CHEMISTRY OF OSAZONES

BY HASSANEL KHADEM

Faculty of Science, Alexandria University, Alexandria, Egypt, U . A . 12.

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
11. Nomenclature. . . . ..................... 140
111. Preparation.. . . . .

3. The Chelated Structure.. .

2. Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1@2
3. Oxidation.. . , . . . . . _ . , . . . . . . . . , . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . 164
4. Osotriazole Formation. , . . . , . . .................................. 166
5. Formation of Formazans.. . . . . , . , . . . . . . . . . . . . . . . , . . . . . . . . , . . . . . . . . . 169
6. Action of Alkalis. . . . . .......................................... 172
7. Conversion into Glyco oses.. . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
8. Transhydrazonation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . 174
9. Anhydro-osazones.. . . . . . . . . . . . . . . . . . . . . . .... .... ... 175
VIII. Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

I. INTRODUCTION'

In the year 1884, Emil Fischerl* prepared the first sugar osazones and
suggested their use for identification purposes. He later used the fact that
D-glucose and D-mannose give the same osazone in his determination of
the structure of these two monosaccharides,z and he accomplished, through
osazone formation, the conversion of aldoses into ketoses.* Since then,
osazone formation has been used in a multitude of syntheses in the carbo-
(1) See E. G. V. Percival, Advan. Carbohydrate Chem., 3, 23 (1948).
(la) E. Fischer, Ber., 17, 579 (1884).
(2) E. Fischer, Ber., 24, 1836 (1891); see C. S. Hudson, J . Chem. Educ., 18, 353 (1941).
(3) E. Fischer and J. Tafel, Ber., 20, 2588 (1887).
139
140 IIASSAN EL KHADEM

hydrate field, as, for example, in the synthesis of L-ascorbic acid,4 as well
as for the analytical purposes for which it was originally intended. This
reaction is, indeed, the simplest way of obtaining 1,a-dicarbonyl deriva-
tives which, because of their high reactivities, may be used as starting
points for many syntheses.

11. NOMENCLATURE
In the early nomenclature, the ending “azone” replaced the final ‘(e”
in the name of the sugar started with, as, for example, D-glucosazone, and,
if the substituent on the hydrazine was other than phenyl, it was preceded
by the substituent as, for example, in p-bromophenyl D-glucosazone. The
syllable “az” shows that nitrogen is present, and the syllable “one” indi-
cates a state of oxidation higher than that of the sugar started with. Later,
the substituent prefixed to the ending “osazone” was put after the sugar
name, and D-glucosazone was thus changed to D-glucose phenylosazone.
To overcome the difficulty arising from the fact that the same osazone is
obtainable from two epimeric aldoses, the related 2-ketose, and a number
of related amino sugars and sugar derivatives, essentially the present
nomenclature was suggested.6 This proposal designated the unsubstitutcd
phenylosazoncs by stating their D or L configuration, followed by the group
configuration of the asymmetric carbon atoms of the sugar residue (for
example, arczbino- or threo-) followed by a term denoting the number of
carbon atoms in the sugar chain, as D-arabino-hexose phenylosazone. It is,
however, more satisfactory to designate the glycose portion by the syste-
matic name5” for the ketose structure from which the osazone may be
derived; thus, the name for D-glucosazone becomes D-arabino-hexulose
phenylosazone. The systematic name is D-arabino-hexosulose bis(pheny1-
hydrazone).

111. PREPARATION
Osazones are usually prepared“ by the action of three molecular propor-
tions of the substituted hydrazine (or its hydrochloride) on one molecular
(4) T. Reichstein, A. Griissner, and R. Oppenauer, Helu. Chim. Acta, 16, 561, I019
(1933); 17, 510 (1934); R. Q. Ault, D. K. Baird, H. C. Carrington, W. N. Haworth,
R. Herbert, E. L. Hirst, E. G. V. Percival, F. Smith, and M. Stacey, J . Chem. Sac.
1419 (1933); D. K. Baird, W. N. Haworth, R. W. Herbert, E. L. Hirst, F. Smith,
and M. Stacey, ibid., 62 (1934); see F. Smith, Advan. Carbohydrate Chem., 2, 79
(1945).
(5) J. C. Sowden, J . Am. Chem. Sac., 69, 1047 (1947).
(5a) M. L. Wolfrom, A. Thompson, and E. F. Evans, J . Am. Chem. Sac., 67, 1793
(1945).
(6) N. K. Richtmyer, Methods Carbohydrate Chem., 2, 127 (1963).
 CHEMISTRY O F OSAZONES 141

proportion of the saccharide in aqueous acetic acid (or sodium acetate,

respectively). For hydrazines disubstituted’ on one nitrogen atom, the
reaction is carried out on the ketose, and, to prepare aliphatic osazones
(such as the carbanioyl derivatives), the reaction is performed with the
glycosulose (“glycosone”) , two molecular proportions of the hydrazine
being used. Electron-attracting groupsg on the benzene ring seem to
facilitate osazone formation, and electron-releasing groups, to retard it;
thus, (nitropheny1)osaeones are among the easiest to prepare, and (alkyl-
pheny1)osazones are formed with more difficulty. The optimum pH of the
reactionlo is 3.2 and the time of reaction should be about one to three
hours. However, some of the less stable osazones, such as those of L-arabi-
nose, may be completely decomposed on prolonged heating, and special
precautions must be taken in their preparation, such as carrying out the
reaction in 2-methoxyethanol. Reducing salts,11J2such as sodium hydrogen
sulfite, have been suggested for suppressing formation of colored oxidation
products, but these have not found general use.

IV. MECHANISM
OF FORMATION

Eighty years after the preparation of osazones by Emil Fischer,la the

mechanism of their formation is still under intensive study, and no satis-
factory scheme has emerged that could be demonstrated by a stepwise
isolation of the intermediates and then be confirmed by studies of the
reaction kinetics. Yet, there is now a greater understanding of the com-
plexity of the problem; and, perhaps, of the need to (a) investigat,e models
of the many hypothetical intermediates which have been suggested and
(b) follow their reactions with tracer elements. As will be seen in the
following pages, radioactive carbon, nitrogen, and bromine, as well as
deuterium and tritium, have been used for following the over-all reaction
between saccharides and substituted hydrazines, but owing to the numerous
side-reactions taking place simultaneously, it has not yet been possible to
reach conclusive results. Similarly, a number of compounds have been
isolated from the reaction mixtures, but, here too, the difficultJyarises as
to whether these are true intermediates of the main reaction-path, by-
products of side-reactions, or, even, degradation products.
The mechanisnis suggested by the various authors for the formation of
a glyculose phenylosazone may be broadly divided into two types; the
(7) C. Neuberg, Ber., 37, 4616 (1904).
(8) M. L. Wolfrom, H. El Khadem, and H. Alfes, J . Org. Chem., 29, 2072 (1964).
(9) G. J. Bloink and K. H. Pausacker, J. Chem. Soc., 1328 (1950).
(10) G. J. Bloink and H.H. Pausacker, J. Chem. Soc., 622 (1951).
(11) M. Wagenaar, Pharm. Weekblad, 71, 229 (1934).
(12) R. H. Hamilton, Jr., J . Am. Chem. Soc., 66, 487 (1934).
142 HASSAN EL KHADEM

first type, which may be termed the Fischer type of mechanism, includes,
a t one stage, a dehydrogenation (oxidation) caused by phenylhydrazine
or its salts. The second type of mechanism may be designated the Amadori
type; it involves an Amadori rearrangement and avoids phenylhydrazine
as an oxidizing agent. There is, of course, no clear-cut differentiation, be-
cause some mechanisms involve both oxidation and Amadori rearrange-
ments, and intermediates common to both types may be found. A unani-
mous agreement prevails that the first step of the reaction is the formation
of the phenylhydrazone by the interaction of one molecular proportion of
the aldose or ketose with one molecular proportion of phenylhydrazine.
This has been demonstrated by the fact that the yield of osazone is the
same regardless of whether the reaction is initiated with a mole of the
free saccharide and three moles of substitut,ed hydrazine, or with a mole
of the phenylhydrazone and two moles of phenylhydrazine.

1. Mechanisms of the Fischer Tgpe

Fischerls suggested that the phenylhydrazone (l), first formed from an
aldose and phenylhydrazine, is oxidized with phenylhydrazine to (2), the
glycosulose 1-(phenylhydrazone) [glycosone 1-(phenylhydrazone)], which
reacts with a third molecule of phenylhydrazine to form the osazone (3).

HC=N-NHPh HC=N-NHPh HC=N-NHPh

I - I
CHOH C=N-NHPh
I I

To overcome the difficulty arising from the assuniption that phenyl-

hydrazine, itself a strong reducing agent, causes oxidation of the secondary
hydroxyl group to a carbonyl group, Kenner and KnightI4 suggested that
it was the phenylhydrazine salt (hydrazonium cation), and not the phenyl-
hydrazine, that was responsible for the oxidation. They based their theory
011 the fact that substituted hydrazine salts are more readily decomposed
reductively by heat than are the corresponding free hydrazines.
Braude and Forbes16found that primary and secondary alcohols contain-
ing two or more ethylenic or aromatic substituents conjugated with the
carbinol group are oxidized by (2 ,4-dinitropheny1)hydrazine salts; they
suggested that the same kind of reaction occurs during osazone formation.
(13) E. Fischcr, Rer., 20, 821 (1887).
(14) J. Kenner and E. C.Knight, Ber., 69, 341 (1936).
(15) E. A. Braude and W. F. Forbes, J . Chem. Soc., 1762 (1951).
 CHEMISTRY OF OSAZONES 143

For the sugar series, the mechanism of Braude and Forbes15may be repre-
sented as follows. The phenylhydrazone (4) forms a coordination compound
(5) with the phenylhydrazonium salt, which dehydrogenates the CHOH
group to the carbonyl compound (6),and this then yields the osazone.

A similar oxidation was reported by Kinsley and Plant,Is who converted

deoxybenzoin mono-[(o-nitrophenyl)hydrazone] into the corresponding
bis(hydrazone) with excess (o-nitropheny1)hydrazine.
Bloink and Pausacker’’ suggested a mechanism similar to that of Braude
and Forbes,15 in which the hydrazonium salt, instead of oxidizing the
hydroxyl group, oxidizes the hydrazino hydrazone formed by the action
of phenylhydrazirie on the phenylhydrazone by way of (7). The hydrazino
hydrazone is also an intermediate in the Weygand scheme B (see p. 145)
for ketoses, where it is produced by an Aniadori rearrangement.

(7)

Bloink and Pausackerl’ supported their mechanism by a study of the

ratio of phenylhydrazine consumed in the conversion of benzoin phenyl-
hydrazone into the bis(pheny1hydrazone) to the amount of ammonia pro-
duced during the reaction. They suggested that, as ammonia is produced
Ph-CH-C-Ph
(Braude and ForbesI5)- #-NNH-ph-t(Bloink and Pausacker17)

PhNHNH, PhNHNH,

Ph- C- C- Ph Ph-CH-C-Ph
II II + PhNH, + NH, I II
0 N-NH-Ph PhNH-HN N-NH-Ph

1
Ph-C-C-Ph Ph-C-C-Ph
I / II II II + PhNH, + NH,
Ph-NH-N N-NH-Ph Ph-NH-N N-NH-Ph

(16) D. A. Kinsley and S. G. P. Plant, J. Chem. SOC.,4814 (1956).

