CA1096377A - Nitro aromatic glycosides - Google Patents

Abstract

E.I. du Pont de Nemours and Company ABSTRACT

Nitroaromatic derivatives of glycosides such as maltotetraose, maltopentaose and maltohexaose are described.
These compounds are useful as standard substrates for the assay of .alpha.-amylase either manually or automatically on suitable instruments.

Description

2'7~

BACKGROUND OE THE INVENTION

Field of the Invention This invention relates to glycosides and more particularly to nitroaromatic derivatives of maltotetraose, maltopentaose, and maltohexaose, useful as standard sub-strates 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 ~-(p-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:

'~:
.,~ ~,.

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(1) Incubation of human urine or saliva samples with ~ nitrophenyl)maltoside at 37 for 16 hr produced 4-nitrophenyl, identified spectrophotometrically by mixing the hydrolyzate with 0.02_ sodium hydroxide. (2) The hydro-lysis was inhibited by protein precipitants such as 10%
trichloroacetic acid and 0.5~ silver nitrate. (3) The hydrolysis was pH-dependent, being most effective at pH
5.9-7Ø They state that this was evidence for "the possible existence o~ an unidentified carbohydrase". ~ -(4-nitrophe-nyl)maltoside is not believed to be useful for an amylaseassay because the cleavage of this compound by ~ -amylase is extremely slow.
SUMMARY OF THE INVENTION
According to the present invention there is pro-vided a compound having the formula:
CH2H , CH20H - CH20H

~ ~L ~ ~OR

wherein n is an integer o~ 2, 3 or 4J and R is a ~ubstituted aromatic radical selected from the group consisting of:

X ~ Y , ~ X , Y Y

wherein X and Y are individually B, N02, halogen3 alkyl of 1 to 4 carbon atoms, ORl or C02Rl, where 1~ .

G37'7 Rl is an alkyl group of 1 to 6 carbon atoms, with the proviso that at least one of X and Y
is N02.
The preferred compounds of the invention are those in which R is nitrophenyl, especially 4-nitrophenyl, i.e., X is NO2 and Y is H, and n is 2 or 3.
The compounds of the invention are useful standard substrates for the assay of serum ~-amylase in the study of pancreatic function.
DETAILED DESCRIPTION OF THE INVENTION
The nitroaromatic glycosides of the invention are ; derived from a series of oligomers and polymers of glucose which are ~[i ->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.

CH2OH CH OH ¦ CH2H CH2H
` 1~~ ~Xo~~ -o~~l HO ~ OH HO ~ ~ ~ OH
OH OH OH OH
n ~-D-Glucose (Gl) n = 0 maltose (G2) n = 1 maltotriose (G3) n = 2 maltotetraose (G4) n = 3 maltopentaose (G5) 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 Chamical Substances for Chemical Abstracts During the Ninth Collective Period (1972-1976)", Chemical Abstracts Service, Columbus, Ohio (1973).

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~63`~"7 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

C~20H CH20Ac Ac . GnO OH ~ GnO OAc n 3 4 Gn=G3(AC)lo or G4(AC)13 1 C6H50H, ZnC12 G O ~ ~ ~O ~IT ATION G ~ AcOC6H5 Gn=G3(AC)lo or G4(AC)13 Gn=G3(AC)lO or G4(AC)13 NaOM 1 MeOH

~0\~
G ~ ~ Gn = MALTO-N-OSIDE RESIDUE~[1-4J
n OH Ac = CH3CO
Gn=G3 or G4 G3(Ac~lo=DEcAAcETyLMALToTRIosyL
Gl~(Ac)l3 = TRIDECAACETYLMALTO-TETRAOSYL

