CA1111417A - Synthesis of 2-amino-2-deoxyglycoses and 2-amino-2- deoxyglycosides from glycals - Google Patents

Abstract

"SYNTHESIS OF 2-AMINO-2-DEOXYGLYCOSES AND
2-AMINO-2-DEOXYGLYCOSIDES FROM GLYCALS"
ABSTRACT OF THE DISCLOSURE
0-acylated-2-azido-2-deoxy glycosyl nitrates are used to prepare 0-acylated-2-azido-2-deoxy glycosyl halides. The halides are useful in the preparation of 0-acetylated-2-azido-2-deoxy glycosides, which in turn can be reduced to 2-amino-2-deoxy glycosides. Of particular interest are the syntheses of 2-amino-2-deoxy glycosides which correspond to the terminal units of the antigenic determinant for the human A blood group. Attachment of these glycosides to a solid support provides immunoabsorbents which effectively and preferentially absorb anti-A-antibodies from blood plasma.

Description

111~417 BACKGROUND OF THE INVENTION
It is well known that carbohydrate structures of various complexities are the antigenic determinants for a wide range of substances.
It is also well established that relatively small molecules, known as haptens, can correspond to the structure of the antigenic determinant.
The hapten, when attached to an appropriate carrier molecule, provides an artificial antigen which,when administered to an animal under appropriate conditions,will give rise to the production of antibodies having a specificity for the hapten. Furthermore, in recent years, much art has developed for the preparation of immunoabsorbents from haptens.
This art involves the attachment of the hapten, normally through covalent .. I
~ bonding but at times through hydrophobic bonding, to a solid, latex or ; gelatinous support. Thus, the hapten is immobilized so that when the resulting i~munoabsorbent is exposed to antibodies with combining sites for the haptenic structure, the antibodies will attach themselves to the surface of the immunoabsorbent and thereby be specifically removed from solution.
Many varieties of solid, latex and gel supports for the preparation of immunoabsorbents have been developed and many ways have been devised for attachment of the hapten to these insoluble structures.
Although improvements in these matters are possible, the main problem remains - of having simple access to the desired hapten in a form convenient for - attachment to the carrier molecule.
It was the original purpose of our work to develop a practical process for the synthesis of =D-galactosamine hydrochloride (XXXVII).
and of =D-lactosamine hydrochloride (XXXIX) and derivatives of these. I
. !
Both galactosamine and lactosamine, usually in the form of their N-acetylated derivatives, are found widespread in nature. They occur in glycoproteins, glycolipids and mucopolysaccharides. As such they are important building units found in the blood group substance antigenic determinants.

(- I
i The main prior art source of D-galactosamine is the acid hydrolysis of chrondroitin sulfate C which is obtained by extracting ; cartilaginous tissues such as tendons, trachea and nasal septa. These yields are uncertain and it is difficult to obtain a crystalline product.
Numerous chemical syntheses exist which include the opening of 1,6:2,3,-dianhydro-~-D-talopyranose with ammonia or with azide ion. However, these methods involve six to eleven separate chemical transformations starting from the simple sugars. Shorter methods depend upon rather rare sugars as starting materials.
Inversion of the C-4 configuration of glucosamine through displacement of a 4-0-sulfonate of 2-acetamido-2-deoxy glucopyranosyl derivatives has also been utilized for the synthesis of D-galactosamine.
However, the elaboration of glucosamine to the necessary starting material is tedious.

.
The synthesis of lactosamine is more difficult as it nec-essarily involves a glycosylation of a galactosyl halide with an elaborate . derivative of 2-acetamido-2-deoxy-glucose. The most recently published method requires nine chemical transformations, starting from 2-acetamido-, . .

2-deoxy glucosamine, prior to the glycosylation step.
In accordance with a feature of the present invention, there is provided a reagent that allows efficient and high yield pre-parations of glycosides which contain the 2-acetamido-2-deoxy-a-D-galactopyranosyl group which is-found, for example, in the antigenic determinant for the human A blood group and the Forssman antigen. The ~` 25 reagent thus claimed useful is 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl chloride (XXIII) prepared simply from D-galactal triacetate (I) in high yield.
It has long been anticipated that the use of a ~-glycosyl halide would tend to yield the a-(1,2-cis)-glycosidic linkage through Walden inversion of the reacting center under Koenings-Knorr reaction conditions when the 2-substituent is so chosen as to not participate in a reaction at the anomeric center. Thus, for example, Wolfrom, Thompson and Linebeck (J. Org. Chem., 28, 860 (1963)) developed tri-0-acetyl-2-nitro-~-D-glucopyranosyl chloride for the purpose of synthesizing a-D-glucopyranosides. Indeed, several papers have appeared in the recent literature which utilize 2-azido-2-deoxy-~-D-glycopyranosyl chlorides such as is reported in processes of this invention leading to the formation of 2-azido-2-deoxy-~-D-galactopyranosides. However, it must be noted that the processes reported by Paulsen and co-workers (Angew.
Chem., Int. Ed., 14, 558 (1975); Tet. Lett., 1493 (1975) and 2301 (1976);
Angew. Chem., Int. Ed., 15, 440 (1975)) are of limited, if any commercial value in view of the extreme difficulty in achieving the synthesis of : the desired 2-azido-2-deoxy reagent; namely, 6-0-acetyl-2-azido-3,4-- O-benzyl-2-deoxy-~-D-galactopyranosyl chloride.
;~ 15 This invention reports a novel process for preparing ef-~i~ ficiently the compound 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl chloride ( XXI I I ) and its engagement in reactions with :: :
alcohol to form 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosides (A) under appropriate Koenings-Knorr type conditions for the condensation.
The invention in AcO