(17) G. J. Bloink and K. H. Pausacker, J. Chem. SOC.,661 (1952).
144 HASSAN EL KHADEM

at a later stage in their mechanism (as compared to that of Braude and

Forbes16), the initial ratio should be higher than two in their mechanism]
and less than two in the mechanism of Braude and Forbes.15Although they
encounteredl7 great experimental difficulty in their estimation, they were
able to show that the ratio of phenylhydrazine to ammonia exceeds 2
initially, then falls below 2, and finally approaches 2 again, and, as these
results are not in accord with Braude and Forbes’s mechanism, the results
were taken to favor their own mechanism.

2. Mechanisms of the Amadori Tgpe

In 1940, WeygandI8 suggested two mechanisms, designated Scheme A
and Scheme B, to explain the formation of osazones through a series of
Amadori rearrangements. He based his theory on the observation that
1-mylamino-1-deoxy-D-fructoses(9) obtained by an Amadori rearrange-
ment of N-aryl-n-glucosylamines (8) in acid media, yield osazones (10)
more rapidly, and in higher yield, than do the free saccharides.
I
CHNHR H,CNHR HC=N-NH-Ph
I I I
HCOH q=0 C=N-NH-Ph
I
H@ H O h 2 PhNHNH,- HOfH
HY:HJ- HCOH
I
- HCOH
I
HCO I
HCOH HLOH
I I I
CH,OH CH,OH CH,OH

(8) (9) (10)

Furthermore, aniline was found to increaselB the rate of formation of
the osazone by ten percent, a fact which cannot be explained in the light
of Fischer’s mechanism, as it would tend to shift the equilibrium away
from the osazone.
I n Scheme A , one molecule of the phenylhydrazone (11) rearranges to
the hydrazino enol (12), which loses aniline to give a keto imine (13),and
the lattter reacts with two molecules of phenylhydrazinc to afford the
osazone (14) and ammonia.
HC=N-NH-Ph
I
CHOH
I
- HC- NH-NH-
II
COH
Ph
- - HC=NH
I
C=O
I
HC=N-NH-Ph
I
C=N-NH-Ph
I
+ NH,

(18) F. Weygand, Ber., 73, 1284 (1940).

(19) E. Knooht and F. P.Thompson, J . Chern. Soc., 126, 222 (1924).
 CHEMISTRY OF OSAZONES 145

In Scheme B, one molecule of the hydrazino enol (12) tautomerizes to

the keto form (15), which reacts with one molecule of phenylhydrazine,
giving a hydrazino hydrazone (16) similar to that of Bloink and Pausacker.l’
This tautomerizes to the -ene bis(hydrazin0) compound (17), and then
loses aniline to afford two isomeric, imino hydrazones (18, 19) which react
with a third molecule of phenylhydrazine to give the osazone (14) and
ammonia.

HC-NH-NHPh
I1
COH
I
- H,C -NH-NHPh
I
c=o
I
- KC-NH-NHPh
I
C=N-NH-Ph
I
II
(12) (15)

HC=N-NHPh
I
C=N-NHPh
I
- HC=N-NHPh
I
C=NH
I
- HC -NH-NHPh
II
C-NH-NHPh
I

I
C= N-NHPh
I
(19)

The choice between Weygand’s Schemes A and B proved difficult, with

authors at one time favoring one Scheme and then the other. An early
attempt to test the validity of the respective Schemes was made by Ruggli
and Zeller,*O using diphenylacetoin (p-bromophenyl)hydrazone and di-
phenylacetoin (p-nitrophenyl)hydrazone, which they treated with phenyl-
hydrazine; they obtained the bis(pheny1hydrazone) in both cases. This
result was taken by them to exclude Scheme B, which, under similar
conditions, should yield a mixed osazone, but this conclusion is now in-
validated by our knowledge of transhydrazonation, that is, the exchange
of hydrazone residues, as in (20) + (21), a possibility. which has caused

Ph- (2%- CHOH PhNHNH, Ph- CH,- C=N-NHPh

I I
Ph-CH2- C = N-NH Ph-CH,- C=N-NHPh

(20) (21)

(20) P. Ruggli and P. Zeller, Helv. Chim. Acla, 28, 747 (1945).
146 HASSAN EL KHADEM

considerable difficulty in the study of osazone formation. Ruggli and

Zeller’s results20 were promptly refuted by Weygand and Reckhaus.21
These authors, using the (p-nitropheny1)hydrazone and treating it with
phenylhydrazine, isolated from the mother liquor, after removal of the
bis(hydrazone), both aniline and (p-nitrophenyl)hydrazine, as would be
expected from Scheme B. However, Scheme A, which should yield only
p-nitroanilinc, was not ruled out completely, but was considered a par-
ticipant in the reaction. Later, Theilacker and TrosterZ2again favored
Scheme A; they showed that benzoin phenylhydrazone (22) is readily
hydrolyzed in boiling acetic acid to ammonia, aniline, and benzil (24),
and suggested that the latter could only be produced from the keto-
imine (23) of Weygand’s Scheme A.

R
I
CHOH
k=N--NHPh
R
I
- PhNH,+
R
I
c=o
I
C=NH
R
I
- R
I
c=o
I
c=o
I
R
+ NH,

(22) (23) (24)

Radioactive t8racerswere then tried, in order to decide which of these

~ ~ tried the use of 82Br,
schemes is correct. Weygand and c o w o r k ~ r sfirst
and treated labeled D-mannose (p-bromo-82Br-phenyl)hydrazone with
unmarked (p-bromophenyl)hydrazine, but rapid transhydrazonation re-
sulted in an equal distribution of radioactive bromine on the hydrazine
residues a t C-1 and C-2, and prevented any correlation from being drawn.
Attempts to solve the problem were then made, with 16N-labeledhydrazines,
by Schemyakin and M a i m h ~ d These
. ~ ~ authors tried to differentiate be-
tween Fischer’s mechanism and Weygand’s Schemes A and B by following
the dist)ribution of 16N in the ammonia and osazone produced by the
action of unlabeled (p-nitropheny1)hydrazine on a (p-nitr~-~~N-phenyl)hy-
drazone (25). According to the Fischer mechanism, they expected all of
the radioactivity to appear in the osazone, but, in Weygand’s Scheme A,
it should all appear in the ammonia. Weygand’s Scheme B should result
in the equal distribution of the labeled atoms between the ammonia and
the osazone. In their first communication,24on benzoin (p-nitropheny1)hy-
drazone, their results seemed to favor Scheme B. Later,26however, the
(21) F. Weygand and M. Reckhaus, Ber., 82, 438 (1949).
(22) W. Theilacker and P. Troster, Ann., 672, 144 (1951).
(23) F. Weygand, H. Grisebach, K. D. Kirchner, and M. Haselhirst, Chem. Ber., 88,
487 (1955).
(24) M. M. Shemyakin and V. I. Maimind, Dokl. Akad. Nauk SSSR,102, 1147 (1055).
(25) E. M. Bamdos, K. M. Ermolaev, V. I. Maimind, and M. M. Shemyakin, Chem.
Ind. (London), 1195 (1959).
 CHEMISTRY OF OSAZONES 147

R
I
Fisc he r

=i 0% of ammonia-15~
100% of o s a ~ o n e - ' ~ N

Ci='5N-NH
-i
Weygand (A) 100% of ammonia-I5N
CHOH 0% of o s a ~ o n e - ~ ~ N
I
R
(25)
Weygand (B)

-150% of ammonia-15N
50% of osazone-l5N

results were reversed and redistribution of label was suppressed by carry-

ing out the reaction in isoamyl alcohol, and removing water from the
reaction niixture by continuous distillation. Experiments on the (p-nitro-
phenyl)hydrazones of D-fructose, 2-hydroxycyclohexanone, and benzoin
showed that, in the early stages of the reaction, the ammonia formed
contains much more than 50% of the label, in agreement with Weygand's
Scheme A. Furthermore, when 2-hydroxycyclohexanone (p-nitropheny1)-
hydrazone was boiled in acetic acid-acetic anhydride, they isolated26 an
acetamido derivative (27) which they believed to have been produced by
way of a keto imine (26) as in Weygand's Scheme A. Similar tracer ex-

periments were carried out by Eppley and Wright,27using 14C-labeled

phenylhydrazine, which was permitted to react with unlabeled D-mannose
phenylhydrazone. One mole of the osazone produced was found to con-
tain radioactivity equivalent to that of 1.2 moles of labeled hydrazine;
this was thought to exclude a mechanism of the Fischer type, which
should lead to no more than one mole. The results were also used for
obtaining a quantitative estimate of the contribution of the two possible
imino-hydrazone intermediates, (18) and (19), of Weygand's Scheme B.
If the reaction proceeds through both intermediates equally, then, out
of 100 molecules of osazone produced, 50 should have two residues of
radioactive phenylhydrazine (from path A) and 50 should have one residue
(26) M. M. Shemyakin, V. I. Maimind, K. M. Ermolwv, and E. M. Bamdos, Dokl.
Akad. N a u k SSSR,128, 564 (1959).
(27) R. Epply and J. C. Wright, Proc. West V a . Acad. Sd.,
33, 73 (1961).
148 HASSAN EL KHADEM

of radioactive phenylhydrazine (from path B). Hence, an osazone contain-

ing 1.5 residues of radioactive phenylhydrazine should be obtained. Since
the results showed that the product contained only 1.2 radioactive resi-
dues, it was concluded that the reaction proceeds preponderantly through
path B.

Tritium has also been used as a label in the study of osazone formation.
Friedberg and Kaplan2*treated n-glucose-1-t with phenylhydrazine without
loss in tritium content, and concluded that an Amadori intermediate
having two hydrogen atoms on C-1, as in Scheme B, is not produced,
because, in that Scheme, a loss of 50y0 in tritium content should have
resulted. These results were later criticized,2Bbecause no consideration

TC=N-NHPh TCH-NH-NHPh TC=N-NHPh HC=N-NHPh

I -----+I - I + I
CHOH c=o C=N-NHPh C=N-NHPh
I I I I

had been given to the isotope effect, which is considerable between T

and H. Weygand and coworkers30showed that, whereas there is, indeed, no
loss in tritiuni when the medium is water, a considerable loss of tritium
occurs when the medium is DzO and the D-glucose is labeled with both T
and D on C-1. This is because the isotope effect between T and D is smaller
than between T and H. In a subsequent publication,31 they showed that
the loss of tritium depends to a large extent on the experimental condi-
tions, especially the proportion of acetic acid present and the concentra-
tions of the reactants. This was apparent when they used one-tenth of
the concentrations employed in their previous experiment, and found
that a loss of tritium also occurs in an aqueous medium but not in the
more concentrated solutions used by Friedberg and Kaplan.2* Weygand’s
experiments seem to favor Scheme B, with some participation of another
scheme, presumably Scheme A.
(28) F. Friedberg and L. Kaplan, J . A m . Chem. Soc., 79, 2600 (1957).
(29) C. J. Collins and M. H. Lietzke, J. A m . Chem. Soc., 81, 5379 (1959).
(30) F. Weygand, H. Simon, and J. F! Klebe, Chem. Ber., 91, 1567 (1958).
(31) H. Simon, K. D. Keil, and F. Weygand, Chem. Ber., 96, 17 (1962).
 CHEMISTRY OF OSAZONES 149

An interesting intermediate, possibly deriving from Weygand's Scheme

B, was isolated by Haas and Seeligera2from the reaction of D-glucose with
phenylhydrazine in acetic acid or hydrochloric acid. This product is
3-(~-arabino-tetrahydroxybutyl)cinoline (30), produced, not by the
hydrolytic decomposition of the osazone, but by the cyclization of the
intermediate glycosulose2-(phenylhydrazone) (28) or the aldimine 2-(phen-
ylhydrazone) (29).

