloroform:acetic acid:water. Material (0.5027 g) from the Rf 0.16-0.29 band was a mixture o~ ~ - and ~ -(4-nitrophenyl)maltotetraoside, approximately 50,~ pure by W. The product was a yellow solid with mp 68-70 and itsstructure was confirmed by: ~max (Nu~ol) 3300 and 1020 cm ; lmax (H20) 35 nm ( 374); ~ D
+ 104 (c 1.06 H20); lH nmr (220 MHz), ~ 8.25 (d J = 10) and 7.35 (d J = 10) (C6H4), 7.91 (t J = 7) and 7.70 (t J = 7) ' 3'7~i (other aromatic material), 5.88 - 5.79 (m) (Hl~ ~nd Hl~ ), 5.30 (m) (OCHO), 4.98 (s) (HOD), and 4.02 - 3.48 ppm (m) (OCH,OCH2).
(G) ~4-Nitrophenyl)maltotetraoside (Alternate Method) CH20AC CH20Ac fH20.Ac NaOCH
0 ~ ~ 0 ~ 0 ~ CH30H
~ OAc ~ ~ Ac ~ K OAc ~ ~ O ~ N02 -AcO ~ ~ J ~ / O - J
OAc OAc 2 OAc CH20H ICH20H ~CH20H

~ >I k~'>~ ~ ~ N2 HO ~ o ~ ~ ~ o_ ~

A mixture of (4-nitrophenyl)tridecaacetylmalto-tetraoside fron part (E) (250 mg, 0.19 mmole) in methanol (2.5 ml) Was treated with a solution of sodium methoxide in methanol (3.3 ml o~ 7.8 x 10 3 M solution) and was stirred for 17 hr at room temperature. Solvent was removed under reduced pressure and the residue, dissolved in a minimal volume o~ methanol (2 ml), was added dropwise to ether (40 ml). The resulting yellow powder was separated, dissolved in methanol, and passed through a 2.5 x 20 cm column of Sephade ~ LH-20 using methanol as eluant. mere was obtained 134 mg (90%) o~ solid product which was re-chromatographed to provide a centre cut (90 mg, 60~) having its structure by ~max (H20) 33 nm (~ 6350), 217 ( 5000);
H nmr (220 MHz), ~ (CD30D) 7.77 (AA~BB~, JAB = 9.5Hz) 4H

(C6H4)~ 5-69 (d J = 4Hz) lH (Hl~), 5.27 = 5.09 (m) 3H (OCHO), 4.87 (s) 13H (HOD), 4.20 - 4.05 (m) (minor impurity), and 4.01 - 3.40 ppm (m) (OCH~ OCH2).

; ~

37~

(H) (4-Nitrophenyl)maltotetraoside (Alternate Method) The procedure described in part (G) was used to prepare a crude sample of (4-nitrophenyl)maltotetraoside, ~max (H2O) 302 nm (~ 5900), 217 (4650); [a]D + 158 (_ 1.1, CH30H). A portion of this sample was purified by fractiona-tion on a Waters Associates ~ Bondapak/Carbohydrate column eluting with an 87:13 mixture of acetonitrile:water. The purified major component of the sample obtained after freeze drying exhibited ~max (H2O) 302 nm ( 9425), 220 (6500).

EXA~PLE 2 (A) Preparation of Maltopentaose ~-Heptadecaacetate CH o~ CH OH CH OH

HO ~ O- ~ O ~ NaOAc/Ac2O

CH2OAc CH2OAc CH2OAc I ~ O\l ~ O\ ~ O\OAc Ac ~ O- ~ O- ~
OAc OAc OAc A mixture of maltopentaose (2.00 g, 2.41 mmole), sodium acetate (2.0 g, 30 mmole), and acetic anhydride (20 ml) was heated at 130 for 2.0 hr. The cooled mixture was added to ice (75 g) and stored at 0. The resulting solid was pulverized and filtered to give an off-white solid (3.35 g, 90%) which was reerystallized from ethanol (40 ml) to provide 20 maltopentaose ~-heptadecaaeetate, 3.16 g (85%), mp 117-120;
whose strueture was eonfirmed by: [a]25 + 122 (c 0.7, CHC13); lH nmr (60 MHz) ~ 5.85 - 3.78 (m) 35H (OCH, OCH2), and 2.34 - 1.83 ppm (series of singlets) 51H (COCH3); lH nmr (220 MHz) ~ 5.76 ppm (d J = 8 Hz), lH (Hla);