AcO ~

OR
A

part concerns the discovery of processes that render compound XXIII a readily available reagent for use in reactions leading to products of type A. Thus, it has become commercially feasible to synthesize the terminal trisaccharide antigenic determinant for the human A blood as is present in structures B for the type 1 and type 2 antigenic determinants for the human A blood group. The trisaccharide is synthesized in a form useful for the preparation of artificial antigens and immunoabsorbents related to the human A blood group.

` (-` - !
111~4~7 , H~ _O

, f o~ ' OH
H
(Type 1 A Determinant) HO
OH
HO NHAc . AcNH ¦ ~ O \ HO
"', ~ 0~o~ I

- ~ OH
HO
(Type 2 Determinant) .'` ~
B
The formation of a-azido-~-nitratoalkanes from the reaction of olefins with sodium azide and ceric ammonium nitrate has been reported by Trahanovsky and Robbins (J.Am. Chem. Soc., 93, 5256 (1971)). However the extension of the above reaction to vinylic ethers or structures as complex as D-galactal triacetate is not obvious. The base of this invention was the discovery that the addition of the azide and nitrate groups to 1,2-unsaturated sugars can be made to proceed in high economical yield to form the 2-azido-2-deoxy glycosyl nitrate.
SUMMARY OF THE INVENTION
In accordance with the basic aspect of the present invention, the treat~ent of protected glycals with azide-ion in the presence of 11.1~17 ,;
ceric ammonium nitrate results in the addition of an azide group and a nitrate group to the C-2 and C-l positions, respectively, of the glycal.
` These novel products, namely the anomeric mixture of 2-azido-2-deoxy glycosyl nitrates, allow entrance into the following classes of compounds: I
(1) the 2-amino-2-deoxy sugars by hydrolysis of the nitrate group and 1, reduction of the azido group, (2) the 2-azido-2-deoxy glycosyl halides by displacement of the glycosyl - nitrate,

(3) the 2-amino-2-deoxy glycosides by reaction of the 2-azido-2-deoxy glycosyl halides.
The virtue of the azido group is that it is a non-participating progenerator of an amino function and as such does not interfere with the synthesis of the 2-amino-2-deoxy-~-D-glycosides.
In accordance with a feature of the present invention, the 2-azido-2-deoxy glycosyl nitrates can be converted to the corresponding 2-amino-2-deoxy sugars by hydrolysis of the nitrate and protecting groups, and reduction of the azido group by methods well known to those skilled in the art. Hydrolysis may precede reduction or vice versa. N-acetylated ; derivatives of the amino sugars can be obtained by conventional methods.
In accordance with a further aspect of the present invention, - the 2-azido-2-deoxy glycosyl nitrates may be treated with a halide salt to effect the displacement of the nitrate group and to produce the 2-azido-2-deoxy glycosyl halides, which are novel compounds.
In a preferred procedure, by treating with iodide ion, an anomeric mixture of the glycosyl nitrates produces the thermodynamically more favorable anomer, 2-azido-2-deoxy-~-D-glycosyl iodide. The ~-glycosyl iodide is readily displaced with one equivalent of chloride ion through - inversion to give in high yields the 2-azido-2-deoxy-~-D-glycosyl chloride.
This route to the ~-halide is advantageous as it allows conversion of the nitrates to a reaction product which comprises predominantly the 2-azido-2-deoxy-~-D-galactosyl chloride, which is useful for the formation of a :-2-deoxy-~-D-glycosjde, an integral unit of the A blood group determinant.
The reagent thus claimed useful-is 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl chloride (XXIII).
The 2-azido-2-deoxy glycosyl halides may be used to prepare 2-amino-2-deoxy glycosides under conditions for glycosidation, such as those generally known in carbohydrate chemistry as Koenings-Knorr conditions. These reactions involve the treatment of the glycosyl halide with an alcohol in the presence of a promoter to effect the replacement of the halogen by the alkoxy group of the alcohol. The 2-azido-2-deoxy glycoside, thus obtained, is reduced by methods well known to persons skilled in the art to obtain the 2-amino-2-deoxy glycosides. In addition the protecting groups can be removed in order to deblock the glycoside.
Specifically, 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl ,.
chloride may be reacted with 8-methoxycarbonyloctyl-2-0-(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-4,6-0-benzylidene-~-D-galactopyranosyl in the presence of a promoter. The trisaccharidic product is deblocked and its azido group is reduced to the amine which is subsequently acetylated to give 8-methoxycarbonyloctyl-3-0-(2-acetamido-2-deoxy-a-D-galacto pyranosyl)-2-0-(~-L-fucopyranosyl)-~-D-galactopyranoside. This latter product corresponds to the antigenic determinant for the human A blood . .; .
; group and can be used to prepare an immunoabsorbent specific for the anti-A antibodies by attachment to an insoluble support. Also, this latter product can be used to inhibit the reaction between anti-A
antibodies and human A erythrocytes. Furthermore, the product can be used to prepare artificial antigens which allow the raising, through immunization, of monospecific anti-A antibodies in test animals. The subsequent isolation of these antibodies using the immunoabsorbent then provides an important and useful reagent for cell and tissue typing.