HO
I
\
/
HN H2N

Two variations of the Weygand mechanism have been suggested which

include both an Amadori rearrangement and an oxidation reaction. The
first was that of Barry and Mitchel1,sa who based their mechanism on the
observation that 1,2-disubstituted hydrazines of the type (31) are readily
oxidized in air to the corresponding hydrazone (32).34They suggested
that, as osazone formation requires the presence of air,36a similar oxidation
air
R-CH,-NH-NH-Ph R-CH=N-NH-Ph
(31) (32)

of Weygand's hydrazino ketose occurs, yielding the glycosulose 1-(phenyl-

hydrazone) (33) and then the osazone. The other mechanism was sug-
HC=N-NHPh
CHOH
I
I
-COH
CH-NH-NHPh
II
I
- GC-NH-NHPh
I
c=o
I
air
-&0
CH=N-NHPh

(32) H. J. Haas and A. Seeliger, Chem. Ber., 96, 2427 (1963).

(33) V. C. Barry and P. W. D. Mitchell, Nature, 176, 220 (1955).
(34) P. Grammaticakis, Compt. Rend., 204, 1262 (1937); 210, 303 (1940).
(35) A. Orning and G. H. Stempel, J . Org. Chem., 4, 410 (1939).
150 HASSAN EL KHADEM

gested by Kitaoka and OnoderaS"; it involves an oxidation of Weygand's

-ene bis(hydrazine) (34), in a manner similar to the formation of quin-
oxaline.

HC'
II
NH
- HC'
,C,
I1
NH

NH
\P;'H2
,,NHPh
- I
HC = N- NHPh
C= N- NHPh
I
t PhNH, + NH,

I
yC\NH II
kHPh
NHPh NHPh

(34)

Micheel and DijongsT have suggested a novel mechanism of osazonc

formation, based on the Amadori rearrangement, not of the hydrazone,
but of its deamination product, the N-phenyl-D-glucosylamine. They
used 4 ,6-O-benzylidene-~-glucose as their model compound, succeeded
in isolating a number of intermediates in crystalline form, and followed
their reaction with l*C-labeled phenylhydrazine. They favored the phenyl-
amino intermediate, because of their previous experience38that Amadori
rearrangements proceed through the formation of 1,1-bis(amino aldoses)
and their observation that the corresponding 1,l-bis(pheny1hydrazino)
compound fails to undergo an Amadori rearrangement because of hydrogen
bonding similar to that found in osazones.
NH-NH- Ph
/
Amadori rearrangement
'\
HC -No
/ \ /"
NH-N-Ph

Intermediates isolated included 1-anilino-4 ,6-0-benzylidene-1-deoxy-

D-fructose and 1-amino-1-deoxy-D-fructosephenylhydrazone. Micheel and
Dijong's mechanism*' may be summarized as follows. Phenylhydrazine is
decomposed to benzene, ammonia, and aniline, and the latter reacts with
the phenylhydrazone (35), causing its deamination to the N-phenyl-D-
glucosylamine (36),which undergoes an Amadori rearrangement through
the 1,l-bis(anilin0) derivative (37), yielding the second isolated inter-
mediate, namely, the 1-anilino-1-deoxy ketose (38),which now reacts with
phenylhydrazine to afford the 1-anilino ketose phenylhydrazone (39).The
latter is dehydrogenated, in its tautomeric form (40), by phenylhydrazine,
and the product (41) reacts with another molecule of phenylhydrazine,
yielding the osazone (42).
(36) S. Kitaoka and K. Onodera, J . Org. Chem., 26, 231 (1963).
(37) F. Micheel and I. Dijong, Ann., 660, 136 (1963).
(38) F. Micheel and I. Dijong, Tetrahedron Letters, 21 (1962); Ann., 668, 120 (1962).
 CHEMISTRY O F OSABONES 151

NHPh
I
HC=N-NHPh
CHOH
I
I
PhNH,
2 7 1
CH-NHPh
CHOH
I 0
--+
HC-NHPh
+OH
- 1
H,C-NHPh
c=o
I
I
(35) (36) (37)
1 (38)
HC=N-NH-Ph
I
C=N-NH-Ph
+-
HCZN-Ph
I
C=N-NHPh C-NH-NHPh
-
---
H,C-
I
NHPh
C =N-NHPh
I I I I
(42) (41) (40) (39)

The mechanism of this dehydrogenation was investigateda* a by study-

ing the distribution of radioactivity using phenyl-W-hydrazine, whereby
it was shown that no intramolecular oxido-reduction takes place, but
rather, that the enediamine form causes hydrogenation of the free phenyl-
hydrazine present in the reaction mixture, to yield aniline and ammonia.
This mechanism was criticized by W e ~ g a n dwhen~ ~ ~this work was pre-
sented, and the problem of intermolecular or intramolecular oxido-reduc-
tion still awaits settlement.

V. STRUCTURE
OF OSAZONES

Fischer’s formulation of phenylosazones in the acyclic structure, such

as (43), was based on the fact that the same osazone is produced from
two epimeric aldoses and the corresponding 2-ketose1 as well as from
their phenylhydrazones, indicating t.hat, in the osazone, the phenyl-
hydrazine residues are attached at C-1 and C-2 of the saccharide chain.
As the asymmetry at C-2 is destroyed during the reaction, it is found that,
for a given substituted hydrazine, there are eight isomeric hexose osazones
(four D and four L isomers) and four pentose osazones (two D and two L).
HC=N-NH-Ph
I
C=N-NH-Ph
Ho&*H
I
HC*OH
I
HC*OH
I
CH,OH

(43)
(38a) F. Micheel and I. Dijong, Abstracts Papers Intern. Symp. Carbohydrate Chem.,
Munster, Germany, 1964, p. 38.
(38b) F. Weygand, oral communication, International Symposium on Carbohydrate
Chemistry, Munster, Germany, 1964.
152 HASSAN EL KHADEM

1. Evidence for the Acyclic Structure

Fischer’s acyclic structure (43) is supported by a considerable weight of
experimental evidence, summarized in the following.
a. Acety1ation.-Acetylation of D-arabino- and D-lyxo-hexulose phenyl-
osazones results in the formation of crystalline tetra acetate^.^^-^^ Wolfrom
and coworkers38have established an analytical technique that diff eren-
tiates between N-acetyl and 0-acetyl groups; the latter were determined
by the procedure of Kunz and They were thus able to show
that all four acetyl groups are 0-acetyl groups. This discovery eliminated
the possibility of cyclic structures, which would have required three 0-
acetyl groups and one N-acetyl group. The infrared spectra of osazone
acetates also support the acyclic structure, aa these compounds show
only one carbonyl band, namely, that of the 0-acetyl groups.
b. Benxoylation.-Fi~cher~~benzoylated a hexose osazone with benzoyl
chloride in quinoline-chloroform, and obtained a hexose phenylosaxone
tetrabenzoate. When the benzoylation is conducted in pyridine (without
chloroform), pentabenzoates are obtained from hexose osazones, and
tetrabenzoates from the pentose and 6-deoxyhexose osazones. 43 These
compounds possess one N-benzoyl group, as well as 0-benzoyl groups, as
is shown by their infrared spectra, which show two carbonyl bands, and
their nuclear magnetic resonance spectra which show one imino proton
(instead of the two in the free osazone). Here again, the presence of four
0-benaoyl groups in hexose osazone benzoates excludes the possibility of
cyclic structures. This evidence indicates that (44) is the structure of fully
benzoylated n-arabino-hexulose phenylosazone.
BZ
I
HC=N-N-Ph
I
C= N-NHPh
I
BzOCH
I
HCOBz
I
HCOBz
bH,OBZ

(44)
c. Formation of 6,B-O-IsopropyZidene Derivatives.-D-arabino-Hexulose
phenylosazone (43), when treated with acidic acetone, yields an iso-
(39) M. L. Wolfrom, M. Konigsberg, and S. Soltzberg, J . Am. Chem. Soc., 58, 490 (1936).
(40) K. Maurer and B. Schiedt, Ber., 68, 2187 (1935).
(41) A. Kunz and C. S. Hudson, J . A m . Chem. Soc., 48, 1982 (1926).
(42) E. Fischer and K. Freudenberg, Ber., 46, 1116 (1913).
(43) H. El Khadem, M. L. Wolfrom, and D. Horton, J . Org. Chem., 30, 838 (1965).
 CHEMISTRY OF OSAZONES 153

propylidene acetal (45) which, on methylation with dimethyl sulfate, is

converted into the N-methyl-di-0-methyl isopropylidene derivative (46). 4 4
Here again, the presence of four substituents on the oxygen atoms of the
sugar residue excludes the possibility of cyclic forms.
Me
I
HC= N-NH-Ph HCTN-NH-Ph HC=N-N-Ph
I I I
C=N-NH-Ph C=N- NH-Ph C=N-NHPh
i I
Me,SO,
HOCH
NaOH
- MeOCH
I
H$!OMe
HLOH
I
HCOH HCO, ,CH,
I I
H,CO/~\CH,
ChOH

d. Periodate Oxidation.-Under mild conditions, D-arabino-hexulose

phenylosazone (43) is oxidized by periodate to niesoxalaldehyde bis(pheny1-
hydrazone) .46 Otherwise, (43) undergoes oxidation and cyclization to
a pyrazolone derivative (47).46Three moles of oxidant are consumed
per mole, and two moles of formic acid and one mole of formaldehyde
are produced. Estimation of the formaldehyde is somewhat difficult147but.,
by removing excess periodate with barium chloride, it can be readily
estimated colorimetrically, with phenylhydrazine and ferricyanide.dE

-
HC=N-NH-Ph HC=N-NH-Ph HC= N- NH-Ph
I I I
C=N-NH-Ph C=N-NH-Ph C=N-NH-Ph
I I I
HOCH 310: HC=O CQH
I
HCOH
- + I.
I 2 HCQH
HCOH + Ph-NH-NZ N
C
'"
I I
JH,OH HCHO O=C-N-Ph

(43) (47)

Micheel and BOhm4*"have used this reaction to prove that, during

the isomerization of 2 , 3 , 4,5-tetra-O-acetyl-aldehydo-~-galactose-l-~~C
6-p-
toluenesulfonate to aldehydo-D-L-galactose heptaacetate (with zinc chlo-
(44) S. Akiya and S. Tejima, Yakugaku Zasshi, 73, 894, 1574, 1577, 1580 (1952).
(45) E. Chargaff and B. Magasanik, J . Am. Chem. SOC.,69, 1459 (1947).
(46) P. Karrer and K. Pfaehler, Helu. Chim. Acta, 17, 766 (1934).
(47) J. E. Courtois, A. Wickstrom, and P. le Dizet, Bull. SOC.Chim. France, 19, 1006
(1952).
(48) L. Hough, D. B. Powell, and B. M. Woods, J. Chem. SOC.,4799 (1956).
(48a) F. Micheel and R. Bohm, Chem. Ber., 98, 1655 (1965).
154 HASSAN EL KHADEM

ride and acetic anhydride), the sequence of the carbon chain is not altered.
They oxidized the osazone after deacetylation, and found that the label
remains in the mesoxaldehyde bis(pheny1hydrazone) and not in the
formaldehyde.
e. Physical Data.-The characteristics of the ultraviolet absorption
spectra of osazones seem to favor the acyclic structure; thus, Enge14Q
found that the ultraviolet absorption spectra of the osazones of trioses and
hexoses are quite similar, and, believing that the former could exist only
in the acyclic form, he considered the hexose osazones to be acyclic. How-
ever, it is now known bhat trioses do exist in a bimolecular, cyclic form
containing a ring of the dioxane type. The cyclic structure, which posessses
a hydrazino hydrazone chromophore instead of the bis (hydrazone) chronio-
phore of the acyclic, triose osazone, would be expected to have a markedly
different ultraviolet spectrum. These results were substantiated by more
elaborate studies of the ultraviolet spectra of osazones by subsequent
aut hors.60-62
The nuclear magnetic resonance ~ p e c t r a ~ofa ~osazones
~~ and their
acetates (see Figs. 1 and 2) afford additional proof of the acyclic structure
of the osazones. Both the acyclic osazone acetates and the free osazone,
such as (43), show two imino protons in their nuclear magnetic resonance
spectra which disappear on deuteration, and, as cyclization as in (48)
would result in the formation of a third imino proton, it may be concluded
that the free osazone exists mainly in the acyclic form (43).

