Full text of DOI:10.1016/S0031-9422(00)89901-X

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9 7 1 , Vol. 1 0 , pp. 2 5 2 9 t o 2 5 3 1 .
Press. Prin t ed in En glan d.
SHORT COMMUNICATION
ISOLATION AND CHARACTERIZATION OF
a -GUAIACONIC ACID AND THE NATURE OF
GUAIACUM BLUE*
J O H N F.
RO BE R T H.
MAR G AR E T K.
and
JOHN M.
Forest Products Laboratory, Forest Service, U.S. Department of Agriculture, and the
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.
(Received 16 November 1970)
Abstract-a-Guaiaconic acid, the constituent of gum guaiac resin that turns blue upon oxidation, was puri-
fied by elution from a polyamide column with aqueous formic acid and shown to be
The blue product, guaiacum blue, is the bis-methylenequinone ob-
tained by twofold one-electron oxidation of a-guaiaconic acid.
GUAIACUM or lignumvitae are names given to timber from a tropical-American genus of
trees
sanctum L. or G.
L.) (Zygophyllaceae). The wood is well known
for its great hardness and density
and oblique fibers and high content (15-30
gr. =
due to its interlocking diagonal
of resin in the green-brown heartwood. The
resin is removed from the logs as a melt or from the chipped wood by boiling in seawater;
it is marketed as brownish lumps, m.p. cu. called gum guaiac.
The resin or the wood itself formerly had many medical and pharmacological applica-
tions, e.g. in treating chronic ailments such as gout or rheumatism, in tests for occult blood
in stains or gastric contents (ether solution turns blue), and in Schonbein-Pagenstecher’s
test paper for detecting traces of HCN (guaiac +
+ HCN give blue color). How-
ever, neither the resin nor the wood is now included in the USP.
Tincture of guaiac turns blue in the presence of many inorganic or organic oxidizing
agents and has long been used to detect oxidative enzymes such as
oxidoreductase, E.C.
or peroxidase (donor:
tyrosinase
:
oxidoreductase, E.C. 1.10.3.
oxidoreductase, E.C. 1.11.1.7). The active principle is a hitherto
unknown compound, classically named a-guaiaconic acid. King and Wilson’ characterized
nine phenolic lignans in gum guaiac by alkylating the whole resin and studying methylated
and ethylated derivatives then isolated; their technique indicated the whereabouts of the
initial phenolic functions, but could not help to establish which compound (if any) was
a-guaiaconic acid. We have now obtained the active constituent.9
Preliminary experiments with TLC and paper chromatography of resin extracts
established that a-guaiaconic acid was a single rather minor constituent of the resin that
This work was supported by funds from the Forest Products Laboratory (Forest Service, U.S. Depart-
ment of Agriculture), Madison, Wis.
Present address, Swift R D Center, 1919 Swift Drive, Oak Brook, Ill. 60521
U.S.A.
Deceased, see
Details of all work with a-guaiaconic acid and guaiacum blue can be found in the Ph.D. thesis of
University of Wisconsin (1969).
9, 679 (1970).
JOHN F.
F. E. KING and J. G.
Chem. 4011 (1964).
2
529