37'~

The details of each step o~ the procedure for preparing the compounds of the invention are as follows:
ACETYLATION 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., 1298 (1953), W.J. Whelan, J.M. Bailey, and P.J.P. Roberts, J. Chem. Soc., 1293 (1953), A. Thompson and M.L. ~olfrom, J. Amer. Chem. Soc., ~, 3612 (1954), M.L. Wolfrom, L.W. Georges, A. Thompson, and I.L. Miller, J. Amer. Chem. Soc., ~ 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 favours the production of compounds where the sub-stituent on the anomeric carbon is in the ~ -configuration (W. Pigman, loc. cit. p. 140-142). In either case a mixture of the ~ and ~ isomers is obtained. The acetylation is con-ducted in acetic anhydride as the solvent and reactant, the amount of acetic anhydride being from 5 to 50 times the weight o~ G4 or G5. me 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 æodium acetate used may be from 1 to 10 molar equivalents per molar equivalent of G4 or G5, 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 1~963~7 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 intermedlate 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 (Hla) 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 9 _ 1~637~

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 DISPLACE~ENT 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~96377 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 (SnCl4), 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 .

1~6377 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., _, 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 sulfurlc acids with nitric acid, or in dichloromethane with a nitronium compound such as nitronium tetrafluoroborate (NO2 BF4 ), nitronium hexafluorophosphate (NO2 PF6 ) or nitronium trifluoromethanesulfonate (N2 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

~963'77 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 metasubstitution 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 ~7 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 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 metha-nol, freed of inorganic ions (if desired) by passage through an acidic ion-exchange column, and recrystallized 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 0-25~C
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 ox bromide of the following structure.

CH2OAc CH2OAc CH2OAc ~co~o~o~x AcO AcO n ~cO

n = 2 or 3 X = Cl or Br .

These halides are prepared either by treatment of the completely acetylated oligosaccharide with anhydrous hydrogen halide, solution of hydrogen halide in mixtures of acetic anhydride and acetic acid, solutions of alumin~ chloride and phosphorus pentachloride or of titanium tetrachloride in chloroform, or from the oligosaccharide itself by treatment with acetyl - chloride (W. Pigman, loc, cit., p. 150-151).
- me halogen atom iæ 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(CH3C00)2), or with iron (III) chloride (Koenigs-Knorr reaction), or with the sodium or -~ potassium salt of the phenol (W. Pigman, loc. cit., p. 194-198).
me rest of the synthesis is the same as described above.
The progress of any of the above reactions can be followed by thin-layer chromatography (TLC) on æilica gel in a suitable solvent system, and by nmr spectroscopy.
e purity of the products of the reactions can be determined by high-performance liquid chromatography (HPLC), by polarimetry, and by ultraviolet and high-frequency (220 MHz) nmr spectroscopy.
It has been found that the nitroaromatic glycosides of the invention are useful substrates for serum Cy-amylase assay. The assay process is illustrated in Scheme II for the preferred compounds o~ the invention, ~-(4-nitrophenyl)malto-tetraoside and ~-(4-nitrophenyl)maltopentaoside. Serum ~ -amylase converts these two compounds to a mixtureof G2 or G3 and ~-(4-nitrophenyl)maltoside. The latter is then hydro-lyzed to glucose and 4-nitrophenyl by ~ -maltase; treatment .~, with dilute alkali p~oduces the 4-nitrophenolate anion which is spectroscopically identifiable and distinguishable from any unreacted glycoside, and which can be related to serum ;~ -amylase levels.
SCHEME II: SERUM ~ -AMYLASE ASSAY

H 1 ~ H

~ OH ~ ~o J \ OH ~ O -~\ OH
HO ~ ~ ~ OR
OH OH _ n OH
~MAX 290_305 nm R 4 02NC6H4 n=2 ~-(4-NITROPHENYL)MALTOTETRAOSIDE
n=3 ~ - (4-NITROPHENYL)MALTOPENTAOSIDE
~ ~-AMYLASE

~0~ ~0 ~ ~0 G2 or G3 + ~ ~ ~ ~ ~ ~
HO ~ ~ ~ ~ ~ ~ OR

R 4 2NC6 4 n=O (4-NITROPHENYL)MALTOSIDE

1 ~-MALTASE

4 or 5 Gl + HO ~ No2 OH-~ 2 4-NITROPHENOLATE ANION ~MAX 410 nm 1~96377 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.