.~
, Found: C, 49.11J 49.28j H 5.54~ 5.71 (B) Preparation of ~- and ~-Phenylhexadecaacetylmatlo-pentaoside ~ 2 C~20Ac ICH20Ac Ac ~ O ~ ~ ~ ~ H

OAc _ OAc OAc CH20Ac CH20Ac -- C~I20AC
~ ~>1 I K~ > o{~>
AcO ~ _ ~ O- ~
OAc OAc 3 Ac A mixture of maltopentaose ~-heptadecaacetate from part (A) (3.00 g, 1.95 mmole), phenol (1.95 g~ 20.8 mmole) and anhydrous zinc chloride (o.46 g, 3.38 mmole) was heated at 100 for 3 hr under an argon atmosphere. The cooled mixture was taken up in dichloromethane (200 ml) and washed with water (2 x 30 ml), 5~ sodium hydroxide (2 x 25 ml), and brine ( 2 x 25 ml). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to give a yellow powder which was treated with sodium acetate (3.0 g) and acetic anhydride (15 ml) and then heated at 125 for 2.0 hr in order to reaacetylate any free hydroxyl groups.
The cooled mixture was added to ice ( 125 g) and stored at 5. me resulting ~ - and ~ -phenylhexadecaacetylmaltopenta-oside solid product was pulverized, separated and air-dried to give 2.45 g of an off-white powder. This material was chromatographed on silica gel (150 g) eluting with a mixture of 97:3 benzene:methanol. The H nmr (220 MHz) of the residue ; from a center cut exhibited S 7.38 - 7.27 (m) 2H and 7.17 - 6.97 i;
~.. `

1~6376 (m) 3H (C6H5), 5-77 (dd ~ = 9.5 Hz), 5.63 (d J - 4 Hz) (Hl~), 5.82 - 3.83 (series of multiplets) 35H (OCH, OCH2) and 2.23 - 1.93 ppm (series of singlets) 48H (CH3CO).
Pure phenylhexadecaacetylmaltopentaoside was ob-tained from the above solid product by HPLC using columns packed with > 40,u silica gel spheres with a mobile phase of 1% methanol, 49% pentane, and 50% dichloromethane. Integra-tion of the 220 MHz lH nmr spectrum showed that this material consisted of ca. 75% ~-phenylhexadecaacetylyaltopentaoside and 25% ~ -phenylhexadecaacetylmaltopentaoside.

(C) Preparation of ~- and ~ -phenylhexadecaacetylmalto-pentaoside (Alternate Method) CH20AC CH20A CH20Ac ZnC12S
/~ O \ /~ O ~ ~o \ Ac 2 k~ ~Y ~ ~ ~ \ + ~ ~ OH
l\ OAc ~ . \ OAc ~ \ OAc ~ \ '' /
AcO `¦ ~ ~ ~ - ~ ~
OAc ~ OAc _ 3 OAc CH20Ac CH20AC CH20Ac ~o~ 1<~ ~I ~ ~ <~>