2
530
F . KRATOCHVIL , ROBE RT H.
MARGARET K.
a n d J OHN M.
fluoresced blue under UV light and could be visualized on chromatograms by spraying with
tyrosinase or KIO,. It autoxidized or photolysed readily on thin layer plates to give
guaiacum blue.
Purified a-guaiaconic acid is readily autoxidizable, especially in alkaline solution,
and gives the characteristic deep blue when aqueous tyrosinase or
alcoholic solution. The IR spectrum of a-guaiaconic acid disk) showed the bands in
OH, 3002(w), and 2940(w) CH, 1612(w), 1596(w), and 15 15(s) Ar, 1468(m),
is added to its
1
430(m), 1402(w), 1363(w), 1325(w), 1280(m), 1263(s),
1177(m), 1119(m), 1041(m), 895(w),
1213(s),
841(w), 818(w),
and
790(m), 718(w), 682(w). The absence of carbonyl absorption and our observation that
a-guaiaconic acid was soluble in dilute alkali, but precipitated on lowering the
indicated that this compound was a phenol rather than an acid.
to 10
A UV spectrum of a-guaiaconic acid in 95
showed intense absorption at 324 nm
(log with peak at 251 nm. Addition of sodium ethoxide shifted the
maximum to 342 nm and the minor peak to 275 nm; acidification returned the maximum
to 324 nm. No shift was observed on addition of sodium acetate. These observations
indicate a highly conjugated phenolic molecule.
The NMR spectrum of a-guaiaconic acid in perdeuteroacetone (100 MHz) showed the
following resonances: methyl
=
vs. TMS,
aromatic
together 3). When
integration ratio
Hz,
was added, the phenol peak dis-
phenolic OH
s,
methoxyl (3.90,
Hz,
d,
=
=
Hz,
d,
=
appeared.
A mass spectrum of a-guaiaconic acid exhibited a stable molecular ion at
= 340
and prominent ion peaks at m/e = 325, 297 and 189. Metastable peaks at m/e =
and 271.41 indicated that the molecular ion lost successively a methyl radical and carbon
monoxide to give the ions at m/e = 325 and 297. This fragmentation is characteristic of
guaiacyl
The ion at m/e = 189 may be produced by loss of 151 mass units
from the molecular ion. A mass unit of 151 corresponds to a hydroxymethoxybenzoyl
radical, indicative of an a-substituted
A
isolated from methylated gum guaiac called dimethylfuroguaiacin (I)
had a UV maximum at 326 nm (log and fluoresced bright blue; the location of
=
the original phenolic group was established by examining diethylfuroguaiacin (II) isolated
from ethylated resin. This plus the facts that furoguaiacin (III) would have a molecular
weight of 340 and that all of our data are compatible with a compound of structure III
suggest that a-guaiaconic acid is ‘furoguaiacin’.
Confirmation of the structure of a-guaiaconic acid was obtained by methylation with
followed by crystallization from ethanol-acetone-water. The white crystals of the
ether produced displayed bluish fluorescence under UV light, had a strong
absorbance at 324 nm (95
of dimethylfuroguaiacin was given as 169-170”. NMR spectrum of our
in
two methoxyl peaks of equal intensity at
ethanol; no base shift), and had a
of
The
derivative
(
showed loss of the phenolic absorption of a-guaiaconic acid and the presence of
and 3.92 A mass spectrum of our I showed
a molecular ion at
of 368 and two significant fragment peaks at
= 353 and 184.
C. S. BARNES a n d J . L.
16, 219 (1963).
J . COLLIN, Bull.
Chim.
69, 449 (1960).
K. H E YN S, R.
a n d H. SCHARMANN, Tetrahedron 22, 2223 (1966).

Isolation and characterization of a-guaiaconic acid and the nature of guaiacum blue
2531
(
I-III)
dimethylfuroguaiacin;
diethylfuroguaiacin;
(IV)
I R =
IV Guaiacum blue.
II R =
III R = H, a-guaiaconic acid;
The pathway leading to the latter is not known, but the ion of mass 353 results from the
loss of a methyl radical and the formation of a stable allylic ion.
Oxidation of the phenol with a variety of enzymes or inorganic oxidants produced the
characteristic blue, water-insoluble pigment guaiacum blue
in benzene 580 nm, in
chloroform 585 nm). Failure to obtain an ESR signal from the pigment, its rapid
tion by acids or base, and the regeneration of color on treatment with weak base of a solution
with HCI strongly suggest that guaiacum blue is the highly conjugated
methylenequinone (IV).
EXPERIMENTAL
Pulverized gum guaiac (25 g) was triturated int he dark with 400 ml of
acidified with 2 ml of
portions of benzene. Repeated
filtered, and re-extracted with a further 100 ml of acidified solvent. The combined extract was
centrifuged, evaporated in
dissolution of the residue in
and dried by evaporation with two
and precipitation with benzene, and subsequent dissolution of the
contents of the supernatant in benzene and precipitation with hexane removed polar constituents of the
exact, and left 11.3 g of red-yellow viscous oil enriched in a-guaiaconic acid. The oil (10 was
graphed in the dark on 120 g of purified
24 g of crude a-guaiaconic acid, which after three
needles, m.p. 149” (yield from resin 1.4%).
Analysis (C, H, 5.99;
using
aq. HCOOH as eluent and yielded about
from gave white
0
18.19; calculated for C20H2 00 5. .C, 7057; H, 5.92;
the molecular weight (340 by mass sprectoscopy), and the NMR data show that a-guaiaconic acid is a
symmetrical guaiamonoepoxylignan.
Acknowledgements-The authors thank Drs. L. Anderson, C.-L. Chen, W. H.
Schnoes for aid and advice.
and H. K.
M. K. SEIKEL , F . D.
and D. B. J OH NSON , Tetrahedron 24, 1475 (1 9 6 8 ).