THE SOLUBLE SPECIFIC SUBSTANCE OF
       PNEUMOCOCCUS.

V. ON THE CHEMICAL NATURE OF THE ALDOBIONIC ACID
     FROM THE SPECIFIC POLYSACCHARIDE OF
          TYPE III PNEUMOCOCCUS.

BY MICHBEL HEIDELBERGER AND WALTHER F. GOEBEL.
(From the Hospital of The Rockefeller Iastitute for Medical Research,
               New York.)

(Received for publication, June 25, i927.)

The so called soluble specific substances of the three fixed types
of Pneumococcus appear identical with three chemically distinct,
serologically type specific polysaccharides produced by these
organisms during growth in culture media (1). The immuno-
logical significance and the chemical nature of these bacterial
carbohydrates have been discussed in detail in earlier publica-
tions from this laboratory. The present communication deals
with the chemical nature of an aldobionic acid, the fundamental
building stone of the polysaccharide derived from Type III
pneumococcus.

This aldobionic acid, the product of hydrolysis of the Type III
specific carbohydrate, has been shown (2) to have the formula
CIIHrsOloCOOH and to be built up from a hexose (glucose) and
a hexose-uranic acid of unknown nature, in such a manner that
the carboxyl group and one aldehydic group remain free.  It is
of interest to extend the investigation of this substance, for it not
only appears unique in the field of sugar chemistry, but important
in that it or its isomers are found among the hydrolytic products
of specific carbohydrates from other microorganisms.  The pres-
ent report aims to identify the hexose-uranic acid which forms
half of the molecule of the aldobionic acid, and to explain the
nature of the glucosidic linkage which binds the sugar to the acid.

613


614   Pneumococcus Specific Polysaccharide

EXPERIMENTAL.

1. Preparatioti of the Aldobionic Acid.

30 gm. of air-dry specific polysaccharide (prepared as in Paper
IV (2)) were dissolved in 120 cc. of 75 per cent sulfuric acid (by
weight) at 0'.  After standing overnight in the ice box the solu-
tion was diluted to 3 liters and boiled 5 hours under reflux.  The
sulfuric acid was then quantitatively removed with highly purified
barium hydroxide and the barium sulfate washed free from reduc-
ing sugars.  The combined filtrates were concentrated to 260 cc.
in Vacua, boiled with a little norit and an excess of calcium carbo-
nate, filtered, and the filtrate concentrated to 100 cc. in vacua.
The solution, which contained small amounts of glucose and the
calcium salt of the aldobionic acid, was poured into 10 volumes
of methyl alcohol.  In this manner the crude calcium aldobionate
was freed from reducing sugars.  The suspension was fltered and
washed two or three times with methyl alcohol.  24 gm. of crude
calcium aldobionate were thus obtained.

2. Preparation of Pure Calcium Aldobionate.

The crude salt was dissolved in twice its weight of water, and
to the solution was added alcohol in smal1 portions.  After each
addition of alcohol the mixture was centrifuged.  Enough alcohol
was added so that after the final centrifugation the supernatant
liquid, still containing a large part of the original calcium salt,
remained as a pale straw-colored solution.  This was decanted
from the deeply coIored lower oily layer and saved.  The Iower
layer was again dissolved in an equal volume of water, and treated
as before with alcohol, the straw-colored supernatant liquid again
being saved. After fractionating the lower layer two or three
times more, a deeply colored oil was obtained which was discarded,
and the combined supernatant liquids were concentrated in vacua
and poured into 10 volumes of methyl alcohol.  18 gm. of purified
calcium aldobionate were finally isolated. The substance gave
the following analysis :

0.1028 gm. substauce: 0.1438 gm. CO, and0.0490 gm. H20.
0.1096 " "   : o.o0!32 " cso.
Calculated for (ClnHlpOln)&a. C 38.33 per cent, H 5.07 per cent, Ca
5.33 per cent.  Found.  C 38.14 per cent, H 5.33 per cent, Ca 6.00 per cent.