A

1~637~

E ~PLE 1 (A) Preparation of Maltotetraose ~-Tetradecaacetate 1~ ~ ~o NaOAc/Ac20 HO O- ' O- J
OH OH OH

CH OAc CH2OAc CH OAc ~ ~ ~ O OAc AcO ~ O- ~ O- ~
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: vmax (CHC13) 1750, 1370, 1230 and 1030 cm 1; ~max (EtOH) 210 nm (~ 740); [~]D + 104 (c 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);
20 Anal. Calcd. for C52H70O35: 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~ -- . , ~;``.`~
_~ I

recrystallization, and the mp was from 122 to 128.

(B) Preparation of q - and ~-Phenyltridecaacetylmalto-tetraosides . _ ~
CH20Ac OAc CH20Ac ~c~o~C+ ~ ~
OAc OAc 2 OAc ~H20Ac CH20Ac C~I20AC
~co~ ~ ~ K,~
OAc OAc 2 OAc A mixture of maltotetraose ~ -tetradecaacetate from 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 mechanically at 100 for 3 hr. The 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~) of crude phenyl derivative aæ a yellow crystalline solid. This material was purified by preparative thin layer and high performance liquid chromatography as follows:
A total amount of 3 . o6 g of crude phenyl derivative was loaded onto 14 2-mm preparative TLC plates and developed 3 times with a mixture of 95:5 benzene:methanol. The Rf 0.13-0.27 band was extracted with chloroform and methanol to give 0.90 g (290 of material which was recrystallized from ethanol, recovery 0.58 g of a mixture of q - and ~ -phenyltridecaacetylmaltotetraosides. Analytical HPLC (polar _ 19 _ ~.

1~637'7 .
silicone microspheres) showed product with retention time 8.99 min and a minor impurity (2.~) at 8.23 min. The structure of the crystalline phenyltridecaacetylmaltotetra- -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);C~12D5 ~ 132 (c-l.OO CHC13); lH nmr (220 MHz)J
~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 o~ singlets), 33H (COCH3);
Anal- Calcd- ~or C56H7234 C~ 52-17; H~ 5-63~

Found: C, 51.44, 51.94, 51.60;
H, 5.35, 5.60, 5.39 A total amount o~ 4.25 g of the crude phenyl derivative was also purified by prepara-tive HPLC on a 1 m x 23 mm Spherosll (44-50) column eluted with a mixture of 1:1 pentane:dioxane (containing 1.5% water) to give 1.07 g (25%
recovery) o~ 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 ~Ac CH20Ac AC~4~ OAC ~} AcOH2S

OAc OAc 2 OAc CH20Ac CE20Ac CH20Ac .. ~~ ~ >I ~~~>
AcO ~ O ~ ~ ~ ~ ~

OAc OAc _~ 2 OAc Maltotetraose ~g-tetradecaacetate (2.75 g, 2.19 ,:

~6377 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 [ ]25 + 135 (c 1.0 CHC13)~
~ 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.

~ 3~t~

(D) Preparation of ~ - and ~-(4-Mitrophenyl)tridecaacetyl-maltotetraoside (Nitric Acid Method) CH20Ac C~20Ac CH20Ac HNO3~

A~k~ ~ H2s04 OAc OAc 2 OAc H20Ac CH20Ac CH20Ac ~ ~I ~ <~}N02 Aco ~ O_ I ~ O_ J ~
OAc OAc Ac A mixture of acetic acid (2.0 ml), acetic anhydride (1.0 ml) and preparative TLC-purified ~ - 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. This sample was approximately 50% 4-nitrophenyl derivative as indicated by ~max (EtOH) 290 nm ( 3100) .
me reaction was repeated on a larger scale (5 g of crude phenyltridecaacetylmaltotetraoside) to give 4.35 g (84~) of crude nitro compound which was chromatographed on silica gel (200 g) (Mallinckrodt SilicA ~ CC-7). The 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 HPL~ 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). me fractions were examined by TLC and UV, and from the middle cuts, O.2557 g A
.