Broadly stated, the invention provides a process which comprises reacting an acylated 2-azido-2-deoxy glycosyl nitrate with a halide salt in a suitable solvent to form an acylated 2-azido-2-deoxy glycosyl halide~
The invention also broadly provides a process which comprises reacting an acylated-2-azido-2-deoxy glycosyl nitrate with an iodide salt in a suitable solvent to form an acylated-2-azido-2-deoxy-~-glycosyl iodide; and reacting said ~-glycosyl iodide with a chloride salt in a suitable solvent to produce the acylated-2-azido-2-deoxy-3-glycosyl chloride.

DESCRIPTION OF THE DRAWINGS
Figure 1 is a formula sheet showing structure formulas and : names for compounds referred to by number in the specification; and Figure 2 is a reaction sheet showing examples of the reactions described in the specification.

.' :

- 7a -114~7 .
DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION
The Azidonitration Reaction:
The Formula Sheet provides structural formulas for compounds I
to LII. Reference is made to these compounds in the course of this des-cription and in specific experimental examples which demonstrate the invention.
Examples I through VII show the reaction of suitably protected glycals with ceric ammonium nitrate and an azide salt to form the I corresponding 2-azido-2-deoxy glycosyl nitrates.
:: j ; 10 The term glycal applies to 1,2 - unsaturated sugars which are characterized by the structural entity ,...................................... ~o :~. \~/
The term protected glycal denotes that the hydroxyl substituents have been masked by blocking groups such as acetyl, propionyl, and benzoyl which, being less reactive than the hydroxyl group, will not participate in subsequent reactions. In this manner,the properties of the glycal other than those of the unsaturation will be retained.
Examples of protected glycals are 3,4,6-tri-0-acetyl-D-galactal, - I; 3,4,6-tri-0-acetyl-D-glucal, VI; 3,4,6-tri-0-benzoyl-~-galactal, X;
hexa-O-acetyl-D-lactal, XIII; and 3,4-di-0-acetyl-D-xylal, XVI.
In the azidonitration of glycals, demonstrated in Examples I
through VII, the protected glycals are reacted with an excess of a 2:1 (mole/
mole) mixture of ceric ammonium nitrate and an azide salt. It is known that these two salts react to form nitrogen gas as a product. The slight excess of reagent is used to compensate for this loss.
Without being bound by the same, the following mechanism is suggested for the azidonitration reaction:

1~11417 Cet4 t N3 Ce 3 + N3 ~, O~c ~ OAc qAc ~ OAc A I QAc ~ I /---OAc AcO ~ ~ ~ ~ L ~ J

~ACO ~ ~ ACO~ ~ OAc ,.ON02 Ce(IV) is a strong oxidizing agent and strips an electron from the negatively - charged azide ion. The resulting azide radical adds across the 1,2-un-saturated bond of the glycal to form an intermediate radical. A second Ce(IV) ion may oxidize the intermediate radical to give an oxycarbonium. The addition of a nitrate ion, to the C-l position, results in the 2-azido-2-deoxy glycosyl nitrate.
The azide salt may be any of the common alkali metal azides.
Sodium azide is used, preferahly for reasons of cost and handling, but the lithium or potassium azides are also suitable.
A 2-azido substituent is desirable as it will not interfere in the subsequent formation of a ~-glycosidic linkage at the anomeric (C-l) center and can be reduced to an amino function by well known methods to produce 2-amino-2-deoxy sugars.
A solvent is- used which is able to dissolYe the three reagents, the nonpolar glycal and the ionic salts, at a level to provide sufficient concentrations of these in the reaction mi`xture. In additi`on, the solvent should be substantially inert to reacti:on and resistant to oxidation by the ceric salt. The preferred solvent is acetonitrile because of its _ g

4~L7 resistance to oxidation and its ahility to proYide appropriate concentra-tions of the reacting speci.es in s.oluti:on. ~ther solvents can be used such as ethyl acetate or acetic'aci'd, but s.ide reactions are rather severe in the case of the latter. The solvent is preferably dried prior to use as the presence of water was found to support side reactions.
'. Due to the dissimilarity in the solubility of the reactants, '. effective sti.rring is required to mai'ntain suffi.cient concentrations in the reaction mixture and to ensure an efficient rate of reaction.
' The preferred reaction temperature range is from -25C to +25C.
' 10 The lower limit was determined by the freezing point of the acetonitrile, the solvent preferentially used; while the upper li:mit was arbitrarily '' chosen as a cutoff above which competing si.de reactions became significant.
Although the reaction kinetics were slower at low.er temperatures, giving rise to longer reaction times, the yields of the desired products were . 15 better.
'' Although the reaction can be performed in air, an inert atmosphere, such as nitrogen, is preferably used.
Examples I and II illustrate two di.fferent techniques, within ~'' the scope of the present invention, for preparing the 2-azido-2-deoxy nitrates of 3,4,6-tri-0-acetyl-D-galactal. The fi'rst is a process which : is attractive to commercial production while the second describes the experiment which led to the discovery. It is wi.thin the scope and spirit of this invention to claim all those variations in the reaction conditions and work-up procedures that are evident to chemists competent to consider and to test the effectiveness of alternate procedures which would involve such variations as changes in reaction and extractïng solvents, modes of addition, stirring rates and temperature range.
EXAMPLE I
The reaction of 2,3,~-tri-0-acetyl-=-galactal.(I) with ceric ammonium nitrate in the.presence.of.sodium azide A three-necked, five liter, round bottom flask equipped with an `~

