Portland State University PDXScholar Dissertations and eses Dissertations and eses 4-1-1968 Bacterial degradation of methyl dehydroabietate Burl C. Carter Portland State University Let us know how access to this document benefits you. Follow this and additional works at: hp://pdxscholar.library.pdx.edu/open_access_etds is esis is brought to you for free and open access. It has been accepted for inclusion in Dissertations and eses by an authorized administrator of PDXScholar. For more information, please contact [email protected]. Recommended Citation Carter, Burl C., "Bacterial degradation of methyl dehydroabietate" (1968). Dissertations and eses. Paper 493. 10.15760/etd.493
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Bacterial degradation of methyl dehydroabietate · 2017. 4. 30. · HU (mass 83) or C6H10 (mass 82). These carbon-hydrogenratios require either unsaturation or cyclization. Evid'ence
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Portland State UniversityPDXScholar
Dissertations and Theses Dissertations and Theses
4-1-1968
Bacterial degradation of methyl dehydroabietateBurl C. CarterPortland State University
Let us know how access to this document benefits you.Follow this and additional works at: http://pdxscholar.library.pdx.edu/open_access_etds
This Thesis is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator ofPDXScholar. For more information, please contact [email protected].
Recommended CitationCarter, Burl C., "Bacterial degradation of methyl dehydroabietate" (1968). Dissertations and Theses. Paper 493.
Dehydroabietic acid has been converted indirectly to
l-ketonordehydroabietane. Treatment with lead tetraacetate gave
the intermediate (C) which upon oxidation yielded the l-keto
compound (D).
3
(C) (D)
Oxidation following condensation of (D) with ethyl formate yielded
the diacid (E). 5
(E)
Mic robial Oxidation
, In view o~ structural similarity, one might 'expect thq.t!
microbial oxidation of methyl dehydroabieiatewould be similar to. .,
. . .
that of phenanthrene. Phenanthrene is end- ring oxidized to a, _!, ' ,
, . .
c1ihydroxy' de rivative (F). Furthe r oxidation (enzy1nati~) of F res ulted
in ring cleavage. Non-enzymatic oxidation of F yielded the diketone
G. 6
4
H.o\ .
Androst- 5-en-7 -one (H) has been oxidized ,by microbes to
'. .' 7the 12-hydroxy derivative (J) and to the 3-hydroxyderivative (K).
Ho
(H) (J)
Recently, B iellmann and co-workers have shown that the
3-keto compound (L) is obtained. upon bacterial oxidation of dehydro-
. d 8abletic aci •
5
(K)
o
(L)
6
DIScussioN
Compound .!!...
1. R. Data:
An intense peak at 5. 73JJindicates the presence of at least one
non-conjugated carbonyl g,roup. The presence of aromatic 6. 21f '
6. 67jJ, and 6. 89,... peaks and a gem-dimethyl doublet (7. 30~, 7. 37)-A )
, indicate that ring C of methyl dehydroabietate has been left intact.
The prominent peaks at 8. 30jJ, 8.39,.,.., and 8. 54jJare undoubtedly those
, *peaks associated with the c-o bond of a methyl ester. The peak of
moderate intensity at 7. OJ--tis probably due to C-fI bending vibrations
of a cyc lie hydrocarbon.
M. S. Data:
The molecular weight of II, determined by mass spectro-
metry, is 260. T he known presence of an isopropyphenyl group
(mass 118 for 3 substituents on the ring, or mass 119 for lsubstit-
uents on the ring), and a carbomethoxyl group (mass 59) accounts for
either 177 or 178 of t~e 260 molecular weight.
'the remaining mass (83 for 2 substituents on the aromatic
ring, or 82 for 3 substituents on the aromatic ring) corresponds to
C 6HU (mass 83) or C 6H 10 (mass 82). These carbon-hydrogen ratios
require either unsaturation or cyclization. Evid'ence supporting
eye lization, i. e. retention of the :B ring of methyl dehydroabietate,
>',< The ac id of II was conve rted to a methyl ester by diazomethane.