~.P~9~7'~

(0.19 moleJ 8%) o~ pure ~ - and ~-(4-nitrophenyl)tridecaacetyl-maltotetraoside was obtained. After recrystallization from ethanol (5 ml) this material had mp 112-114. me structure of the material was confirmed by ~maX (CHC13) 1745, 1580, 1$20, 1420, 1370 and 1220 cm 1; ~max (~tOH) 290 nm (~ 7180); Ld~D5 156 (c 0.525 CHC13); lH nmr (220 MHz), ~ 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 (COCE3);
Anal. Calcd. for C56H71N036: C~ 5 Found: C, 49.95; H, 5.54 (E) ~- and ~ -(4-Nitrophenyl)tridecaacetylmaltotetraoside Nitronium Tetrafluoroborate Method) CH20Ac , CH20Ac CH20AC
~c~~ <~> ~
OAc Ac2 OAc CH20AC CH20AC _ ' CH20AC

.. ~ Q>l ~ >~ ~ `> {~N02 AcO ~ O I ~ O_ J ~
OAc OAc ~ 2 Ac A slurry of nitronium tetra~luoroborate (1.91 g, 14.5 mmole) in dry methylene chloride (8 ml) was treated with a solution o~ phenyltridecaacetylmaltotetraoside ~rom part (C) (1.24 g, o.96 mmole) i~ dichloromethane (10 ml), and the resulting mixture stirred ~or 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 sul~ate and evaporated under reduced pressure to provide a yellow glassy solid (1.30 g) of ~ - and ~ -(4~nitro-phenyl)tridecaacetylmaltotetraoside product. Hal~ of this 3~7~7 product was recrystallized from ethanol to provide a cream-colored solid, o.48 g (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~, OCH5 OCH2), and 2.25 - 1.95 serie~ o~ singlets) 39H (COCH3)~ ~max (Et~H) 288 nm ( 5840)~
25 + 152 (c 0-5 CHC13) (F) Preparation of ~ - and ~3 -(4-Nitrophenyl)maltotetraoside CH20Ac CH20Ac l CH20Ac NaOCH/

¦<OAC >I 1~ >~ ~>~0--~--N0 AcO ~ O- ~ O_ I ~
Ac _ Ac _ 2 OAc H$~o ~ \~ ~\ o{~o A mixture of column-chromatographed 4-(nitrophenyl)-tridecaacetylmaltotetraoside from part (D) (6.76 g, 5.1 mmole), methanol (50 ml) and sodium methoxide (50 mg) was left at 25 overnight. The mixture was filtered to remove a small amount o~ 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 plstes 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 of ~ - and ~ -(4-nitrophenyl)maltotetraoside, approximately 50% pure by W. me product was a yellow solid with mp 68-70o and its structu~e was cOnfirmed by: ~ max (Nu~ol) 3300 and 1020 cm 1; ~ max (H20) 35 nm (E 374); ~ D5 + 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) ~ ~ - 24 -. . . ~.