AcO ~ o_ J -~ ~ O - J
OAc I _ OAc 3 OAc A mixture of maltopentaose ~-heptadecaacetate from part (A) (3.00 g, 1.95 mmole) and phenol (1.95 g, 20.8 mmole) in a 3-neck flask under nitrogen was treated with a solution o~ zinc chloride (0.49 g) in 2.0 ml of a mixture of 95:5 acetic acid:acetic anhydride, and the reaction mixture was slowly warmed. When the reaction mixture became homogeneous, the internal pressure was gradually reduced to 23 mm and the mixture was stirred at 100 for 2.5 hr. me residue was treated with ben~ene (200 ml) and 18% aqueous sodium chloride solution. The organic phase was washed twice with 2.5~ sodium h~droxide (50 ml) and twice with saturated sodium chloride (50 ml). The organic layer was dried over sodium sulfate, evaporated, and the residue treated with anhydrous sodium acetate (3.0 g) and acetic anhydride (15 ml). The mixture was heated at 120 for 1.0 hr, cooled, and treated with ice water (200 ml). me solid product which formed upon standing at 0~ was pulverized, filtered~ and air dried to give 2.97 g of an of~-white powder. This material was chromatographed on a silica gel column eluting with a mixture of 97:3 benzene:
methanol. There was obtained a total o~ 2.00 g (65~) of pure - and ~-phenylhexadecaacetylmaltopentaoside, mp 119-125;
~?D5 + 137 (c 1.0, chloro~orm);
Anal. Calcd for C68H8842 C~ 5 Found: C, 51.62, 51.13; H, 5.70, 5.47 (D) Preparation of (4-Nitrophenyl)hexadecaacetylmalto-pentaoside _ _ CH20Ac T20Ac CH20Ac ~ ! ~ OAc ~ 02+~F4 a~ ~12 AcO O_ J ~ Q ~ ~ ) OAc _ OAc_ 3 bAc ~H20Ac CH20Ac CH20Ac ~c ~ ~ o _ ~ ~ 2 OAc OAc 3 OAc A slurry of nitronium tetrafluoroborate (0.85 g,

6.4 mmole) in dry dichloromethane (10 ml) under an argon atmosphere was treated with a solution of phenylhexadecaacetyl-maltopentaoside from part (C) (1.00 g, o.64 mmole) in dichloro-methane (12 ml). The resulting mixture was stirred for 20 min at room temperature~ poured into stirred, cold, saturated sodium chloride solution (70 ml), and the separated organic ~ 7 ~

layer dried over magnesium sulfate. Evaporation of solvent under reduced pressure gave a yellow solid (1.05 g) which was recrystallized from ethanol (30 ml) to provide o.87 g of an off-white powder. The product structure was confirmed b~ ~ maHxH 290 nm ( 5550), 210 nm (~ 7750); lH nmr spectrum (220 MHz) ~ 7.75 (AA'BBt, JAB = 9 5 Hz) 4~ (C6H4)~ 5-75 (d J = 4 Hz) (Hl~), 5.78 - 3.85 (series of multiplets) (OCH, OCH2), and 2.22 - 1.93 ppm (serie~ of singlets) 48H (CE3CO).
Two recrystallizations of the crude (4-nitrophenyl)-hexadecaacetylmaltopentaoside from ethanol gave a sample with ~ max (CH30H) 290 nm (~ 6650) and 212 (7700).
(E) Preparation of (4-Nitrophenyl)maltopentaoside CH OAc CH OAc CH OAc ~ ~ >1 1~ ~ ~O<~N0 AcO 0- ~ ~ O _ I ~
OAc _ OAC_ 3 OAC

~0 ~ 0 ~ 0 ~ ~~ ~ ~2 _ A solution of (4-nitrophenyl)hexadecaacetylmalto-pentaoside from part (D) (430 mgJ 0.27 ~mole) in methanol (5 ml) was treated with a solution of sodium methoxide in methanol (4.7 ml of 7.8 x 10 4 M), and then stirred for 17.5 hr at room temperature. The solution was concentrated under a stream of dry nitrogen, and the stirred residue was treated with ether (40 ml) to precipitate a solid which was filtered and air-dried to provide a slightly yellow powder (247 mg~ 98~ max (H20) 33 nm (~ 5980)J 215 (5530). HPLC
(Waters Associates /u Bondapak/Carbohydrate, 80~ acetonitrile/
water, 254 nm W detector) of the product showed two equally ; - 3 . ~, 37~

intense peaks with retention times 10.9 and 12.3 min corresponding to 4-(nitrophenyl)maltopentaoside and an unidentified material.