CJ
HCZN-NH-Ph
l
C=N-NH-Ph
I CHEN-NHPh
HOCH
I
HCOH
HO NH-NHPh
HAOH
I HO
CH,OH
(43) (48)

2. Evidence for the Existence of Cgclic Structures

The strongest evidence for the existence of cyclic forms of osazones
came from the work of the P e r ~ i v a l swho
, ~ ~ showed that, on methylation,
D-arabino-hexulose phenylosazone (49) yields an N-methyl-tri-0-methyl
(49) L. L. Engel, J . Am. Chem. Soc., 67, 2419 (1935).
(50) P. Grammaticakis, Compl. Rend., 223, 1139 (1946).
(51) V. C. Barry, J. E. McCormick, and P. W. D. Mitchell, J . Chem. SOC.,222 (1955).
(52) G. Henseke, G. Hanisch, and H. Fischer, Ann., 643, 161 (1961).
(63) M. L. Wolfrorn, G. Fraenkel, D. R. Lineback, and F. Komitsky, Jr., J . Org. Chem.,
28, 457 (1964).
(54) E. E. Percival and E. G. V. Percival, J . Chem. Soc., 1398 (1935).
 CHEMISTRY OF OSAZONES 155

FIQ. 1.-Nuclear Magnetic Resonance Spectra of D-l&-co-Hcxulose Phenylosazone

Tetraacetate in CDCls: (1) before deuteration; (2) after deuteration.

I I I I I
-2 0 2 4 6 C

FIQ.2.-Nuclear Magnetic Resonance Spectra of D-lyzo-Hexdose Phenylosazone in

Methyl Sulfoxide: (1) before deuteration; (2) after deuteration.
156 HASSAN EL KHADEM

derivative (50) which, on treatment with p-nitrobenzaldehyde, gives a

3,4,5-tri-O-methyl-~-arabino-hexosulose(51). The structure of the latter
compound was determined by reduction to the D-fructose derivative (52)
followed by methylation to the known 1,3,4,5-tetra-O-methyl-~-fructose
(53), thus indicating the involvement of the hydroxyl group a t C-G in
ring formation.

Me
I
HC=N-NH-Ph HC=N-N-Ph
I I HI I
-C-NHNHPh O=C-COH
I I
MeOCH MeOCH
Me,SO, I p -1iitrobenz- I
+
HCOMe HCOMe
+ NaOH I aldehyde I
HCOH HCOMe HCOMe
I I
OCH, -OCH, -OCH,

(49) (50) (51)

I MeOH,C-{OH

Indirect evidence for the possible existence of cyclic forms of an osazone

in solution appears from the different ways in which the osazones and
their acetates react with nitrous acid, yielding the glycosulose in the
first case and the osotriazole in the second.66An explanation of this be-
havior was provided by the assumption that the osazones exist as equi-
librium inixturcs coniposed mainly of the acyclic form but also containing
a small proportion of the cyclic forms (54). The glycosulose (55) was
considered to be produced by the more rapid interaction of nitrous acid
with the cyclic form, causing the equilibrium to shift in that direction
until completion of the reaction, whereas the osotriazole (57) was thought
to be produced by the interaction of nitrous acid with the acyclic form
(56) of the acetate, which is incapable of cyclization.
I n summation, it may safely be stated that the free osazone exists
preponderantly in the acyclic form, such as (43), and that the cyclic forms,
(55) M. L. Wolfrom, H. El Khadem, and H. Alfes, J. Org. Chem., 29, 2072 (1964).
 CHEMISTltY OF OSAZONES 157

HC=N-NH-Ph - HC=N-NH-Ph
I
HNO~ HC=O
I
rT-NH-NHPh
I
C=N-NH-Ph - c=o
I I
Free osazone (54) (55)

(56) (57)
Osazone acetate

such as (49), if present, exist as minor constituents of the equilibrium

mixture.

3. The Chelated Structure

The FiesersS6suggested, on theoretical grounds, that osazones exist in
tautomeric, chelated forms which stabilize these unsaturated compounds
and prevent the reaction of hydrazines with saccharides from proceeding

NHPh NHPh
IICII IIDII

The Fiesers’ Formulas

(56) L. F. Fieser and M. Fieser, “Organic Chemistry,” D. C. Heath and Co., Boston,
Mass., 1944, p. 351.
158 HASSAN EL KHADEM

k
Henseke and Kbhler’s Formula

beyond C-2. Two pairs of chelated tautomers were suggested, one pair,
“A” and (‘B,’’having the imino proton of the hydrazone residue at C-1
involved in ring formation; and the other, “C” and “D,” having the
imino proton of the hydrazone residue at C-2 involved in chelate forma-
tion. Later, Henseke and K6hler5’ suggested the presence of another
chelated ring, involving the hydroxyl group at C-3.
Of the four chelated structures suggested by the Fiesers, the resonance
pair “A” and “B” suffice to explain the fact that only the hydrazone
residue at C-1 can be methylated or benzoylated, because the imino group
of the hydrazone residue at C-2 is involved in hydrogen bonding. Struc-
tures “C” and “D” would be expected to undergo alkylation on the hydra-
zone residue at C-2 and must, therefore, either be eliminated from con-
sideration or be regarded as minor constituents. This leaves the resonance
pair “A” and “B” as the most probable of the Fiesers’ structures.
The chelated structure suggested by Henseke and Kohler differs from
structures “A” and “B” in that the hydrazone residue at C-2, instead of
forming a chelate ring with a nitrogen atom of the hydrazone at C-1, is
chelated with the oxygen atom of the hydroxyl group at C-3. This struc-
ture was suggested in order to explain the mutarotation of Percival’s
dianhydro-osazone (see p. 177), which cannot form a chelate ring of the
Fieser type. The existence of this type of chelated ring needs further
confirmation, however, especially in view of the fact that this structure
does not explain why osazone formation proceeds from the hydrazone
stage to that of the osazone, since the sugar hydrazone can itself be stabi-
lized by forniation of a similar, chelated ring.

Evidence for the Chelated Rings

(a) Unsymmetrically disubstituted hydrazines, which yield osazones
that are unable to form chelated rings, react with aldopentoses (58),
tetroses, and trioses to yield a l k a z ~ n e sIn
. ~ these
~ compounds, the osazone
reaction does not stop at C-2, but proceeds along the whole sugar chain,
giving such products as (59), as predicted by the F i e s e r ~ . ~ ~
(57) G.Henseke and H. Kohler, Ann., 614, 105 (1958).
(58)0. L. Chapman, W. J. Welstead, T. J. Murphy, and R. W. King, J . Am. Chem.
Soc., 86, 732 (1964).
 CHEMdISTRY OF OSAZONES 159

HC=N-N, /Me
Ph

C=N-N
'Ph

C=N-N

-
HC=O 'Ph
I Me
I
CH(oH)
I PhNNH,
CH(OH) Ph
I
CH(OH)
I
CH,OH HC=N-N
\Ph
(58) (59)

(b) The fact that osazones react with diazotized aniline to give forma-
zanslbein strongly alkaline media only, was taken by MesteF to indicate
that such media are necessary for breaking the chelate ring shown in (60),
prior to the formation of the formazan (61).

YHPh
N=N-Ph

(c) The mutarotation of osazones, fist observed by Levene and Jacobs,Bo

and confirmed by others, was attributed to an equilibrium between t,he
different cyclic structures. Later, however, Henseke and Kohler67showed
that osazones of disubstituted hydrazines (which are unable to form
chelated rings) do not mutarotate, and that mutarotation of osazones takes
place in such basic solvents as pyridine but not in neutral solvents, sug-
gesting that the equilibrium shifts from the chelated form to the un-
chelated one.
(d) Polarographic studiese1.62of osazones in acid and alkaline media
have shown the presence of hydrogen bonding, due to chelation, in both
the 1 ,2-bis(phenylhydrazone) and the 1-(2-methy1-2-phenylhydrazone)
2-(phenylhydrazone) of D-urubino-hexosulose. The chelation disappears in
(59) L. Mester, J . A m . Chem. SOC.,77, 4301 (1955).
(60) P. A. Levene and W. A. Jacobs, Ber., 42, 3247 (1909).
(61) B. Jambor and L. Mester, Actu Chim.Acad. Sci. Hung., 9, 485 (1956).
(62) B. Jambor and K. Kisban, Acla Chim.Acad. Sci. Hung., 9, 493 (1956).
160 I-IASSAN EL KHADEM

alkaline media, giving rise to a different type of reduction-potential curve.