M. Heidelberger and W. F. Goebel

615

The free aldobionic acid was obtained by adding a little less
than the calculated amount of oxalic acid to a 5 per cent solution
of the calcium salt, filtering off the calcium oxalate, concentrating
the filtrate to dryness in vacua, and dissolving the residue in
methyl alcohol. The, alcoholic solution of the aldobionic acid
was atered from the small amount of insoluble calcium aldobio-
r&-&e, and the filtrate was evaporated to dryness in vacua.  The
sugar, if properly manipulated, will puff up into a spongy mass,
and when completely dry it may be readily broken up and scraped
from the flask.

3. Oxidation of the*Aldobionic Acid with Barium Hypoiodite.

5.0 gm. of aldobionic acid were dissolved in a small amount of
water and the solution was oxidized with barium hypoiodite by
a method previously described (3).  The solution of the oxidation
product, after being freed completely from inorganic constituents,
was boiled with caSun carbonate and a small amount of norit.
It was then filtered and concentrated to 30 cc. in vaeuo.  By the
gradual addition of alcohol a precipitate of the calcium salt of
the oxidized aldobionic acid, which will be termed glucurono-
gluconic acid, separated out.  This calcium salt is far less soluble
than is that of the aldobionic acid, 25 cc. of alcohol sufficing to
remove it from solution.  The calcium salt was filtered off and
weighed. 5.2 gm. were recovered.

0.1000 gm. substance: 0.1284 gm. CO* and 0.0426 gm. H1O.
0.1368 " "   : 0.0188 " CaO.
Calculated for CJL~O,(COO)S Ca.  C 35.12 per cent, H 4.42 per cent,
Ca 9.76 per cent.  Found. C 35.01 per cent, H 4.77 per cent, Ca 9.82 per cent.

    4. Properties of the Glucuronogluconic Acid.

This dicarboxylated acid, when free from calcium, forms a
white amorphous powder soluble in methyl alcohol, and less
soluble in ethyl aIcoho1.  It is soluble in hot glacial acetic acid,
and, on cooling, dropIets of oil separate which do not crystallize
on standing.  The free carboxylated sugar is a strong acid, and
an aqueous solution colors Congo red paper a vivid blue.  The
oalcium salt shows lc&, = - 7.5".

The acid itself gives a strong naphthoresorcinol test and on
distillation with 12 per cent hydrochloric acid it yields 17 per cent


616   Pneumococcus Specific Polysaccharide

of furfural. The acid is non-reducing, but when boiled with
strong hydrochloric acid reducing sugars appear.  On hydrolyz-
ing for 15 hours with normal hydrochloric acid it yields a maxi-
mum of 24.8 per cent reducing sugars (calculated as glucose).  It
was thought possible to hydrolyze the acid with sodium amalgam
as Levene and La Forge (4) hydrolyzed chondrosin, but experi-
ments in this direction failed. The acid is extremely stable to
hydrolysis, both by acids and alkalies.

5. Hydrolysis of the Aldobionic Acid with Bromine and
       Hydrobromic Acid.

2.0 gm. of aldobionic acid were dissolved in 50 cc. of N hydro-
bromic acid and to the solution was added 0.5 cc. of bromine.
The mixture was boiled under a reflux for 20 hours. It was
necessary to replace the bromine from time to time.  At the end
of the hydrolysis the solution gave only a very faint naphthoresor-
cinol test and showed no reduction. The solution was then
evaporated in vacua to remove hydrobromic acid and bromine.
The remaining traces of acid were removed with silver sulfate and
the silver bromide was filtered off.  The silver ion in the filtrate
was removed with hydrogen sulfide and after filtration the sulfate
ion was removed quantitatively with barium hydroxide. A
filtrate free from inorganic constituents was thus obtained.  This
filtrate was evaporated to 2 cc. in vacua, made strongly alkaline
with 50 per cent potassium hydroxide, and then acidified with
glacial acetic acid.  The solution was seeded with a small crystal
of potassium acid saccharate and was placed in the ice box.
After standing 24 hours crystals of potassium acid saccharate
(0.20 gm.) were filtered from the mother liquor.  The crude salt
was recrystallized from 1 cc. of boiling water.