In Table II, when the phenol of Examples 1 and 2 is replaced as the reactant by the substituted phenols listed in Column A, the ~-(substituted aryl)polyacetyl glycosides listed in Column B are obtained by the procedures of Examples l(B) or (C) or 2(B) or (C). Nitration of the products of Column B
by the procedures of Examples l(D) or (E) or 2(D) gives the ~-(substituted nitroaryl)polyacetyl glycosides listed in Column C. Deacetylation of the products of Column C by the procedures of Examples l(F), (G) or (H), or 2(E) gives the ~-(substituted nitroaryl)glycosides listed in Column D.
Maltohexaose ~-eicosaacetate (G6Ac20) can be prepared by acetylation of maltohexaose according to the procedures of Examples l(A) and 2(A). Maltotetraose, malto-pentaose and maltohexaose are prepared as described earlier.
In Column A of Table II, the following starting materials are available commercially: 2-, 3-, or 4-methyl-phenol (o-, m-, and p-cresol), 2-, 3- and 4-chlorophenol, 2-fluorophenol, 3-bromophenol, 4-iodophenol, 2-, 3- and 4-nitrophenol, 4-methoxyphenol, 2-isopropyl-5-methylphenol (thymol), methyl salicylate, 3-ethylphenol, 4-t-butylphenol, 1- and 2-naphthol, 4-chloro-1-naphthol, and 4-hydroxybenzoic acid (for the preparation of hexyl 4-hydroxybenzoate). The following starting materials are all described in "Dictionary of Organic Compounds`', 4th Ed., edited by I. Heilborn, Oxford University Press (1965) on the pages indicated: 2-methyl-5-isopropylphenol (carvacrol, p. 568), 2-methoxyphenol(guaiacol,

7'~i p. 1549), and 3-methoxyphenol (p. 2857). Hexyl 4-hydroxy-benzoate can be made by the acid-catalyzed esterification of 4-hydroxybenzoic acid with l-hexanol using the procedure given for methyl salicylate by A. I. Vogel "Practical Organic Chemistry", 3rd Edition, Longmans Green, London, 1959, p. 782.

_ t` .

1~9~37G

TABLE II
cL-(Substituted Nitroaryl)Glycosides Example Column A Column B
3 2-CH3C6H40H G4(Ac)13(2-cH3c6H4) 4 3-CH3C6H40H G4(AC)13(3-cH3c6H4) 4-CH3C6H4oH G5(Ac)16(4-CH3c6H4) 6 2-ClC6H40H G5(Ac)16(2-clc6H4) 7 3-ClC6HLjOH G5(AC)16(3-clc6H4) . 8 4-ClC6H40H G4(Ac)13(4-Clc6H4) 9 2-FC6H40H G4(AC)13(2-Fc6H4) 3-BrC6H40H G5(Ac)16(3-BrC6H4) 11 4-IC6H40H G4(AC)13(4-Ic6H4) 12 2-02NC6H40H G4(AC)13(2-o2Nc6H4) ; 13 3-02NC6H40H G5(Ac)16(3-02NC6H4) 14 4-02NC6H40H G5(AC)16(4-o2Nc6H4) 2-CH30C6H40H G4(AC)13(2-cH3oc6H4) 16 3-CH30C6H40H G5(AC)16(3-cH3oc6H4) ; 17 4-CH30C6H40H G4(AC)13(4-cH3oc6H4) 18 2-cH3-5-(cH3)2cHc6H3oH G4(Ac)13(2-CH3-5-(CH3)2cHc6H3) 19 2-(cH3)2cH-5-cH3c6H3oH G5(AC)16(2-(cH3)2cH-5cH3c6H3) 2-CH302CC6H40H G4(Ac)13(2-cH3o2cc6H4) 21 3-C2H5C6H40H G5(Ac)16(3-C2H5c6H4) 22 4-t-C4HgC6H40H G4(AC)13(4-t-c4H9c6HL~) 23 OH G4(AC)13(l-c1oH7) 24 OH G4(AC)13(2-c1oH7) X~