D-arabino-Hexosulose 1,2-(2-methyl-2-phenyl)osazone,T\ hich is not che-
lated, shows the same reduction-potential curve regardless of whether the
medium is acid or alkaline.
( e ) The nuclear magnetic resonance spectra of saccharide osazones and
their acetates are characterized by two imino protons which disappear on
d e ~ t e r a t i o n . ~ ~The ~ ~ ~low
~ ~ field
~ ~ ~ *resonance,
*~ chelated, imino proton
appears a t about r -2.5, and the unchelated iniino proton at about r 1.5;
the latter disappears more rapidly on deuterationne2& As D-arabino-hexulose
1-(2-methyl-2-phenyl)-2-phenylosazoneshows only the low field, chelated,
imino p r ~ t o n , ~ ~ , ~ ~
it ahas
, e *been
b concluded that the imino proton of the
hydrazine residue a t C-2 in the previous osazones is involved in chelation,
as shown in the Fiesers’ structure “A” (see p. 157).
( f ) X-ray analysis62cof D-arabino-hexulose phenylosazone also agreed
with the arrangement of carbon and nitrogen atoms of the Fiesers’ struc-
ture “A”.ll

VI. PHYSICAL
PROPERTIES
The osazones are yellow, crystalline compounds that melt with decom-
position. They exhibit mutarotation~~es-64; this had previously been at-
tributed to a variety of causes, ranging from c y ~ l i z a t i o nto
~ ~partial hy-
. ~ ~ present view, however, is that mutarotation of osazones is
d r o l y ~ i sThe
~ ~ explanation was devised because it was found
due to c h e l a t i o ~ i .This
that mutarotation occurs only with osazones capable of forming chelated
rings67; the disubstituted osazones, such as D-arabino-hexulose 2-methyl-2-
phenylosazone, do not mutarotate. Furthermore, mutarotation takes place
only in such basic solvents as pyridine, which might cause a partial break-
down of the chelated ring.
Chapman and co-workerssZbfollowed the nuclear magnetic resonance
spectra of osasones in methyl sulfoxide during the process of mutarotation,
and observed that the spectra become constant when the optical rotation
reaches the equilibrium values. They found that, during the course of
mutarotation, new NH resonances appear in the region of the unchelated
(62a) L. Mester, E. Moczar, and J. Parrello, Tetrahedron Letters, 3223 (1964); J . A m .
Chem. SOC.,87, 596 (1965).
(62b) 0. L. Chapman, R. W. King, W. J. Welstead, Jr., and T. J. Murphy, J . A m .
Chem. Soc., 86, 4968 (1964).
(62c) K. Bjamer, S.Dahn, S.Furberg, and C. S.Petersen, A d a Chem. Scand., 17, 559
(1963).
(63) P. A. Levene and F. B. LaForge, J . Biol. Chem., 18, 319 (1914); 20, 429 (1915).
(64) E. Zerner and R. Waltuch, Monafsh., 36, 1025 (1914).
(65) L. Mester and A. Major, J . Am. Chem. Soc., 79, 3232 (1957).
 CHEMISTRY OF OSAZONES 161

imino proton and that a down-field shift of the proton of the hydroxyl
group a t C-3 takes place. They concluded that, during mutarotation, the
chelated ring is opened, and a new ring is formed which involves the
hydroxyl group a t C-3 as follows.

These findings are consistent with the fact that, during mutarotation, a
shift of the ultraviolet absorption maximum to a shorter wave-length takes
place, and that the starting isomer is recovered from the equilibrium
mixture. Another interesting phenomenon in the optical properties of
osazones is their thermomutarotationBB;the optical rotations of these
compounds are greatly influenced by temperature.
The ultraviolet spectra of osazones have been intensively s t ~ d i e d , ~ ~ - ~ ~
and, as shown earlier (see p. 154), the characteristics favor assigning of an
acyclic structure to osazones. The spectra of osazones are characterized
by three maxima, a t about h256, 308-314, and 395-399 nip, which aid in
their r e ~ o g n i t i o n . ~ ~
The infrared spectra of phenylosazones have also been studied in de-
and have been suggested as a means of identification of the different
saccharide osazones. Saccharide osazones show the characteristic hydroxyl
bands a t 3 p, the C=N band of the hydrazone residues a t 6.3 p, and three
bands attributed to the phenyl ring. In the case of acetylated osazones,
the 0-acetyl band appears a t 1735 cm.-l and N-acetylated derivatives show
the N-acetyl band atG8-69a about 1640 cm.-'. These characteristics have
greatly simplified the differentiation of 0-acetyl from N-acetyl groups in
acetylated osazones. Also, for benzoylated osazones, the differentiation
offers no difficulty; the 0-benzoyl band appears at 1725 cm.-' and the
N-benzoyl band a t 43,69a 1685 cni.-l.
The nuclear magnetic resonance spectra of o s a ~ o n e s ~show,~ J ~ besides
(66) G. Henseke and H. J. Binte, Chem. Ber., 88, 1167 (1955).
(67) W. Otting, Ann., 640, 44 (1961).
(68) H. El Khadem and M. M. Mohammed-Ali, J . Chem. Soc., 4929 (1963).
(69) IS. El Khadem, Z. M. El-Shafei, and M. M. Mohammed-Ali, J . Org. Chem., 29,
1565 (1964).
(69a) H. El Khadem, Z. M. El-Shafei, and M. M. A. Abdel Rahman, Carbohydrate Res.,
1, 31 (1965).
162 HASSAN EL KHADEM

the protons of the sugar residues (which do not differ much from those
of other sugar derivatives), two imino protons: one at low field (about
T-2), due to the chelated proton of the hydrazone at C-2, and one at a
higher field, due to the imino proton a t C-1.
The apparent dissociation constant7O of different substituted osazones
has been measured in acetic acid, and shown to follow Hammett’s equation.
The reduction potential has been followed polarographically61~82 and used,
as noted previously (see p. 159), to demonstrate the chelated nature of
osazones.
Finally, to facilitate the identification of sugar osazones, crystallographic
studies have been made.

VII. REACTIONS

A striking feature in the reactions of osazones is the fact that the two
hydrazone residues are nonidentical in their reactivity. The hydrazone
residue at C-1 is readily a l k ~ l a t e dand
~ ~ a ~ y l a t e d , ~ ~but * ~ ~at
f ~ that ~ sC-2
is not. This behavior has been attributed to chelation of the imino proton
of the hydrazone at C-2. Furthermore, among the hydroxyl groups of the
sugar residue, the one on C-3 seems to be very reactive, readily undergoing
dehydration and, in some cases, inversion; this is followed in reactivity
by the primary hydroxyl group (on C-6 of hexoses), which readily engages
in the formation of 3,6-anhydrides.
The most important reactions of osazones are treated in the following
Sections.

1. Salt Formation
Owing to the basic nature of their imino groups, the osazones can be
converted into saltsV2by concentrated acids. Such disubstituted osazones
as D-arabino-hexulose 1,2-(2-methyl-2-phenyl)osazonereadily give crystal-
line salts that can be hydrolyzed to the glycosulose l-hydrazone.eEMono-
substituted-aryl osazone salts are less stable.

2. Reduction
Fischer’* reduced D-arabino-hexulose phenylosazone (43) with zinc
and acetic acid, and obtained l-amino-l-deoxy-D-fructose (62) (“isoglu-
cosamine”) as its crystalline acetate. He used this reaction as a means of
(70) D. Wegmann and W. Simon, Helv. Chin. Acta, 46, 962 (1962).
(71) V. H. Morris, J . Am. C h m . Soc., 64, 2843 (1932).
(72) E. Fischer, Ber., 22, 87 (1889).
(73) E. Fischer, Ber., 10, 1920 (1886).
 CHEMISTRY O F OSAZONES 163

converting aldoses into ketoses, and obtained D-fructose by treating 1-

amino-1-deoxy-D-fructose (62) with nitrous acid.8 The yield of l-amino-l-
deoxy-D-fructose has been increased to 60% by reducing with hydrogen
over palladium-carbon catalyst in acetic acid. 7 4 * 76
HC =N-NH-Ph
I p.:
- HYoH
C=N-NH-Ph
I
HOCH HOCH
I I
HCOH HCOH
I I
HrioH
CKOH CqOH

(43) (62)
By the catalytic reduction of the phenylosazone from lactose, Kuhn
and Kir~chenlohr~~ obtained both P-D-galactopyranosyl-(1-+4)-l-amino-l-
deoxy-&fructose and ~-~-ga~actopyranosy~-(1-+4)-2-amino-2-deoxy-~-glu-
cose. The N-acetyl derivative of the latter was found to be identical with
a disaccharide obtained from the partial hydrolysis of a blood-group sub-
stance. The structure of a disaccharide obtained from the partial hydrolysis
of heparin was likewise confirmed by comparison of the disaccharide with
a synthetic product obtained by catalytic hydrogenation of the osazone
from maltose. Here, too, both the 1-amino-1-deoxy and the 2-amino-
7 7 3 7 s

2-deoxy compounds were obtained.

Another interesting reduction was carried out by Wolfrom and cowork-
ers79; using hydrogen, Raney nickel catalyst, and D-arabino-hexulose
phenylosazone (43) , they obtained 1,2-diamino-1,2-dideoxy-~-mannitol
(63).They laters0 improved the yield, and obtained some of the epjmeric
HC=N-NH-Ph C H, NH, yJWH2
I I
C =N-NH-Ph GNCH HCNH,
I I I
HOCH HOCH
I - 1 H°CH + I
HCOH HCOH HCOH
I I
HJoH HCOH HCOH
I I I
CH,OH CGOH CH,OH

(43) (63) (64)

-
(74) K. Maurer and B. Schiedt, Ber., 68, 2187 (1935).
(75) J. Druey and G . Huber, Helv. Chim. Acta, 40, 342 (1957).
(76) R. Kuhn and W. Kirschenlohr, Chem. Ber., 87, 1547 (1954).
(77) M. L. Wolfrom, H. El Khadem, and J. R. Vercellotti, Chem. Znd. (London), 545
(1964).
(78) M.L. Wolfrom, H. El Khadem, and J. R. Vercellotti, J . Org. Chem., 29,3284 (1964).
(79) M. L. Wolfrom, F. Shafizadeh, J. 0. Wehrmuller, and R. K. Armstrong, J . Org.
C h m . , 23, 571 (1958).
(80)M. L. Wolfrom and J. L. Minor, J . Org. Chem., SO, 841 (1965).
164 HASSAN EL KHADEM

1,2-diamino-1 ,2-dideoxy-~-glucitol (64) as well, by carrying out the

reduction in 2 N alcoholic potassium hydroxide (to break the chelated ring
and facilitate the reduction).
1,2-Diamino-l, 2-dideoxy-~-mannitol (63) was obtained by Henseke, 81
also, from D-arabino-hexosulose 1-(2-methyl-2-phenyl)hydrazone 2-oxime
(65) by reduction with hydrogen in the presence of platinum oxide.
Me
Hy=N-&-Ph yWH,

-
C =NOH H,NCH
I I
HOCH HOCH
I I
HCOH HCOH
I I
HYOH HCOH
I
CH,OH ChOH

(65) (63)

3. Oxidation
Diels and coworkers8*have shown that, when D-arabino-hexulose phenyl-
osazone is oxidized with oxygen (or air) in an alkaline medium, a dehydro-
osazone is obtained which possesses two hydrogen atoms fewer than the
parent osazone. On acetylation, the compound gives a triacetate, and its
hydrazine residues could not be removed with p-nitrobenzaldehyde, sug-
gesting their involvement in ring formation. The authors supported this
view by the findings that dehydro-osazones cannot be obtained from
niethylphenylosazones or from the Diels anhydro-osazone (which, at that
time, was believed to be a pyrazole compound).
HC=N,
1 ,N-Ph
C-N

Later, Mester and Moczar83 oxidized the dehydro-osazone (66) with

periodic acid and found that one mole of it consumes one mole of oxidant
(instead of two, as would be expected from Diels’ formula). When the alde-
(81) G. Henseke and R. Neinass, Chem. Ber., 97, 733 (1964).
H. J. Stephan, and R. Konig, Ber., 71, 1189 (1938).
(82) 0. Diels, E. C~USS,
(83) L. Mester and E. Mocsar, Chem. Ind. (London), 554 (1962).
 CHEMISTRY OF OSAZONES 165

hyde obtained (67) was boiled with phenylhydrazine, it gave glyceralde-

hyde phenylosazone (68) and the phenylhydrazide of formylglycolic acid
(69). As it gave no formazan, they represented the dianhydro-osazone
and its oxidation as follows.