inlet tube, exhaust tube and an effic;ent mechanical stirrer was charged with solid ceric ammonium nitrate (899.90 9, 1.64 mole) and solid sodium azide ~53.37 9, 0.82 mole) and cooled to -15C under a nitrogen atmosphere.
2,3,4-tri-0-acetyl-D-galactal(I)(150 9, 0.551 mole) was dissolved in anhydrous acetonitrile (3.4~) in a three-necked, four-liter flask equipped with an inlet and an outlet tube. This soluti~on was cooled to -15C
while sweeping with nitrogen. By applying a positive pressure of nitrogen "' the acetonitrile solution was pumped into the vessel containing the solid '- reactants vi'a an inert tube. After camplete addition of the acetonitrile solution (approximately 1 minute ), mechanical sti:rring was commenced and ' continued for approximately l5 to 20 hours or until such time as no glycal remained on examinati'on of the reacti'on mi'xture by thin layer chromatography (t.l.c.) on silica gel eluted with hexane-ethyl acetate (v/v~ 6:4. At :
', that time toluene (1~) and cold water (1~) were added and the reaction ' 15 vessel was removed from the cooling bath. This mixture was transferred to a ten-liter container and after addition of toluene (2~) the organic layer was separated and transferred to a separatory funnel. This solution was washed with cold water (3 x lR). The organic layer was filtered through toluene-wetted filter paper and the filtrate was concentrated in vacuo at a temperature below 40C to a syrup (200 9~. The proton magnetic resonance (p.m.r.) spectrum of this syrup showed it to be composed mainly of 2-azido-2-deoxy nitrates. The composition of the product was 37% of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl nitrate (II), 55% of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl nitrate (III) and 8h of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-P-talapyranosyl nitrate (IV).
The low yield of compound IV indicates that the azidonitration reaction is highly stereoselecti`ve at the C-2 positi`on.
Tri'turation of a portion of the syrupy product (21.0 9~ with cold ethyl ether gave compounds II and ~V ~8.3 9) whi'ch co-crystallized.
The mother li'quor contained almost pure ~-D-nitrate, III, (12.6 9).

-` 1111417 '~ Compound III could not he crystallized. The infrared (i r.) spectrum (film) ' of compound III displayed absorbances at 2120 cm 1(N3) and 1650 cm 1 (ON02); its partial p.m.r. spectrum in CDC13 was, ppm 5.71 ~d, 1, Jl 2 9.0 Hz, H-ll, 5.42 (q, 1, H-4), 5.08 (q, 1, J3 4 3.2 Wz, H-3), 3.87 (q, 1' J2 3 10.8 Hz, H-2), 2.18, 2.10, 2.03 (3s, 9, 3 OAC).
Compound II`, free of the talo azide (lV), was obtained by anomerization of the ~-D-ni'trate, III, with ni~trate ion. A solution of the syrupy ~-D-nitrate, III, (9.50 9, 25.5 mmole~ and anhydrous lithium - nitrate (3.50 9, 50.1 mmole) in 4:1 (v/v) acetoni'trile:dimethylformamide (35 ml) was stirred for 42 hours at ambi~ent temperature, after which time it was diluted with dichloromethane (250 ml) and washed with ice cold water (3 x 125 ml). The organic solution was dried and evaporated to give a syrup (9.0 9). The p.m.r. spectrum of this syrup showed it to be a mixture of 63% ~- and 37% ~-~-ni'trates, II and III. Crystallization from ethyl ether gave the ~-D-nitrate, II, (6.2 g), m.p. 103 - 104C, [~]D5 + 125 (c 1, chloroform). The infrared spectrum (film) of compound II displayed absorbances at 2120 cm 1 (N3) and 1650 cm 1 (ON02); its partial p.m.r. spectrum in CDC13 was, p.p.m. 6.34 (d, 1, Jl 2 4.1 Hz, H-3), 4.12 (q, 1~ J2 3 11.5 Hz, H-2), 2.18, 2.09, 2.02 (3s, 3 OAC).
A minor side product (<10%) of the reaction could be isolated i~
either by chromotography on si'lica gel of the reacti'on mixture or, in some cases, by evaporation of the three aqueous washings obtained during the reaction product workup described above. The compound readily crystallized - from the washings by evaporation or upon tri'turati'on with ethyl ether, and ~: 25 was shown to be N-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-=D-galactopyranosyl) acetamide (V); m.p. 142-143.5C, [~]D5 + 68.0 (c 1, chloroform). Its ' partial p.m.r. spectrum in DMS~-db was,p.p.m. 9.83 (d, 1. JNH 1 9-5 Hz, NH),

5.78 (q, 1~ Jl 2 5.5 Wz, H-l), 5~48 ~1~ q~ 32 3 11.3 Hz, H-3), 5.22 (1, d, J3 4 3.5 Hz, H-4), 4.2~ (q, 1, H-2~.