~t3963~

~ABLE II (eontinued) Example _ Column A Column B
OH G4(AC)13(4-cl-l-c1oH7) Cl 26 4-C6H13C02C6H40H G4(Ac)13(4-C6H13c02c6H4) 27 3-CH3C6H40H G6(AC)19(3-cH3c6H4) 28 2-ClC6H40H G6(AC)19(2-clc6Hv) 29 3-02NC6H40H G6(Ae)19(3-02NC6H4) 2-CH30C6H40H G6(Ac)19(2-cH3oc6H4) 31 OH G6(AC)19(l-c1oH7) ~b 32 C6H50H G6(Ac)1s(C6Hs) Example Column C Column D
3 G4(Ae)13(2-CH3-4~o2Nc6H3) G4(2-CH3-4-02Nc6H3) 4 G4(Ae)13(3-CH3-4~02NC6H3) G4(3-CH3-4-02NC6H3) G5(Ae)16(4-CH3~2~02NC6H3) G5(4-CH3-2-02NC6H3) 6 G5(AC)16(2-cl-4-o2Nc6H3) G5(2-Cl-4-02NC6H3) 7 G5(AC)16(3-cl-4-o2Nc6H3) G5(3-Cl-4-02NC6H3)

8 G4(AC)13(4-cl-2-o2Nc6H3) G4(4-Cl-2-02NC6H3)

9 G4(AC)13(2-F-4-o2Nc6H3) G4(2-F-4-02NC6H3) G5(Ae)16(3-Br-4-02NC6H3) G5(3-Br-4-02NC6H3) ll G4(Ae)13(4-I-2-02NC6H3) G4(4-I-2-02NC6H3) 12 G4(Ae)13[2,4-(02N)2C6H3] G4[2,4-(02N)2C6H3]
13 G5(AC)16[3~5-(o2N)2c6H3] G5[3,5-(02N)2C6H3]
14 G5(AC)16[2~4-(o2N)2c6H3] G5[2,4-(02N)2C6H3]

~' 1~63~

TABLE II (continued) Example Column C Column D
GLI(Ac)13(2-cH3o-4-o2Nc6H3) G4(2-cH30-4-o2Nc6H3) , 16 G5(Ac)16(3-CH30-4~02NC6H3) G5(3-CH30-4-02NC6H3) 17 G4(Ac)13(4-CH30-2-o2Nc6H3) G4(4-CH30-2-02NC6H3) 18 4(Ac)13(2 CH3-5-(CH3)2CH-4-o2NC6H2) G4(2-cH3-5-(cH3)2cH-4-o2NcH2) l9 G5(Ac)16(2-(CH3)2cH-5-cH3-4-o2Nc6H2) G5(2(CH3)2CH-5-CH3-4 2NC6H2) G4(Ac)13(2-CH302C-4~o2Nc6H3) G4(2-CH302C-4-02NC6H3) 21 G5(Ac)16(3-C2H5-4-02NC6H3) G5(3-C2H5-4-02N6H3) 22 G4(Ac)13(4-t-c4Hs-2-o2Nc6H3) G4(4-t-C4Hg-2-02NC6H3) 23 G4(Ac)13(4~02N~l~C1oH7) G4(4-02N~l~C1oH7) 24 G4(Ac)13(l-o2N-2-c1oH7) G4(l-02N-2-C1oH7) G4(AC)13(4-cl-2-o2N-l-c1oH7) G4(2-N02-4-Cl-l-c1oH7) 26 G4(AC)13(2-o2N-4-c6H13co2c6H3) G4(2-02N-4-C6H13c02c6H3) 27 G6(Ac)19(3-CH3-4-02NC6H3) G6(3-CH3-4-02NC6H3) ; 28 G6(AC)19(2-cl-4-o2Nc6H3) G6(2-Cl-4-02NC6H3) '' G6(Ac)19(3,5-(02N)2C6H3) G6(3,5-(02N)2C6H3) G6(AC)19(2-cH3o-4-o2Nc6H3) G6(2-CH30-4-02NC6H3) 31 G6(AC)1g(4-o2N-l-c1oH7) G6(4-02N-l-C10H7) 32 G6(Ac)19(4-02NC6H~ 6(4-o2NC6H4) ~7~ii SUPPLE~NTARY DISC10SURE

The process for the preparation of the ~ and ~ nitro-aromatic glycosides described hereinbefore may be further illustrated as follows:

(A) Preparation of ~ - and ~ -(4-nitro~henyl)maltopentaoside CH20AC CH20Ac 20Ac C ~ NaOCH3/