7
is found in the mas s spectrometry fragmentation patterns of II. The
base peak (most intense peak) of It, corresponding to the most stable
ion or ion fragment, is at 159. Dimethyl substituted 1, 2, 3, 4-tetra
hydronaphthalene* (with one abstracted H) is a reasonable choice,
especiatty in view of the stability requirement. Mo~eover, there are
fragments corresponding to the los s of H and one methyl (mas s 143,
16. 70/0 relative intensity), two methyls (mass 129, 28.5% relative
intens ity), and two methyls and H (mas s 128, 21. 80/0 relative intensity)
f~om the dimethyl substituted 1, 2, 3, 4-tetrahyd~onaphthalene
**fragment. In view of this, four isomers can be drawn.
IIA
IIC
IIB
IID
*1, 2, 3, 4-tet~ahydronaphthaleneskeleton is obtained byremoval of ring A of methyl dehyd~oabietate.
~.c~~A fifth isomer~ II E, is conside~ed unlikely since no reasonable fragmentation can be drawn fo~ the P-27 peak; also, due to thefact that a P-29 (231) peak (loss of CZH 5) is absent in fragmentationof II.
The fragmentation patterhs of II require a structure that can satisfy
the following conditions: a structure that can lose mass 27, mas s 41,
mas s 55, and mas s 59 with apparent rearrangeme nt such that H
migration gives rise to a P-60 fragnie nt.
Bond cleavage seems to occur predominately between the
most substituted carbons. 9
Cleavage of the C(5)-C(6) and C(7)'-C(8) bonds followed by
rearrangeme nt (where hydrogen migrat~es', from the 28 mass, frag-
ment) would account for the P-27 peak for the four 'previously
mentioned isome rs.
I~
IIB
P -27; 27
1\
, \ .. - , - - .. p- 27' '27. Ii 3C0 C:O H ' J,
IIC
*Podocarpane numbers are used here and on aU subsequent, I
structures.
9The most probable rearrangement to occur is one similar to the
McLafferty rearrangement of esters whereby a hydrogen atom on the ,"
carbonVto the carbonyt group migrates to the oxygen of the carbonyl
10group. Onl! lIB,11.
Cand 11
Dcali assume the proper steric
position to accommodate such 3: rearrangetne nt. Cleavage of the
C(9) -COO) and C(5) -C(tO) bonds of 1IB with the same type of re'
arrangeme nt would account for the P- 27 fragme nt. lIn could also
lose 2? a. tn. u. by C(5)-C(10) and C(5) -C(6) bond cleavage with
the same rearrangeme nt.
P-27;27 P-41;41
...,,,P-55;55
Moreover, only lID' by very similar fragme ntation, can lose 41, and
55 a. m. u.
Los s of 59 and 60 a. m. u. follows much the same scheme.'
p- 59 corresponds to the los s of carbomethoxyl and P- 60 corresponds
to the loss of carbomethoxyl plus proton where the hydrogen on the
ester carbonyl oxygen comes from the carbon Y to the carbonyl
group. See table M. S. - tie
10
Fraction III
I. R.
F'raction III is a mixtu~e of at least two compounds (s ee
gas' chromatograph). A lthough no positive identif ication could be
made, some interesting points are suggested by the infrared spectrum. ,
Two peaks are present in the region of carbonyl absorbtion. One
peak of high intensity is at 5. 77 jJ. and the second peak of lowe r
intensity is at 6. 03 jJ. A carbonyl absorbing near 6.03 Jl couid be
either a carbonyl adjacent to the aromatic ring, 01" a carbonyl of an
, 11ester chelated by the enol form of a keto-ester. A weak O,-,H peak
at 2. 9)J supports the tatter.
11
Compound IV
The infrared spectrum of IV is very similar to that of methyl
dehydroabietate. r he 6.03 Jl peak was suspected to be due to an un-
resolved minor cotr1[Jonent. The G. L. C. retention times of IV and
methyl dehydroabietate were the same, thus affording identification•
of the major component of IV. The presence of peaks at 260 a. m. u.