HOhC

HO
d r ) - N H - N H P h - p+ HoCI 4
C-NH-NH-Ph

O=C-C-N=NPh
H
N=N-Ph

(66) (67)

PhNHNH,

N-NHPh NH-NHPh
It I
HC c=o
t I
+=N-NH-,, C=N-NH-Ph
I
CH,OH HC=N-NH-Ph

(68) (69)

The same authorsg4later corrected the configuration of the hydroxyl

group at C-3; they found that the same dehydro-osazone is obtained from
D-arabino-hexulose phenylosazone and D-ribo-hexulose phenylosazone, but
that the dehydro-osazones from D-lyxo-hexulose phenylosazone and L-XY~O-
hexulose phenylosazone are enantiomorphous, suggesting that the hydroxyl
group at C-3 of one of them undergoes a transposition so that it may acquire

o -arabino D-ribo 0 -1yxo L-xylo

Same d e h y d r o - Enantiomorphous
osazone dehydro-osazone

(84) L. Mester and E. Moczar, J. Org. Chem., 29, 247 (1964).

166 HASSAN EL KHADEM

a stable configuration. From nuclear magnetic resonance studies, they

concluded that the hydroxyl group at C-3 is axial, and they represented
the D-urubino-hexulose derivative as having structure (70)
HN- P h

It
Ph-N

4. Osotriuzole Formationa6
In 1944, Hann and Hudsona6found that, when refluxed with cupric
sulfate, osazones are converted into colorless triazole derivatives which
they designated osotriazoles. They determined the structure of the D-
arubino-hexulose derivative by means of the following reactions: (1) it
gave a tetraacetate and a tetrabenzoate which could be saponified to the
parent osotriazole, indicating the presence of four free hydroxyl groups
therein; (2) on periodate oxidation, 3 moles of oxidant were consumed
per mole of (71), giving 1 mole of formaldehyde, 2 moles of formic acid,
and a known compound, 4-formyl-2-phenyl-l , 2,3-triazole (72).

HC=N-NH-Ph HC=N, HC=N\

I I N-Ph I ,N-Ph
C=N-NH-Ph C=N’ C=N
I I
HOCH HOCH 3 HIO, Hk=O
I cuso, I
HCOH - HCOH (72)
I I
HCOH
I
HCOH
I
+
CHIOH ChOH 2 HCQH + HCHO

(43) (71)

The osotriazoles, which possess sharp melting points and show no inuta-
rotation, have been used for the characterization of 0sazones.~7-~6Owing
(85) The chemistry of osotriazoles has been reviewed; see H. El Khadem, Advan. Carbo-
hydrate Chem., 18, 99 (1963).
(86) R. M. Hann and C. S. Hudson, J . A m . Chem. Soc., 66, 735 (1944).
(87) W. T. Haskins, R. M. Hann, and C. 8. Hudson, J . A m . Chem. Soc., 67, 939 (1945);
68, 1766 (1946); 69, 1050, 1461 (1947); 70, 2288 (1948).
(88) D. A. Rosenfeld, N. K. Richtmyer, and C. S. Hudson, J . A m . Chem. Soc., 73, 4907
(1951).
(89) L. C. Stewart, N. K. Richtmyer, and C. S. Hudson, J . Am. Chem. SOC.,74, 2206
(1952).
 CHEMISTRY OF OSAZONES 167