`` 1111417 , EXAMPLE II
The reaction of 3,4,6-tri-0-acetyl-D-galactal (I) with ceric ammon;um `'' nitrate i'n the presence of sodium azi''de~
,, Distilled 2,3,4-tri-0-acetyl-D-galactal (I~ ('21.1 9, 0.007 M) (b.p. 147-155 at 0.1 mm) was dissolved in dry acetoni-trile (420 ml) and cooled to -25C under a nitrogen atmosphere i'n the dark. A mixture ~- of solid ceric ammonium ni'trate (lOa.2 9, Q.182 mole) and soli-d sodium '~ azide ~6.043 9, 0.092 mole) was added all at once and the resulting ; suspension was stirred for 15 hours at -25C~ At this time cold ethyl ~- ~ 10 ether (40Q ml) was added and the resulting mixture filtered to remove any - .
' solids. The filter cake was washed with diethyl ether (2 x 100 ml) and the combined filtrate was poured into i-ce water (500 ml). The organic solution was separated and washed with i-ce cold water (3 x 500 ml), dried - over anhydrous sodium sulfate, fi'ltered and evaporated to give a syrup - 15 (21.0 9), which corresponded to a 73% yield of the crude nitrates (II and III). Thin layer chromatography examinati-on on silica gel developed ; with 6:4 (v/v) hexane: ethyl acetate showed no remaining starting material.
P.m.r. examination showed the product to be essentially identical to the -' syrupy product obtained in Example I.
Because of the reactivity of glycosyl nitrates in general, '; care must be exercised in the handling of these compounds so as to not ~-' effect undesired decomposition or solvolytic reactions. The mixture of a- and ~-nitrates obtained may vary since, as is demonstrated in Example I, the compounds are readily interconverted in the presence of nitrate ion. The mixture is as useful as either of the pure products for the purposes of this invention, as will be demonstrated later. In general, no effort is made to separate the compounds (I'I and III). However, it was found that the ~-anomer (I-II`) i`s readily obtai`ned in the crystalline state and if this substance i's desired, the yi`eld can be improved by anomerization of the ~-anomer which is- the thermQdynaml~cally leas stable compound.

-,- - 13 -.

~: -`; 1111417 ' The azidonitration reaction demonstrated in Example I is not restricted to the acetylated galactal, I, but finds useful application with suitably 0-protected glycals in general. This is demons;trated by Example ` III wherein the selected reactant is tri-0-acetyl-~-glucal, (VI), a different hexal, and further exemplified through the use of hexa-0-acetyl-D-lactal - (XIII), having a disacchari'de structure, i`n Example IV and of 3,4-di-0-acetyl-D-xylal (XVI), a pental, in Example'Y.
Further, Example III illustrates that the temperature at which the reaction is conducted may be varied although product puri'ty decreases at reaction temperatures above 0C. TKe use of potassium azide is also demonstrated.
EXAMPLE III
The reaction of 3,4,6-tri-0-acetyl-D-glucal (VI) with ceric ammonium nitrate in the presence of potassium azide !
Treatment of 2,3,4-tri-0-acetyl-D-glucal ~VI) ~5.86 g, 21.5 mmole) with ceric ammonium nitrate (27.8 9, 50.7 mmole~ and potassium azide (2.39 9, 25.7 mmole) at 25C by the method of Example I for tri-0-acetyl-D-galactal, gave a mixture of 2-azido nitrates in 60% yield. Of the azido nitrate products, this mixture was shown to be composed of 3,4,6-tri-0-- 20 acetyl-2-azido-2-deoxy-~-D-glucopyranosyl nitrate (VIII~ 42.~%, 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-glucopyranosyl nitrate (VII) 24~, ' and 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-mannopyranosyl nitrate (IX~
33%. This composition was based on the relative intensities of the p.m.r.
'' anomeric signals assigned to compounds VII, IX, and VIII which were at 6.4 p.p.m., J=4.0 Hz, 6.28 p.p.m., J=1.8 Hz and 5.72 p.p.m., J-8.8 Hz, re-spectively.
' EXAMPLE IV
The reaction of hexa-0-acetyl-D-lactal (XIII) with ceric ammonium nitrate in the presence of sodium azide The azidonitration of hexa-0-acetyl-D-lactal (XIII) serves as a new route to the important disaccharide'known as a lactosamine and which is a building block of oligosaccharides which form the core structure of f~
41'7 ' oligosaccharides found in human milk and the antigenic structures of the human blood group substances.
. I
Treatment of hexa-0-acetyl-=D-lactal ~XIII) (1.0 9, 1.7g mmole) with ceri:c ammonium n;trate ( 2.45 9, 4.48 mmole) and sodi'um azide (0.174 g, ; 5 2.685 mmole) by the method of Example ~I gave a mixture of the 2-azido '- nitrates (0.89 9) in greater than 75% yield. P.m.r. examination showed signals at 6.3a p.p.m. (d, 4.25 Hz) and 5.56 p.p.m. ('d, 8.5 Hz) which were ass-igned to the anomeri`c protons of the 2-azi`do nitrates XIV and XV, respectively. Trituration of this s~yrup w;tfi ethyl ether gave crystalline 3,6-di-0-acet~1-4-0-~2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl~-2-azido-2-deoxyl-~-D-glucopyranosyl nitrate ~XV) (0.5 9) in 42% yield: m.p.
69 ~ 70; [~]D5 + 15 (c 1, chloroform). The infrared spectrum (nujol mull) of compound XV di'splayed absorbances at 2120 cm 1~N3) and 1650 cm 1 (ON02); it partial p.m.r. i`n CDC13 was, p.p.m.' 5.56 ~d, 1, Jl 2 8.5 Hz, lS H-l), 3.56 ~q, 1, J2 3 8.25 Hz, H-2).
Column chromatography of the mother liquor, after the removal of crystalline compound XV, on si1i'ca gel developed with hexane-ethyl acetate-ethanol (v/v) 10:10:1 afforded addi`ti`onal quantities of compound XV (0.05 9) and 3,6-di-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-~-galuctopyranosyl)-2-- 20 azido-2-deoxy-~-D-glucopyranosyl nitrate ~XIV) (0.31 9) which was crystal-lized from ethyl ether: m.p. 138 - 140; [~]2D5 + 69.7(c 1, chloroform).
The infrared spectrum (nujol mull) of compound XIV displayed absorbances at 2120 cm 1 (N3) and 1650 cm 1 (ON02); its partial p.m.r. spectrum in CDC13 was, p.p.m. 6.30 (d, 1, Jl 2 4.25 Hz, H-1),3.72 (q, 1, J2 3 10.5 Hz, H-2).
EXAMpLE y ' The reaction of 3, 4-di-0-acetyl-D-xylal'~XYI;) with ceric ammonîum nitrate .
in the presence of sodium azide~' Example V shows that the appli`cati`on of the process of azido : 30 nitration can be extended to the pentopyranoglycals.