37~

(other aromatic material), 5.88 ~ 5.79 (m) (Hl~ and Hl~), 5.30 (m) (OCHO), 4.98 (s) (HOD), and 4.02 3.48 ppm (m) (OCH, OCH2).
(G) (4-Nitrophenyl)maltotetraoside (Alternate Method) _ _ .' IH20AC CIH20Ac CH20Ac NaOCH3/

Ac ~ O ~ ~ ~~ ~ 2 OAc OAc OAc ~ ~ ~
~ OH ~ I< ÇH ~ k OH ~ O ~ N02 HO ~ O_ I ~ O_ 1 ~ \~_____~

- A mixture of (4-nitrophenyl)tridecaacetylmalto-; tetraoside from 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 of 7.8 x 10 3 M solution) and was stirred for 17 hr at room temperature. Sol~ent was removed under reduced pressure and the residue, dissolved in a minimal volume of methanol (2 ml), was added dropwise to ether (40 ml). The resulting yellow powder was separated, dissolved in methanolJ and passed through a 2.5 x 20 cm column of Sephade ~ LH-20 using methanol as eluant. There was obtained 134 mg (900 of solid product which was re-chromatographed to provide a center cut (90 mg, 60~) having its structure by ~ max (H20) 33 nm (~ 6350), 217 (~ 5000);
H nmr (220 MHz), ~ (CD30D) 7-77 (AA'BBI, 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)J and 4.01 - 3.40 ppm (m) (OCHJ OCH2).

~ - 25 -. ,.p.~ -1~6377 (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); []D + 158 (c 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).

(A) Preparation of Maltopentaose ~-Heptadecaacetate CH2~ CH OH CH OH
,~0 ~ L~O NaOAC/Ac20 HO ~ ~O- ~ O

CH OAc CH OAc CH OAc O~OAc AcO - ' 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 recrystallized from ethanol (40 ml) to provide maltopentaose ~-heptadecaacetate, 3.16 g (85%), mp 117-120;
whose structure was confirmed by: []D5 + 122 (c 0.7, CHC13); 1H 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 (Hl);

~t,,~

37'~

64 86 43 , 9 8 ; H, 5.6 ~ound: C, 49.11, 49.28; H, 5.54, 5.71 (B) Preparation of ~ - and ~-Phe~ylhexadecaacetylmalto-pentaoside CH20Ac CH20Ac CH20Ac ~ >I ~ >I ~ \~oAc~}oH ZnC12 AcO ~ - I ~ O ~

OAc OAc OAc CIH20Ac CH20Ac CH20Ac Ac ~ ~ ~ O ~ \ OAc ~
~ O_ I ~0_ \~/
OAc OAc OAc , _ 3 A mixture Or 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. me 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 sulrate and concentrated under reduced pressure to give a yellow powder which was treated with sodlum acetate (3.0 g) and acetic anhydride (15 ml) and then heated at 125 for 2.0 hr in order to reaacetylate any ~ree hydroxyl groups.
The cooled mixture was added to ice (125 g) and stored at 5. The resulting ~- and ~ -phenylhexadecaacetylmaltopenta-oside solid product was pulverized, separated and air-dried to give 2.45 g of an of~-white powder. This material was chromatographed on silica gel (150 g) eluting with a mixture of 97:3 benzene:methanol. The lH nmr (220 MHz) of the residue ~rom a center cut exhibited ~ 7.38 - 7.27 (m) 2H and 7.17 - 6.97 . - 27 -i37'7 (m) 3H (C6H5), 5-77 (dd J = 9.5 Hz), 5.63 (d J = 4 Hz) (Hl~), 5.82 - 3.83 (series of multiplets) 35H (OCX, 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 ~ 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% ~-phenylhexadecaacetylmaltopentaoside and 25% ~-phenylhexadecaacetylmaltopentaoside.