to the stability of the triazole ring toward acids, they have been used
for determining the structure of diasccharidesg7-100 and anhydro-osa-
zones.101-104They have also been used as analytical tools10”108and to intro-
duce stable sugar residues into various organic molecules.lOs-lll
Although the exact mechanism of osotriazole formation is not clear,
it seems that an oxidation is involved a t one stage; this is because only such
salts as those of Fe3+ and Cu2+,in their higher state of valency, can bring
about osotriazole formation.ll2 Similarly, such mild oxidants as nitrosodi-
sulfonatella and the halogens chlorine,l14-l16b r o n 1 i n e , ~ ~ 2and
J ~ ~iodine114
~~~~
convert osazones into osotriazoles. With chlorine and bromine, simul-
taneous halogenation of the benzene ring takes place a t the para position,

(90) J. W. Pratt, N. K. Richtmyer, and C. S. Hudson, J. A m . Chem. SOC.,74, 2210

(1952).
(91) J. V. Karabinos, R. M. Hann, and C. S. Hudson, J. A m . Chem. SOC.,76, 4320,
4324 (1953).
(92) A. Thompson and M. L. Wolfrom, J. A m . Chem. SOC.,76, 5173 (1954).
(93) P. P. Regna, J . A m . Chem. SOC.,69, 246 (1947).
(94) V. Ettel and J. Liebster, Collection Czech. Chem. Commun., 14, 80 (1949).
(95) J. F. Carson, J.A m . Chem. Soc., 77, 1881 (1955).
(96) E. Hardegger and H. El Khadem, Helv. Chim. Actu, 30, 900, 1478 (1947); 34,
253 (1951).
(97) W. Z . Hassid, M. Doudoroff, and H. A. Barker, Arch. Biochem., 14, 29 (1947).
(98) W. Z. Hassid, M. Doudoroff, A. L. Potter, and H. A. Barker, J . A m . Chem. SOC.,70,
306 (1948).
(99) F. H. Stodola, H. J. Koepsel, and E. S. Sharpe, J.A m . Chem. Soc., 74, 3202 (1952).
(100) F. H. Stodola, E. S. Sharpe, and H. J. Koepsel, J. A m . Chem. SOC.,78, 2514 (1956).
(101) E. Hardegger and E. Schreier, Helv. Chim. Actu, 36, 623 (1952).
(102) H. El Khadem, E. Schreier, G. Stohr, and E. Hardegger, Helv. Chim. Aciu, 36,
993 (1952).
(103) S. Bayne, J. Chem. SOC.,4993 (1952).
(104) E. Schreier, G. Stijhr, and E. Hardegger, Helv. Chim. Acta, 37, 574 (1954).
(105) C. T. Bishop, Science, 117, 715 (1953).
(106) J. C. Bevington, E. J. Bourne, and C. N. Turton, Chem. Ind. (London), 1390
(1953).
(107) S. P. Rao, J. N. Gaur, and S. K. Sharma, Naturwissenschuften, 48, 98 (1961).
(108) S. P. Rao and J. N. Gaur, Indian J. Chem., 1, 378 (1963).
(109) H. El Khadem and M. H. Meshreki, Nature, 194, 373 (1962).
(110) B. B. Bishay, H. El Khadem, Z . M. El-Shafei, and M. H. Meshreki, J. Chem. SOC.,
3154 (1962).
(111) H. El Khadem, G. H . Labib, and M. H. Meshreki, J. Chem. Soc., 3528 (1963).
(112) H. El Khadem and Z. M. El-Shafei, J . Chem. Soc., 3117 (1958).
(113) H. J. Teuber and G. Jellinek, Ber., 86, 95 (1952).
(114) H. El Khadem, Z. M. El-Shafei, and M. H. Meshreki, J. Chem. SOC.,2957 (1961).
(115) H. El Khadem, A. M. Kolkaila, and M. H. Meshreki, J. Chem. Soc., 3531 (1963).
(116) H. El Khadem, M. H. Meshreki, and G. H. Labib, J. Chem. SOC.,2306 (1964).
(117) H. El Khadem and Z. M. El-Shafei, J. Chem. Soc., 1655 (1959).
(118) H. El Khadem, Z. M. El-Shafei, and Y. S. Mohammed, J. Chem. Soc., 3993 (1960).
168 HASSAN EL KHADEM

when this is free. Another reagent used for closing to the triazole ring is
nitrous acid. This reagent is known to convert osazones, such as (43), into
the glycosulose 1-(phenylhydrazone) (73) and then into the glycosulose
(74); but, with osazone acetates, such as (75), it yields the osotriazole
acetate (76),66
HC=N-NH-Ph
I
C=N-NH-Ph
H’i=N-NH-p HC=O
I
c=o
I ?=O I
HOCH
I
HCOH
I
HNQ 5
HOCH
HC‘OH
I
HN02
--
HOCH
I
HCOH
I
HCOH HCOH HC OH
I I I
CH,OH ChOH ChOH

(73) (74)

HC=N-NH-Ph HC=N,
I N-Ph
+=N-NH-Ph C=N/
I
ACOCH ACOCH
I I
HCOAc HNo2 = HCOAc
HCOAC
I
CH,OAc

(75) (76)

The problem of determining whether, during osotriazole formation,

aniline is removed from the hydraeone residue of C-1 or of C-2 was solved
independently by Weygand and Henseke and their associates. The first
groupz3 made use of the fact that when D-arabino-hexulose (p-bromo-
pheny1)osazone is treated with szBr-labeled (p-bromophenyl) hydrazine,
transhydrazonation proceeds unequally, so that most of the label appears
on C-1. This was demonstrated by treating the osazone (77) with periodic
acid and cleaving the resulting pyrazolone derivative (78) with stannous
chloride, to yield p-bromoaniline having 23% of the label from the hydra-
zone at C-2. When the unequally labeled osaeone (77) was converted into
the osotriazole (79), 18% of the label remained, indicating that the hydra-
zine residue at C-1 had been split off. Henseke and coworkers,11gon the
other hand, prepared a number of mixed osazones having either the phenyl-
hydrazine residue a t C-1 and another substituted hydrazine residue at
C-2, or the phenylhydrazine residue at C-2 and the other substituted hydra-
zine residue at C-1. These were treated with cupric sulfate, and it was
(119) G . Henseke and M. Winter, Chem. Ber., 93,45 (1960).
 CHEMISTRY OF OSAZONES 169

HC=N-NH a B r *
I
C=N-NH
I
HOCH
HCOH
I
-
HIO, B r o k
N-CH
,
Yi
'
,CH-N=N a B r

HCOH

1
I
CQOH

(77)
I snc1,
cuso,

HC=N
I
Q N e B r *
> a B r *
C=N (23% of label)
I
HOFH
HCOH
I
HCOH
I
CQOH

(79)

(18% of label)

found that, invariably, the hydrazine residue at C-1 was split off during
formation of the triazole.
Correlations between the configuration of the sugar residue in osotria-
zoles and the sign of their rotation have been made independently by the
author120and by Mills,121in the form of an osotriazole rule. This rule states
that, when the hydroxyl group attached to C-3 of the sugar residue is to
the right in the Fischer projection formula, the sign of rotation of the de-
rived osotriazole is positive, and when to the left, it is negative. This rule
was laterlZ2generalized to correlate the sign of rotation with the con-
figuration of the asymmetric carbon atom attached to a heterocyclic or
aromatic ring.

5 . Formation of Formazans12a
In 1955, Mester showedK9that diazotized aniline couples with D-arabino-
hexulose phenylosazone (43) in alcoholic potassium hydroxide to give an
(120) H. El Khadem, J . Org. Chem., 28, 2478 (1963).
(121) J. A. Mills, Australian J . Chem., 17, 277 (1964).
(122) H. El Khadem and Z. M. El-Shafei, Tetrahedron Letters, 1887 (1963).
(123) See L. Mester, Advan. Carbohydrate Chem., 13, 105 (1958).
170 HASSAN EL KHADEM

osazone forniazan (80). The reaction could not be carried out in the ab-
sence of alkali, owing to chelation of the osazone (which chelation was
broken by the alkali). The structure of the osazone formazan was estab-
blished by treating123D-arabino-hexosulose 1-(phenylhydrazone) (81) with
diazotized aniline and converting the glycosulose hydrazone forinazari
(82) into the same osazone formazan (80) by treatment with phenyl-
hydrazine. Furthermore, the osazone formazan (80) gives a tetraacetate,
indicating an acyclic structure.
N=N-Ph
I
HC=N-NH- Ph GIN-NH-Ph
I
C=N-NH-Ph +=N- NH- Ph
HOCH
I
HCOH
- PhN-N' <i
+ HO'H
HFOH
HCOH H$OH
&%OH C%OH

(43 1

N=N-Ph
I
HC=N-NH-Ph C=N-NH-Ph
I I
c=o c=o
I I
HOCH PmGNO
HOCH
I - I
HCOH HCOH
I I
HCOH HCOH
I
&%OH CbOH

(81) (82)

The osazone forniazans are readily converted into the osotriazole forma-
zans by the action of boiling acetic acid. Mester and W e ~ g a n d l followed
*~
this reaction with 14C-labeled phenylhydrazine in an ingenious manner.
They prepared three types of labeled osazone formazan having, respec-
tively, the labcl on (1) the phenylhydrazorie formazan a t C-1; (2) the
hydrazone residue a t C-2; and (3) the diazotized aniline of the osazonc
formazan. They found that formazans prepared according to Scheme I,
when converted into the osotriazole formazan, lose 50% of their label,
whereas those having the label on the hydrazine residue at C-2 retain the
label (as expected from their formulation). It was also found that a dif-
(123a) L. Mester and A. Major, J . C L m . SOC.,3227 (1956).
(124) L. Mester and F. Weygand, Bull. SOC.Chim. France, 350 (1960).
 CHEMISTHY OF OSAZONES 171

(Scheme I)

HC= N- NH-Ph
I
c=o
I
- *Ph-N-N
0

C=O
JT=N

A\N-N
/Ph*
+H
/
\
- A
C=O N-N
N=N
/Ph
\
I Ph I , Ph*

PhNHNH, PhNHNH,

x
N=N
-c- cr = N h -c-c/
// \ /
// \\ /
N N-N N\ N-N
\
\ N--If Ph*
/
Ph p/h

(50% o r less of t h e
l a b e l retained)
HCO,H

y=N-Ph*
C=N\
I N- Ph
C=N’
I

(Scheme 2)

Ph
/
0
-
HC=N-NH-Ph Ph-N-N ,N=N
I
c=o A\ /“H
I C E O N-N
I \
Ph

’
*PhNHNH,

Ph
N=N-Ph /
N=N
I
T?-?\ ’ X
C=N, (100% of t h e
I N-Ph* a c t i v i t y retained) I
C=N’ N N-N
\ \
I N-I4 Ph
/
Ph*
172 HASSAN EL KHADEM

ference occurs, in label retention in Scheme 1, between saccharide osazones

and such simple bis(hydrazones) as those of glyoxal and pyruvaldehyde.
This was attributed to chelation with the hydroxyl group at C:-3, but in a
subsequent publication, M e s t e F found that 3,6-anhydro-~-ribo-hexulose
phenylosazone, which does not possess a free hydroxyl group at C-3,
also shows this behavior.
(Scheme 3)

Ph*
/

N\ N-N
\
F-If Ph

6. Action of Alkalis
The osazones are unstable toward alkalis and are rapidly degraded
thereby. From the reaction mixture of D-arabino-hexulose phenylosazone
and ethanolic potassium hydroxide, Diels and coworkersv2"isolated glyoxal
phenylosazone and oxalic acid. Under similar conditions, the osazone from
cellobiose yielded a colorless compound C17HzrOsN2, formulated by Diels
as (83), a structure which needs further confirmation.

HZ+-rph
I
HC
J%OH

(83)
(125) L. Mester, Bull. SOC.Chim.France, 381 (1962).
(126) 0. Diels, R. Meyer, and 0. Onnen, Ann., 626, 94 (1936).
 CHEMISTRY OF OSAZONES 173

7. Conversion into Glycosuloses'21

Fischer128showed that the substituted hydrazine residues of osazones
can be hydrolytically split with concentrated hydrochloric acids, to give
the corresponding glycosulose (glycosone) (86). This reaction has been used
for converting aldoses into ketoses by subsequent reduction of the glycosu-
lose,I2 and in the synthesis of L-ascorbic acid4 and its homologs. The
conversion of osazones into glycosuloses (86) has been carried out with
such aldehydes as b e n ~ a l d e h y d e ~and
~ ~ Jo-nitrobe~izaldehyde,~~~
~~ as well
as with nitrous a~id'~ZJ33and, in the case of disubstituted osazones such as
(M),with cupric sulfate.'S4 In the latter two instances, it was possible to
stop the reaction at the glycosulose 1-hydrazone stage (85) and to isolate
these compounds (which have recently received considerable attention
because of their use in the preparation of mixed osazones).
HC=N-N(Me)Ph HC=N-N(Me)Ph HC=O
I I I
C =N-N(Me) P h c=o c=o
I I I
HOCH HOCH HOCH
I - 1 - 1
HCOH HCOH HCOH
I I I
HCOH HCOH HYOH
I I
CbOH CGOH CH,OH

(84) (85) (86)

The position of the hydrazone residue in D-arabino-hexulose 1-(2-methyl-

2-phenylhydrazone) (87) was established by Henseke and Hantschel,lS5
HC=N-N(Me)Ph
HF=N-N(Me)Ph
-
I
c=o HOCH
I I
HOCH
I
HCOH
H H°FH
HCOH
I I
HCOH HCOH
1 I
CqOH ChOH

(87) (88)
(127) The chemistry of glycosuloses was reviewed by S. Bayne and J. A. Fewster,
Advan. Carbohydrate Chem., 11, 43 (1956).
(128) E. Fischer, Ber., 21, 2631 (1888).
(129) E. Fischer and E. F. Armstrong, Ber., 36, 3141 (1902); 44, 1898 (1911).
(130) S. Bayne, Methods Carbohydrate Chem., 2 , 421 (1963).
(131) R. S. Morrell and A. E. Bellars, J . Chem. Soc., 87, 280 (1905).
(132) H. Ohle, G. Henseke, and Z. Czyzewski, Chem. Ber., 86, 316 (1953).
(133) G. Henseke and M. Winter, Chem. Ber., 89, 956 (1956).
(134) H. El Khadem, J. Chem. Soc., 3452 (1953).
(135) G. Henseke and H . Hantschel, Chem. Ber., 87, 477 (1954).
174 HASSAN EL KHADEM

who catalytically reduced (87) and obtained D-inannose 1-(2-methyl-2-

phenylhydrazone) (88), thus demonstrating that the hydrazone residue in
(87)is attached to C-1.
A large number of glycosulose l-(Zsubstituted) hydrazones have been
prepared by Henseke and coworkers136from the osazones by action of
nitrous acid. These products, such as (89), were then treated with other
substituted hydrazines, to give inixed or with
hydrazine and other carbonyl reagents, to obtain ketazines and a z i ~ i e s ~ ~ O J ~ ~
hydrazone thiosemicarba~ones,~~~J~~and hydrazone
HC=N-N(Me)Ph HC=N-N(Me)Ph

-Hot
I
+=0 C=N-R
HOCH
HAOH H I OH
HCOH
H+OH I
CKOH CH,OH

(89)

( R = O H , NH,, NHPh, NHCONH,, NHCSNH,, etc.)

Hexosulose hydrazones and the mixed osazones react with diazotized
aniline to give f ~ r m a z a n s ,and
~ ~ with
~ J ~ hydrogen
~ peroxide to give pentonic
acids.lq6Unlike the niixed osazones obtained by transhydrazonation, the
derivatives obtained from the glycosulose hydrazones are pure, uncon-