.
~ - 15 -!
1~11417 ;:'"' ~ Treatment of di-0-acetyl-D-xylal (XVI) (29~ (0,472 9, 2.36 ., I
mmoles) with ceric ammonium nitrate ( 4.39 9, 8.0 mmoles) and sodium azide (0.260 9, 4.0 mmoles) by the method des-cribed in Example II gave a mixture of 2-azido-nitrates in 88% yield. P.m.r. exami'nation of the product mixture showed signals at 5.70 p.p.m. (d, 7.5 Hz), 68%, 6.28 p.p.m.
(d, 4.0 Hz), ~16%, and 6.56 p.p.m. ~d,' 4.5 Hz~, ~16%. The major product was shown to be 3,4-di-0-acetyl-2-azido-2-deoxy-~-D-xylopyranosyl nitrate XVII by double i'rradiation experi'ments whi'ch showed the presence of a quartet at 3.70 p.p.m., wi'th J2 3 ~' 8.75 Hz and Jl 2 = 7-5 Hz, which was assigned to H-2 of compound XVII. The products comprising the remaining 32% of the mixture of 2-azido-2-deoxy-ni'trates must be the ~ and ~-=D-lyxo anomers XVIII as anomerization of tne mi'xture of ni'trates, by the method described in Example I for compound III, caused the appearance of a new signal in the p.m.r. spectrum of this product mi'xture, at 6.31 p.p.m.
(d, Jl 2 3.65 Hz). This signal is attri'buted to the anomeric proton of '-: 3,4-di-0-acetyl-2-azido-2-deoxy--=D-xylopyranosyl nitrate (XVIX).
EXAMPLE VI
' The reaction of 3,4,6-tri-0-benzoyl-=D-galactal (X) with ceric ammonium , :, - - ............ ..
nitrate in the presence of sodium azi-de.

The azidonitration reacti-on i's not restricted to acetylated -glycals but can be applied to any sui'tably protected glycal. For example, the blocking groups may be propionyl or benzoyl. This is demonstrated in :; this example wherein 3,4,6-tri-0-benzoyl-D-galactal (X) is used as the -~ starting material.
Treatment of 3,4,6-tri-0-benzoyl-=-galactal (X) (7.18 9, 12.2 mmole) with ceric ammonium nitrate (20.2 9, 36.6 mmole) and sodium azide (1.18 9, 18.1 mmole) by the method descri~ed i'n Example I for tri-0-acetyl-D-galactal gave a mixture of 2-azido-2-deoxy-nitrates (7.5 9~ in 75% yield. Examination of the p.m.r. spectrum of tfie crude product in CDC13 showed i-t to be composed of 2-azi`do-3,4,6-tri-_-benzoyl-2-deoxy-~-.~

f , I
11114~7 `.'' I

D-galactopyranosyl nitrate (X11'(30%~ and 2-azido-3,4,6-tri-0-benzoyl-2-deoxy-~-D-galactopyranQsyl nitrate ~XII~ (45%). The anomer;c si'gnal of the a-D-ni'trate was observed at 6.67 p.p.m. with Jl 2 = 4.6 Hz.' Although the anomeric signal of the ~-D-anomer was masked, the H-2 signal was observed at 4.20 p.p.m. as a large tri'plet with Jl 2 = 9-5 Hz.
EXAMPLE VII
Reaction of 3,4,6-tri-0-acetyl-D-galactal wi`th sodium azide and ceric , ammonium nitrate in ethyl acetate.
Although acetoni'trile i's the preferred solvent, the azidonitration ; lO reaction is not restricted to the choice of this solvent. This is demon-strated by this example wherein ethyl acetate is used as the solvent.
Treatment of tri-0-acetyl-D-galactal (I) ~0.30 9, l.09 mmole) '~ with ceric ammonium nitrate (l.4l 9, 2.57 mmole) and sodium azide (0.084 9, ; l.29 mmole) in ethyl acetate (5 ml) by the method described in Example I
gave a mixture of the 2-azido nitrates in greater than 60% yield. P.m.r.
' examination of the product showed the 2-azido-nitrate composition to be similar to that described in Example I. However, examination by thin layer chromatography on silica gel, developed with 6:4 (v/v) hexane:ethyl acetate, - gave evidence that more side reactions had occurred in this solvent.