(C) Preparation of ~ - and ~ -Phenylhexadecaacetylmalto-pentaoside (Alternate Method) CH20Ac C ~ Ac CIH20Ac Acn~H//

+ ~ OH _ 2 _~
AcO - J O l OAc OAc OAc _ _ 3 CH20Ac ~ CH20Ac - CH~OAc ~o~ ~ ~~ 1~~ {~>
AcO ~ o_ J ~ o_ ~
OAc Ac Ac _ _ 3 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 of zinc chloride (0.49 g) in 2.0 ml of a mixture of 95:5 acetic acid:acetic anhydride, and the reation 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 benzene (200 ml) and 18% aqueous sodium chloride solution. The organic phase was washed twice with 2.5% sodium - 2~ -i ~

iO~3177 hydroxide (50 ml) and twice with saturated sodium chloride (50 ml). The organic layer was dried over sodium sul~ate, evaporated~ and the residue treated with anhydrous sodium acetate (3.0 g) and acetic anhydride (15 ml). The mixture was heated at 120 ~or 1.0 hr, cooled, and treated with ice water (200 ml). The solid product which formed upon standing at 0 was pulverized, filtered, and air dried to give 2.97 g of an o~f-white powder. This material was chromatographed on a silica gel column eluting with a mixture of 97:3 benzene:
methanol. mere was obtained a total of 2.00 g (65~) of pure d - and ~ -phenylhexadecaacetylmaltopentaoside, mp 119-125;
~D5 + 137 ~c 1.0, chloroform);
Anal. Galcd for C6 ~8842 Found: C, 51.62, 51.13; H, 5.70, 5.47 (D) Preparation of (4-Nitrophenyl)hexadecaacetylmalto-pentaoside c ~ Ac ~

Ac ~ - ~ ~ Q ~ o ~ +N02+BF4 OAc OAc Ac CH20Ac C1120Ac CH20Ac C~2C12 ~ ~ ~ ~ ~ 2 O- _ _ OAc OAc 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 gg o.64 mmole) in dichloro-methane (12 ml). me resulting mixture was stirred ~or 20 min at room temperature, poured into stirred, cold, saturated sodium chloride solution (70 ml), and the separated organic ~ .
~.,~ ., l~Ci37 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 0.87 g o~ an off-white powder~ The product struCture Was confirmed by ~CH30H 290 nm (~ 5550), 210 nm ( ~7750), lH nmr spectrum max (220 MHZ)~ 7.75 (A~IBB~, JAB = 9-5 HZ) 4H (c6H4)~ 5-75 (d J = 4 HZ) (Hl~), 5r78 - 3.85 (series of multiplets) (OCH, OCH2), and 2.22 - 1.93 ppm (series of singlets) 48H (CH3CO).
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 CH20Ac CH20Ac CH20Ac NaOCH3/
~ O ~ ~ O ~ ~ ~ ~ 2 AcO ~ O_ J ~ O_ I ~
OAc - OAc 3 OAc H ~ O ~ ~ ~ N02 A solution of (4-nitrophenyl)hexadecaacetylmalto-pentaoside from part (D) (430 mg, 0.27 mmole) 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 nitrogenJ and the stirred residue was treated with ether (40 ml) to precipitate a æolid Which Was filtered and air-dried to provide a slightly yellow powder (247 mg~ 98%), ~maX (H20) 33 nm (~ 5980), 215 (5530), HPLC

(Waters Associates ,U Bondapak/Carbohydrate, 80~ acetonitrile/
water, 254 nm W detector) o~ the product showed two equally ~ , - 30 -..-. . ~

637';' - 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-- 30 isopropylphenol (carvacrol, p. 568), 2-methoxyphenol(guaiacol, X

1~96377 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.

. ~,, . ~

1~637'~

TABLE II
-(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) 2-ClC6H40H G5(Ac)16(2-clc6H4)

7 3-ClC6H40H 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) ll 4-IC6H40H G,~(Ac)13(4~IC6H4) 12 2-02NC6H40H G4(AC)13(2-o2Nc6H4) 13 3-02NC6H40H G5(AC)l6(3-o2Nc6H4) 14 4-02NC6H40H G5(AC)16(4-o2Nc6H4) 2-CH30C6H40H G4(Ac)l3(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-c2Hsc6H4) 22 4-t-C4HgC6H40H G4(Ac)13(4-t-c4Hsc6H4) - 23 OH G4(AC)13(l-c1oH7) 24 OH G4(AC)13(2-c1oH7) ~1' .