Ac ~ ~ O _ ~ O_ ~ ~ O ~ NO CH30H
Ac OAc 3 Ac CH2GH CH20H` I CH2H`

H~o ~o ~ ~}~2 (4-Nitrophenyl)hexadecaacetylmaltopentaoside (6.1 g, 3.8 mmole, prepared as described in part (D) of Example 2, was mixed with methanol (150 ml) and treated with 4.1 ml o~ 0.15 M sodium methoxide in methanol (0.62 mmole). The resulting yellow solution was neutralized with 1~ hydrogen chloride in methanol and the solvent was partially removed under reduced pressure to initiate precipitation. The remaining solution was added slowly to 370 ml diethyl ether with vigorous magnetic stirring. The mixture was stirred for an additional hour to allow for complete precipitation. Fil-tration and suction drying under an atmosphere of nitrogen pro-vided 3.24 g (90~) of cream~colored solid. ~ mHaOX 303 ( 7310) 217 (~ = 5385). HPLC (propylethylenediamine modified silica (8~u particles), 4.1 mm i.d. x 25 cm, 25/75 H20/CH3CN, 254 nm detector) showed two major components, 56~ and 30~, identified as ~ and ~ isomers of (4-nitrophenyl)maltopentaoside in 65/35 ratio.
_ 36 1~9G37G

A portion of thiæ material wa~ chromatographed on SEPHADEX* G-15 and eluted with water to provide a center cut which was lyophilized to give a cream-C~lored solid. mis samples exhibited ~ m ~ 303 (~ = 9160), 219 ( = 6590) . HPLC
showed two ma~or components, 66~ and 30~. lH nmr (220 MHz) recorded in D20:7.72 (center of aryl AA'BBI pattern, JAB =
9.5 Hz), 5.76 (d, J = 4 Hz, Hl/ of a ~ isomer), 5.45 - 5.27 (m, _C(OR)(ORI)), 4.23 - 3.32 (m, C_ and CH2). me ~/~ratio was ca. 62/38.

* denotes trade mark

Claims (9)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing .alpha. and .beta. nitroaromatic glycosides comprising:
(a) contacting an acetylated glycoside of the formula:

wherein Ac is an acetyl group, and n is an integer of 2, 3 or 4, with a phenol selected from the group consisting of , and wherein X and Y are individually H, NO2, halogen, alkyl of 1 to 4 carbon atoms, OR' or CO2R' where R' is an alkyl group of 1 to 6 carbon atoms, with the proviso that only one of X
and Y is NO2, in the presence of a catalyst at a temperature in the range of about 80-120°C;
(b) nitrating the product of (a) by contacting said product with:

(i) nitric acid contained in a mixture of acetic acid and sulfuric acid, or (ii) a nitronium compound selected from nitronium tetrafluoroborate, nitronium hexafluoro-phosphate and nitronium trifluoromethane-sulfonate contained in dichloromethane, chloroform or 1,2-dichloroethane; and (c) deacetylating the product of (b) by contacting said product with:
(i) a catalytic amount of an alkali metal lower alkoxide contained in the corre-sponding alcohol, or (ii) a solution of anhydrous ammonia or HC1 in methanol.

2. The process of Claim 1 wherein the phenol is

3. The process of Claim 2 wherein n is 2 or 3.

4. The process of Claim 3 wherein the catalyst in step (a) is p-toluenesulfonic acid, or an anhydrous covalent metal chloride.

5. The process of Claim 4 wherein the anhydrous covalent metal chloride is zinc chloride.

6. The process of Claim 4 wherein the temperature in step (a) is in the range of about 100-110°C.

7. The process of Claim 4 wherein the nitration reaction of step (b) comprises contacting the product of step (a) with nitronium tetrafluoroborate contained in dichloromethane, chloroform or 1,2-dichloroethane at a temperature of about 25°C, the molar ratio of nitronium tetrafluoroborate to step (a) product being in the range of 1-20:1.

8. The process of Claim 7 wherein the nitronium tetrafluoroborate is contained in dichloromethane and the molar ratio is about 10:1.

9. The process of Claim 4 or Claim 7 wherein the deacetylation reaction of step (c) comprises contacting the product of step (b) with about 0.01-0.1 molar equivalent of sodium methoxide contained in methanol at a temperature in the range of about 0-25°C.