- l7 -417 ' ; Conversion of Azidon;trates to Aminosugars The acylated 2-azido-2-deoxy nitrates can be converted to the corresponding 2-amino-2-deoxy sugars by hydrolysis of the nitrate and acyl groups and reduction of the azido group by methods well known to those skilled in the art. Hydrolysis may precede reduction or vice versa. The aminosugars, in particular galactosamine and lactosamine and their N-acetylated derivatives are important building units for the blood group substance antigenic determinants. The N-acetylated derivatives are pre-pared from the aminosugar by methods well known to those skilled in the art.
The aminosugars may also be used to prepare the 2-acetamido-2-deoxyglycoses.
Reduction ot azido groups to amino groups is well known and can be conducted in virtually quantitative yield under a wide variety of conditions including reductions with metals such as sodium or zinc, reduction by catalytic hydrogenation using such catalysts as nickel, platinum or palladium, reduction using hydrides such as sodium borohydride, ; borane and lithium aluminum hydride, electrolytic reductions and reduction by hydrogen sulfide under alkaline conditions.
; Broadly stated, the invention provides a process for converting an acylated 2-azido-2-deoxy glycosyl nitrate to a 2-amino-2-deoxy glycose which comprises reducing the azido group to an amino group and hydrolyzing - the acyl and nitrate groups.
- The nitrate group of the acylated 2-azido-2-deoxy-nitrate may be displaced with an acyl group by conventional methods prior to - hydrolysis or reduction. For example, the nitrate compound may be treated with sodium acetate in acetic acid as illustrated in Examples XIV - XVI.
EXAMPLE VIII
Preparations of the anomeric 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-D-galactopyranoses (XXVII) and (XXVIII).
A solution of the pure 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-; 30 galactopyranosyl nitrate (III) (0.15 g, 0.40 mmQle) and sodium acetate (0.65 g, 0.80 mmole) in glacial acetic acid (2 ml) was heated to 100 - !

for 15 minutes at which time examination by thin layer chromatography of silica gel developed with 6:4 (v/v) hexane:ethyl acetate showed one homogeneous spot of lower Rf than compound III. The solution was diluted with dichloromethane (5 ml) and washed with ice cold water (5 ml).
Evaporation of the solvent, after drying over sodium sulfate and filtration, gave a syrup (0.134 9, 90% yield), which spontaneously crystallized upon trituration with ethyl ether.
Recrystallization from ethyl ether or cold ethanol gave an analytically pure sample of l,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-D-galactopyranose ~XXVII), m.p. 114 - 115, [~]D5 + 91.70 (c 1.05, chloroform), i.r. (film) 2120 cm (-N3).
The p.m.r. spectrum of compound XXVII in CDC13 showed, in part, p.p.m. 6.38 (d, 1, Jl 2 3-7 Hz, H-l), 5.50 (q, 1, J3 4 3 Hz, H-4), 5.36 (q, 1, J2 3 7 Hz H-3), 3.97 (q, 1, H-2).
" ~ .
A solution of the crude 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl nitrate II (1.01 9, 2.70 mmole) and sodium acetate (0.43 9 5.20 mmole) in glacial acetic acid (10 ml) was heated to 100 for 20 minutes. The reaction solution was then diluted with dichloromethane - (50 ml) and washed with ice cold water (250 ml). Evaporation of the solvent, after drying over sodium sulfate and filtration, gave syrup (1.0 9).
. . i ~; Inspection of this syrup by p.m.r. spectroscopy showed it to be composed of , compound XXVII (30%) and 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-D-. .
~ galactopyranose XXVIIr (60%). The anomeric proton of the ~-anomer - (XXVIII) was assigned to a doublet, with J=8.5 Hz, at 5.61 p.p.m.
Compounds XXVII and XX~III were obtained in a near 3:1 `, mixture by acetolysis in acetic acid containi`ng sodium acetate of the mixture of compounds II and I:II obtained by way of the process described ~:- in Example II.
,:~