X

~6377 TABLE II (continued) Example Column A Column B
OH G4(AC)l3(4-cl-l-cloH7) Cl 26 4-C6H13C02C6H40H G4(Ac)l3(4-c6H13co2c6H4) 27 3-CH3C6H40H G6(AC)l9(3-cH3c6H4) 28 2-ClC6H40H G6(Ac)19(2-clc6H4) : 29 3-02NC6H40H G6(AC)l9(3-o2Nc6H4) 2-CH30C6H40H G6(AC)l9(2-cH3oc6H4) 31 OH G6(AC)l9(l-cloH7) 32 C6H50H G6(Ac)1s(c6Hs) Example Column C Column D
3 G4(AC)13(2-cH3-4-o2Nc6H3) G4(2-CH3-4-02Nc6H3) 4 G4(AC)l3(3-cH3-4-o2Nc6H3) G4(3-CH3-4-02NC6H3) G5(Ac)16(4-CH3-2~o2Nc6H3) G5(4-CH3-2-02NC6H3) G5(Ac)16(2-Cl-4-02NC6H3) G5(2-Cl-4-02NC6H3) , 7 G5(Ac)16(3-Cl-4-o2Nc6H3) G5(3-Cl-4-02NC6H3) 8 G4(Ac) 13 (4-Cl-2-02NC6H3) G4(4-Cl-2-02NC6H3) 9 G4(Ac)13(2-F-4-02NC6H3) G4(2-F-4-02NC6H3) G5(AC)16(3-Br-4-o2Nc6H3) G5(3-Br-4-02NC6H3) 11 G4 (Ac) 1 3 (4-I-2-02NC6H3) G4 (4-I-2-02NC6H3) 12 G4(Ac)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]

1~6377 TABLE II (continued) Example Column C Column D

G4(Ac)l3(2-CH30-4~02NC6H3) G4(2-CH30-4-02NC6H3) 16 G5(AC)16(3-cH3o-4-o2Nc6H3) G5(3-CH30-4-02NC6H3) 17 G4(AC)l3(4-cH3o-2-o2Nc6H3) G4(4-CH30-2-02NC6H3) 18 G4(AC)13(2-cH3-5-(cH3)2cH-4-o2Nc6H2) ~4(2-cH3-5-(CH3)2cH-4-o2NcH2) 19 Gs(Ac)16(2-(CH3)2cH-5-cH3-4-o2Nc6H2) G5(2(CH3)2CH-S-CH3-4 2NC6H2) G4(Ac)13(2-CH302C~4~o2Nc6H3) G4(2-CH302C-4-o2Nc6H3) 21 G5(AC)16(3-c2H5-4-o2Nc6H3) G5(3-C2H5-4-02N6H3) 22 G4(AC)13(4-t-c4H9-2-o2Nc6H3) G4(4-t-C4Hg-2-02NC6H3) 23 G4(Ac)13(4-o2N-l-c1oH7) G4(4-02N-l-C1OH7) 24 G4(Ac)13(1-02N~2~C1oH7) G4(1-02N-2-CloH7) G4(Ac)13(4-Cl~2~02N~l~C1oH7) G4(2-N02-4-Cl-l-C1oH7) 26 G4(Ac)l3(2-o2N-4-c6H13co2c6H3) G4(2-o2N-4-c6H~3co2c6H3) 27 G6(Ac)19(3-CH3-4~02NC6H3) G6(3-CH3-4-02NC6H3) 28 G6(AC)19(2-cl-4-o2Nc6H3) G6(2-C1-4-02Nc6H3) 29 G6(Ac)19(3,5-(02N)2C6H3) G6(3,5-(02N)2C6H3) G6(AC)19(2-cH3o-4-o2Nc6H3) G6(2-cH30-4-o2Nc6H3) ! 31 G6(Ac)19(4-o2N-l-c1oH7) G6(4-02N-l-C1OH7) 32 G6(Ac) 19 (4-02NC6H4) G6 (4-02NC6H4) ,, .~