taminated compounds, and the positions of their hydrazone residues are
well established. Thus, they afford remarkable possibilities, not only in
the preparation of new types of compound, but also in enabling the reac-
tion of osazones to be followed more closely, as in the case of triazole
forniation,llgand in studying the structure of anhydro-osazones.139

8. Transhydrazonation
The excharige of the hydrazone residues in osazones has been termed
transhydrazonation and, sometimes, transosazonation. The phenomenon
(136) The chemistry of glycosulose hydrazones has been reviewed by G. Henseke, Acla
Chim. Acad. Sci. Hung., 12, 173 (1957).
(137) H. Beyer, 0. Henseke, and W. Liebenow, Chem. Ber., 86, 10 (1953).
(138) G. Henseke and W. Liebenow, Chem. Ber., 87, 1068 (1954).
(139) G. Henseke and M. Bautze, Chem. Ber., 88, 62 (1956).
(140) G. Henseke and H. J. Binte, Ann., 612, 205 (1958).
(141) G. Henseke, H. J. Binte, and S. Schwerin, Ann., 640, 37 (1961).
(142) G. Henseke and U. Kruger, Chem. Ber., 88, 1640 (1955).
(143) G. Henseke and G. Badicke, Chem. Bey., 89, 2910 (1956).
(144) G. Henseke and H. W. Pelz, Chem. Ber., 97, 725 (1964).
(145) G. Henseke and M. Winter, Chem. Ber., 92, 3156 (1959).
(146) S. Tejima, Yukugaku Zusshi, 77, 673 (1957).
 CHEMISTRY OF OSAZONES 175

had early been recognized by Neuberg,l*' who found that, when L-arabinose
phenylhydrazone is heated with p-bromophenylhydraeine, it is converted
into the corresponding (p-bromopheny1)osazone. This reaction was closely
investigated by Voto~ek,148-160who experimented with a wide variety of
substituted osazones and hydrazines, and found that transhydrazonation
occurs only if the osazone formed is less soluble than the starting osazone.

Furthermore, he used the reaction to obtain a number of mixed osazones;

these compounds are, however, best prepared from the glycosulose hydra-
zones. A detailed study of transhydrazonation was made by Mandel,lK1
who confirmed VotoEek's finding that the reaction proceeds best when the
products have low solubilities, as with the (nitro- and dinitro-pheny1)-
osazones.

9. Anhydro-osazones

The reactions discussed in this Section are those involving the removal
of the elements of water from osazones, and not merely the preparation of
osazones from anhydro saccharides. There are, at present, three types of
anhydro-osazone and, in all three types, the elements of water are elim-
inated from the hydroxyl group at C-3 and another group of the osazone,
accompanied in some cases by inversion of configuration a t C-3.
a. 3 ,6-Anhydro-osazones.-This type of anhydro-osazone was first re-
ported by Fischer13; it was later studied by Diels and M e ~ e r , who
' ~ ~ ob-
tained such compounds by refluxing osazones in methanol containing some
sulfuric acid. Several structures were proposed for the derivative obtained
by way of (43) from D-glucose, often called "Diels' anhydro-osazone,"
until, finally, Hardegger and C O ~ O ~ ~ showed~ ~ Sit to ~ 3~,6-anhydro-
~ be J ~ ~ J ~ ~

(147) C. Neuberg, Ber., 32, 3384 (1899).

(148) E. Votorek and F. Valentin, Collection Czech. Chem. Commun., 3, 432 (1931).
(149) E. VotoEek and F. Valentin, Arhiv. Hem. i. Farm., 6, 155 (1931).
(150) E. Vot,oEek and R. VondraEek, Ber., 37, 3848 (1904).
(151) I. Mandl, Arch. Biochem., 26, 109 (1950).
(152) 0. Diels and R. Meyer, Ann., 619, 157 (1935).
(153) E. Hardegger and E. Schreier, Helv. Chiim. Ada, 36, 232 (1952).
176 HASSAN EL KHADEM

n-ribo-hexulose phenylosazone (90). Evidence for this structure is as

follows.
HC =N-NH-Ph HC =N-NH-Ph
I I
C=N-NH-Ph C=N-NH-Ph
I
HOCH
I
HCOH
I
HCOH
I I
CQOH
(43) (90) (91)

(a) It, (go), can be converted into an anhydro-osotriazole (91)’O’ and an

anhydroglycos~lose,~~a indicating that the hydrazone residues are not
involved in anhydride formation. (b) It, (go), gives a diacetate, and so does
its osotriazole.lO’(c) Diels’ anhydro-osazone (90) may also be obtained
from D-ribo-hexulose phenylosazone,lbabut it differs from 3 ,6-anhydro-~-
arabino-hexulose phenylosazone. (d) It, (90), forms a f ormazan
During anhydride formation from n-urabino-hexulose phenylosazone,
3,6-anhydro-~-urabino-hexulose phenylosazone is also produced in small
yield.1°2
The anhydrophenylosazone derived from n-galactose,1~8~104~16~~164-16~ on
the other hand, does not undergo inversion at C-3 to any appreciable ex-
tent, such inversion being confined to n-glucose, disaccharides having
n-glucose as the reducing component, and D-gulose.loaSimilar 3 ,6-anhydro-
osazones can be prepared from (2-methyl-2-pheny1)osazone acetates by
d e a c e t y l a t i ~ n ~ *with
~ J ~ sodium
~ hydroxide in dilute acetone. For the
phenylosazone acetates, this deacetylation leads to dianhydro-osazones,
as will be seen later (see p. 177). Disaccharide phenylosazones also yield
anhydro-osazones on deacetylation. The 1actoselb8and cell0biose~~9 deriva-
tives are identical with those obtained by boiling with methanolic sulfuric
acid, and have been identified as P-D-galactopyranosyl-(1 4 - 3 ,6-anhydro-
n-ribo-hexulose phenylosasone and P-D-glucopyranosyl-(1-+4)-3,6-anhydro-
n-ribo-hexulose phenylosazone, respectively.lo8The osazone acetate from
maltose, on the other hand, yields two monoanhydro derivatives, whereas,
by Diels’ method, a mono- and a di-anhydro-osazone are obtained; the
structure of these compounds needs further investigation.
(154) E. G. V. Percival, J. Chem. Soc., 783 (1945).
(155) A. N. O’Neill, J. Am. Chem. Soc., 77, 2837 (1955).
(156) H. Zinner, K. H. Stark, E. Michalzik, and H. Kristen, Chem. Ber., 96, 1391 (1962).
(157) E. E. Percival and E. G. V. Percival, J . Chem. Soc., 750 (1941).
(158) E. E. Percival and E. G. V. Percival, J. Chem. SOC.,1320 (1937).
(159) J. R. Muir and E. G. V. Percival, J . Chem. Soc., 1479 (1940).
 CHEMISTRY OF OSAZONES 177

3 ,6-Anhydro-osazones are also obtained when osazones are refluxed with

acetic acidag; thus, di-0-acetyl-3 ,6-anhydro-~-ribo-hexulose I-N2-acetyl-
phenylosazone (92) is formed, together with the dianhydro-osazone (de-
scribed on p. 178), from D-arabino-hexulosephenylosazone (43) and boiling
acetic anhydride. On deacetylation, compound (92) yields Diels’ anhydro-
osazone (go), and (92) can be prepared from the latter by acetylation with
boiling acetic anhydride.

Ac
I
HC=N-NH-Ph HC=N-N-Ph HC=N- NHPh

-
I
I
&N-NH-Ph b=N-NHPh $!=N-NHPh
I I ,
HOCH AGO
I
HCOH (boiling) HCOAc Ac20

J
I
HrioH
CH20H “O
CH20
ACl HfoH
CH20

(43)

b. Dianhydro-osazones.-(1) Percival’s Anhydro-osazone.-Percival1O0Ja1

showed that, when a hexose phenylosazone acetate is deacetylated with so-
dium hydroxide in dilute acetone, it is converted into a dianhydro-osazone.
This compound exists in two isomeric forms-one obtained from hexoses
of the D series, and its enantiomorph from those of the L series. It gives a
monoacetate and a monomethyl ether and was assigned the tricyclic
structure (93). Henseke and coworkers102were able to remove one hydrazine

E;Y--..
Ph-N-N=CH
I

HCOH
CH,

(93)

residue from this dianhydro-osazone by the action of nitrous acid, and

obtained the hexosulose dianhydro-phenylhydrazone.They concluded that
one of the hydrazine residues is not involved in anhydride formation, and
formulated the glycosulose hydrazone and Percival’s dianhydro-osazone as
(160) E. G. V. Percival, J . Chem. Soc., 1770 (1936).
(161) E. G. V. Percival, J . Chem. Soc., 1384 (1938).
(162) G . Henseke, V. Miiller, and G . Badicke, Chem. Ber., 91, 2270 (1958).
178 HASSAN EL KHADEM

(94) and (95), or their tautomeric forms. The tautomer of (95), which
possesses two imino protons, must be discarded, since the nuclear magnetic
resonance spectrum of the dianhydro-osazone shows only one imino proton
and agreeslBZBwith formula (95).

I I
HOCH- CH, HOCH-CH,

(94) (95)

(2) Dianhydro-osazones Having Pyrazole Rings.-Another dianhydro-

osazone was obtained as the acetate (96) by the author and ~ o w o r k e r s ~ ~ ~ ~ ~
by acetylating phenylosazone (43) with boiling acetic anhydride. On
de-0-acetylation with methanolic ammonia, the hexose compound (96)
yields the N-acetyl-dianhydro-osazone (97), from which (96) can be pre-
pared by acetylation. The de-0-acetylated dianhydro-osazone (97) was

HCZN- NH-Ph Ac Ac

-
I I
y=N-NH-Ph HC=N-NPh
HF=N-hPh I
HOCH Ac 0 C=N, C=N,
I A I ,N-Ph I
HC=C
,N-Ph
HCOH (boiling) HC=C I I
I
HCOH H~OAC HCOH
I I
CH,OH
&%OH CKOAc

oxidized with one mole of periodic acid per mole, yielding formaldehyde
and a pyraeole aldehyde (98) ; on oxidation with potassium permanganate,
(97) yields the known l-phenylpyrazole-3,5-dicarboxylicacid (99) and
so it was given structure (97). Compound (97), which exists in only two
enantiomorphous modifications, differing in the configuration of the
hydroxyl group a t C-5 of the hexose precursor, affords a direct means of
recognizing the D or L configuration of hexoses from the sign of the optical
rotation.r22 Similar dianhydro-osazones have been obtained from 6-de-
oxyhexoses and pentoses; the latter give an optically inactive derivative.
The structure of these compounds was unequivocally confirmed by nuclear
magnetic resonance data (see Fig. 3) . I 6 3 Disaccharide8 having 1,6-1inkages,
such as isomaltose and gentiobiose, also give dianhydro-osaz~nes.~~~
(162a) H. El Khadem and M. M. A. Abdel Rahman, J . Org. Chem., SO, in press (1966).
(163) H. El Khadem, J . Org. Chem., as, 3072 (1964).
I

20Ac
'ZA

I
I
C-N,
H C -0Ac

rn
nc-c'
H-C-OAC I
I '
H-t-OAC
I
s c -0t.c

NAc
L.4
TMS

J-*"- dhb

FIG.3.-Nuclear Magnetic Resonance Spectrum of 5-(D-glf/CwO-l, ZDiacetoxyethyl)-3-formyl-l-phenylpyrazole NZ-Acetyl-N*-phenyl-

hydrazone.
180 HASSAN EL KHADEM

Ac
I
HC=N-NPh
I
C=N\
I ,N-Ph
HC=<i

HC=N-NPh
I
N-Ph

I N-Ph
CQOH
I
CO,H
(97)
(99)

VIII. USES

Osazones have mainly been used for the identification of saccharides,

either through their melting points or by comparing their crystalline
forms.1s4Je6For the latter purpose, methods have been devised to carry
out osazone formation on microscope s l i d e ~ ,so~ as
~ ~toJpermit
~ ~ permanent
mounting of authentic osazones thereon.las Osazone mixtures can be
chromatographed on calcium carbonate,l69 using ethanol-chloroform or
ethanol-acetone for elution, or on aluminalO*or silica gel,l7O using benzene-
dioxane or toluene-ethanol. Partition chromatography on paper impreg-
nated with 0.1 M potassium tetraborate has also been used, with dioxane-
hexane-water as the so1vent.l71 Several spot-tests have been suggested for
osazones.
Finally, osazones have been suggested for a variety of uses, such as
fungicides172arid antiviral agent^,^'^ additives in photographic films (to
increase the ~ensitivity'7~)~
and additives to glycerides176 and polye~ters'7~
(164) W. Z. Hassid and R. M. McCready, Ind. Eng. Chem. Anal. Ed., 14, 683 (1942).
(165) C. van Duijn, Mikrokosmos, 36, 16 (1942).
(166) L. Rosentheler, Pharm. Acta Helv., 21, 217 (1946).
(167) H. F. Schaeffer, J . Chem. Educ., 26, 20 (1948).
(168) D. E. Kidder and W. D. Roberston, School Sci. Rev., 31, 346 (1950).
(169) P. F. Jorgensen, Danek Tidsskr. Farm., 24, 1 (1950).
(170) E. F. L. J. Anet, J. Chromatog., 9, 291 (1962).
(171) B. Arreguin, Anal. Chem., 31, 1371 (1959).
(172) R. E. Miller and V. R. Gaertner, U. S. Pat. 2,844,505 (1958).
(173) B. D. Tiffany, J. B. Wright, R. B. Moffett, R. V. Heinzelman, R. E. Strube, B. D.
Aspergren, E. H. Lincoln, and J. H. White, J . Am. Chem. Soc., 79, 1682 (1957).
(174) M. A. Akhmedzyanov, M. S. Khaikin, V. A. Kukhtin, A. V. Borin, and V. 1.
Slesareva, U.S.S.R. Pat., 133,340 (1960).
(175) S. Shappirio, U. S. Pat., 2,430,031 (1947).
(176) R. Wendling and J. Sotiropoulos, French Pat., 100,141 (1955).
 CHEMISTRY OF OSAZONES 181

as stabilizers. D-arabino-Hexulose 6-N-(n-octyl)carbamate was also sug-

gested as being useful as a surface-active compound.177Owing to the
change in color which they undergo in alkaline media, osazones have been
tested for use as indicator~,'7~J7~
and have been suggested for use in spot
tests.180-1ss
(177) E. Ulsperger and W. Gerhardt, J . Prakt. Chem., [4] 16, 308 (1962).
(178) L. M. Kulberg, Nauchn. Ezhegodnik Saratow Univ., 512 (1955).
(179) N. V. Chugreeva, Zh. Obshch. Khim., 27, 3136 (1957).
(180) F. Feigl, Mikrochim. Acta, 1, 127 (1937).
(181) L. Rosenthaler, P h r m . Acta Helv., 26, 365 (1950).
(182) F. Feigl, Anal. Chem.,33, 1118 (1961).
(183) F. Feigl, V. Anger, and G. Fischer, Milcrochim. Acta, 878 (1962).