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(~
1~11417 EXAMPLE IX
Preparations of 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-=D-gluco and manopyranoses (XXIX and XXX).
A mixture of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-=D-mannopyranosyl nitrate (IX) and the ~- and ~- anomers of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-=D-glucopyranosyl nitrate (VII and VIII), obtained as described in Example III, was treated with a solution of sodium acetate (0.350 9, 4.26 mmoles) in acetic acid (10 ml) at 100C for one hour. Work up of the product mixture by the method of Example II gave a foam (0.70 9).
Column chromatography (30 x 2 cm~ on silica gel (70 9) eluted with hexane-; ethyl acetate-ethanol (v/v) 10:10:1 afforded the separation of the gluco-(XXIX) and mano(XXX) 2-azido-2-deoxy acetates, 0.340 9 and 0.310 9 respectively.
Pure 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-=D-glucopyranose (XXIX) (0.211 9, 21%) was obtained by recrystallization from ethyl ether;
m.p. 117 - 118C, [~]D5 + 128 (c 0.9, chloroform). The partial p.m.r.
spectrum of compound XXIX in CDC13 gave, p.p.m. 6.29 (d, 1~ Jl 2 3.5 Hz, H-l), 5.45 (t, 1, J3 4 9.0 Hz, H-3), 5.08 (t, 1, J4 5 9.Q Hz, H-4), 3.65 (q, 1~ J2 3 9 0 Hz, H-2~.
Pure 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-=D-mannopyranose (XXX) (0.220 9, 22%) ~as obtained by recrystallization from ethyl ether;
m.p. 131 - 132C, [~]D5 + 78.6 (c 1.02, chloroform). The partial p.m.r.
spectrum of compound XXX in CDC13 gave, p.p.m. 6.09 (d, 1, Jl 2 1.8, H-l).
EXAMPLE X
Preparations of the anomeric forms of 1,3,6-tri-0-acetyl-4-0-(2,3,4,6-.-. i tetra-0-acetyl-~-=D-galactopyranosyl)-2-azido-2-deoxy-=D-glucopyranose (XXXI and XXXII) Treatment of an anomeric mixture of 3,6-di-0-acetyl-d-0-(2,3,4,6-tetra-0-acetyl-~-galactopyranosyl)-2-azido-2-deoxy-=D-glucopyranosyl nitrate (XIV and XV) comprising about 70% of the ,r-~-anomer (XY) (3.50 9) with sodium acetate (2.16 9, 26.3 mmole) in acetic acid by the method described in Example XV gaYe crystalline 1,3,6-tri-' O-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-g-=D-galactopyranosyll-2-azido-2-deoxy-a-=-glucopyranose (2.48 9) (XXXI) in 73% yield. Recrystallization from ethyl acetate-pentane gave the pure ~-anomer ~XXXI), m.p. 77 - 78C, [~]2D5 ~ 55.4 (c 1, chloroform). The partial p.m.r. spectrum of compound XXXI in CDC13 was, p.p.m. 6.22 (d, 1~ Jl 2 3.65, H-l), 3.46 (q, 1~ J2 10.5, H-2).
Similar treatment of the pure a-nitrate, XIV, in the manner described above gave crystalline 1,3,6-tri-0-acetyl-4-n-(2,3,4,6-tetra-; 0-acetyl-~-@-glucopyranose (XXXII) in good yield (70%).
- Excellent yields of compound XXXII were also obtained by the treatment of 3,6-di-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-=D-galactopyranosyl)-2-azido-2-deoxy-~-=D-glucopyranosyl chloride (XXV) (Q.264, 0.414 mmoles) or of the corresponding ~-bromide, XXVI, with silver acetate (0.137 9, ; 1.656 mmoles) in acetic acid (5 ml) at ambient temperature for one hour.
At that time, the reaction solution was diluted with dichloromethane (20 ml), filtered and washed with water (2 x 20 ml). The organic layer was dried and evaporated to give a white foam (0.25Q 9~. Crystallization of this material from hot methanol gave 1,3,6-tri-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl~-2-azido-2-deoxy-~-=-glucopyranose `~, (XXXII). The partial p.m.r. spectrum of compound XXXII in CDC13 gave, ~ p.p.m. 5.51 (d, 1, Jl 2 8.75 Hz, H-l), 3.57 (q, 1~ J2,3 I EXAMPLE XI
Preparations of the 2-acetamido-1,3,4,6-tetra-Q-acetyl-2-deoxy-~- and ~-D-galactopyranoses ~XXXIV and XXXV~
This example provides an effi:cient process, based on reduction by zinc, for the conversion of the mixture of anomeric 1,3,4,6-tetra-0-acetyl-2-azida-2-deoxy-D-galactoses, XXV M and XXVrII, o~tained in , , 11114~7 Example YIII to an anomeric mixture of the 1,3,4,6-tetra-0-acetyl-2-acetamido-2-deoxy-galactopyranoses, XXIV and XXV, and how this m;xture is useful for the preparation of D-galactosamine hydrochloride (XXXVII).
Glacial acetic acid (200 ml) and sodium acetate (8.2 9, û.l mole) were added to the a- and ~-anomer;c mixture of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-=D-galactopyranosyl nitrates (II and III) (32 9, 0.08 mole) prepared by the method of Example II and the mixture was stirred for one hour at 100. Zinc metal (12.8 9, 0.2 mole) was then added to the solution cooled to 60 and stirred for 15 minutes. Acetic anhydride (17 ml) was added and the mixture heated on the steam bath (100) for one hour and filtered. The solution was poured into 100 ml of water and stirred for one hour. Then 300 ml of water was added and the mixture extracted ; three times with dichloromethane (100 ml). The extracts were combined, filtered through dichloromethane-wetted paper and evaporated to a thick syrup which hardened to a crystalline mass on trituration with ether.
The p.m.r. spectrum of this product was in agreement with that expected for