.377 SUPPLEMENTARY DISCLOSURE
In a preferred embodiment~ the glycosides of the present invention are in the form of a mixture of isomeric ~
and ~ glycosides. Such a mixture is easy to prepare and dif-ficult to separate. In addition both isomers can be utilized in the ~-amylase assay discussed hereinbefore.
m e present invention is further illustrated by the following exampleO

(A) Preparation of ~ - and ~ -(4-nitrophenyl)maltopentaoside _ _ , C~20Ac CH20Ac CH20Ac NaOCH3/
O~ ~ O~ ~ O ~ ~_____~ CH30H
~ OAc ~ < OAc ~ < OAc ~ ~ N02 AcO ~ O _ ~ O_ ~

;i OAc O~c OAc :~ _ _ ., C:EI20H CH20H CH20H

~0 ~ 0 ~ 0 ~ ~0 ~ N2 OH OH

(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 of 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. me mixture was stirred for an additional hour to ,~ .

3,77 allow for complete precipitation. Filtration and suction drying under an at sphere of nitrogen provicled 3.24 g (90%) of cream~colored solid. ~ ~0 303 ( 7310), 217 (ç 5385), HPLC
(propylethylene-diamine modified silica (8,u particles), 4.1 mm i.d. x 25 cm, 25/75 H20/C~ CN, 254 nm detector) showed two ma~or components, 56% and 30%, identified as ~ and ~isomers of (4-nitrophenyl) maltopentaoside in a 65/35 ratio.
A portion of this material was chromatographed on SEPHADEX G-15 and eluted with water to provide a center cut which was lyophilized to give a cream-colored æolid. mis sample exhibited ~ H20 303 (~ 9160), 219 (~ 6590), HP~C æhowed max two ma~or components, 66% and 30%. lH nmr (220 MHz) recorded in H20 7.72 (center of aryl AA'BB~ pattern, JAB = 9-5 Hz), 5.76 (d J = 4 ~z~ Hl, of a ~ iæomer), ~.45-5.27 (m, _C(OR) (ORt)), 4.23-3.32 (m, CH and CH2). me ~/~ ratio waæ ca 62/38.

~..

Claims (12)

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

1. A compound having the formula:

wherein the configuration of the substituent -OR on the anomeric carbon is .alpha.- or .beta.-, n is an integer of 2, 3 or 4, and R is a substituted aromatic radical selected from the group consisting of:

, , wherein X and Y are individually H, NO2 halogen, alkyl of 1 to 4 carbon atoms, OR1 or CO2R1, where R1 is an alkyl group of 1 to 6 carbon atoms, with the provisio that at least one of X and Y
is N02.

2. The compound of Claim 1 wherein R is .

3. The compound of Claim 2 wherein X is NO2 and Y is H.

4. The compound of Claim 1 wherein R is 4-nitro-phenyl.

5. The compound of Claim 4 wherein n is 2.

6. The compound of Claim 4 wherein n is 3.

7. A composition of matter comprising a mixture of the .alpha. and .beta. compounds of Claim 1.

8. A composition of matter comprising a mixture of the .alpha. and .beta. compounds of Claim 1 wherein R is .

9. A composition of matter comprising a mixture of the .alpha. and .beta. compounds of Claim 1 wherein X is NO2 and Y
is H.

10. A composition of matter comprising a mixture of the .alpha. and .beta. compounds of Claim 1 wherein R is 4-nitrophenyl.

11. A composition of matter comprising a mixture of the .alpha. and .beta. compounds of Claim 1 wherein n is 2.

12. A composition of matter comprising a mixture of the .alpha. and .beta. compounds of Claim 1 wherein n is 3.