0.0474 gm. substance gave 0.0168 gm. J.&SO,.
   Calculated for COOK(CHOH)JOOH, K 15.75 per cent.
     Found.                    `( 15.89 " "
On substituting glucose for the aldobionic acid, a repetition of
the above experiment gave no potassium acid saccharate.

DISCUSSION.

Since it had previously been shown that the aldobionic acid,
the chief hydrolytic product of the specific polysaccharide of Type


M. Heidelberger and W. F. Goebel    617

III pneumococcus, is composed of glucose and a hexose-uranic
acid of unknown nature, united in such a manner that one aldehy-
die group remains reactive in the bionic acid molecule, it. must
necessarily be assumed that the sugar and sugar acid are combined
in glucosidic linkage either through the aldehydic group of the
glucose or through that of the hexose-uranic acid.  If the linkage
were of the first type, then the product obtained by oxidation of
the free aldehydic group with barium hypoiodite would obviously
be a true glucoside of a dibasic hexose acid; if the linkage were
through the aldehydic group of the hexose-uranic acid, the latter
would be intact after oxidation, whereas the free aldehydic group
of the glucose would be oxidized.  Since the glucuronogluconic
acid, obtained by the oxidation of the aldobionic acid, still gives a
naphthoresorcinol test and still yields the same amount of furfural
on distillation as it did before oxidation, one must assume that
the hexose-uranic acid does remain intact and that the free
aldehydic group of the aldobionic acid is actually the reducing
group of the glucose half of the molecule and not that of the
uranic acid.  It has been shown above, that the aldobionic acid
yields saccharic acid on hydrolysis in the presence of bromine.
Since glucose does not yield thit acid under these conditions, one
must necessarily assume that the saccharic acid formed in the
above experiment owes its origin to the hexose-uranic acid part
of the aldobionic acid molecule and that the hexose-uranic acid
is therefore glucuronic acid.

The formula (I)

       COOH
     I
    HO-C-H
     I
YCH
     I
0      HCOH

I

  HO&H
     I
L-       0-CHrC-C-C-C
        H

I.


618   Pneumococcus Specific Polysaccharide

ia in accord with the results of the experiments which have been
performed, whereas the isomeric formula (II)

  OH
I
HI 9
HCOH
I 0
HOCH
I  I  r-O-7
AC--l
I   I  OHH OH
HC---0-C-C-C-C-C-CH,OH
1    /   H OHH H
      H
COOH
           II.

is not.

Whether the linkage between glucuronic acid and glucose is
through carbon atom (6), in formula (I), or through one of the
other carbon atoms, remains to be determined.

SWMMARY.

The aldobionic acid CIIHloOlaCOOH isolated from the hydro-
lytic products of the specific polyaaccharide of Type III pneumo-
coccus has been shown to be a compound of glucuronic acid and
glucose, combined in glucosidic linkage through the aldehydic
group of glucuronic acid and one of the carbon atoms of glucose.

In conclusion the writers wish to express their gratitude to
Dr. P. A. Levene for his many helpful suggestions, particularly
his suggestions on the manipulation of sugar acids.

BIBLIOGRAPHY.

1. Dochez, A. R., and Avery, 0. T., J. Exp. Med., 1917, xxvi, 447.  Heidel-
  berger, M., and Avery, 0. T., J. Ezp. Med., 1923, xxxviii, 73; 1924,
  xl, 301. Heidelberger, M., Goebel, W. F., and Avery, 0. T., J. Exp.
  Med., 1925, xlii, 727. Avery, 0. T., and Heidelberger, M., J. Exp.
  Med., 1923, xxxviii, 81; 1925, xlii, 367.

2. Heidelberger, M., and Goebel, W. F., J. Sol. Chem., 1926, lxx, 613.
3. Goebel, W. F., J. Biol. Chem., 1927, lxxii, 801.
4. Levene, P. A., sndLaForge, F. B., J. Biol. Chem., 1913, xv, 69.