(70/0 intensity) and 233 a. rrt. u.(4. 50/0 intensity) in the mass spectro-
metry fragmentation pattern of IV (peaks lacking in the fragmentation
pattern of methyl dehydroabietate) confirmed the suspicion that a
minor component was pres ent in IV.
Since IV was obtained ori acid extraction, methyl dehydro-
abietate must have been hydrolyzed during the growth period. A
,contr~l was run in order to determine whether hydrolysis was due to
chemical action or due to bacterial action. A 1. 0 gram sample of
methyl dehydroabietate was subjected to the same condition found
during growth except that the ,media was not irtoc'ulated with the, .
bacteria. Upon acid extraction, ' a very small amount (less than'l mg)
of material was obtained. An infrared spectrum showed the material
to be methyl dehydroabietate with a very smaH amount of impurity.
It was concluded that hydrolysis 'of the ester was du~ to bacter.iat '
actionrathe r than chemical action.
12
Fraction V
G. L. C.'
The presence of one peak suggests a reasorl;ably pure frac-
tion, althoug,h mass spectral d~ta indicate a small 'amount of impur-
>:citie s.
I. R.
Two intens e peaks in the infrared spectrum at' 5.. 73).J and
5. 77 )J indicate t.he pres ence of at least two non- conj ligated carbonyl
I
gro,ups. Also present are the peaks associated with the aromatic
ring (6. 21)..1, 6. 67}Jand 6. 89)J ) and the isopropyl ,group (7,. 30 }.J
and 7. 37 )J) indicating that ring C and the is op'ropyl group of methyl
dehydroabietate have been left intact.
M. S.
The molecular weight of V was determined by mass spec-
trometry to be 328 a. m. u. T his corresponds to the introduction of
one oxygen atom into methyl dehydroabietate (me w. 314) and loss of
two protons. Since infrared data indicates non-benzilic oxidation,
the following sites are possible:
'(A) Ketones: C{l)' C(2)' C ,C ; (B) Aldehydes: C ',C, (3) (6) . , (16) (17)
Absence of an intense mass spectral peak at 327 (P-.l)
indicates that introduction of oxygen led to formation o'f a keto~e
rather than an aldehyde. (Aldehydes have large P-1 mass spectral
~.cPeaks 314, 312, 297, 296 requiring very unusual fragmentation of the parent ( P 328) a:re cons idered to be due to impurities.
13 .
peaks due to cleavage of the, aldehydic proton). 12
A comparison of the mass spectrum of V with that of methyl
dehydroabietate reveals that both have peaks at 128, 129, and 141
a. m. u. corresponding to the foHowing:
H
H
H
H
H
This would suggest that oxidation occurred in ring A bf met hyl
dehydroabietate, and that oxidation occurred at eit~er C(2) or C(3)
since the fragments (128, 129, 141) are a result of ring A cleavage.
A ketonic carbonyl at C(l) would result in ring B cleavage lipon electron
13impact. The refore, the ketone carbonyl is eithe1" at C (2)' or ·C(3)·
Microbial oxidation of dehydroabietic acid results ~n oxidation at ·the
14. .. .3-position. The structure of V is proposed to be:
I'
Hfog
14
'EXPERIMENTAL >~
, >'c >'cBacterial Isolation:' I
Bacteria were isolated by the enrichment technique using
bark and lor dry needles of lodgepole pine as the inoculum source.
A minera1 salt me dium consisting of:
(1 ) KHZP04 1.7 grams I liter
(2) KZHP0
44.4 grams lliter
(3) NH4Cl Z grams lliter
(4) MgS04• 7HZO· ,0.04 grams Ilite~
(5) MnS°4 ·'HZO 0.05 grams /liter I '
(6) FeS04·7HZO 0.05 gran-is lliter
(7) 'GaC1Z·2H ° 0.01 grams lliter. Z
(8) NaMo04 0.01 grams I liter
(9) , *>'c* 1. ,0 grams lliterMethyl abietate I
, was used for tlfe enrichment procedure and for subsequent growth to
obtain de gradation products.
>:cJ3acteria were aerated with a New B'runswick Gyrotory shakermodel G-25. Removalof cells was accomplished with·a Serv~ll
model RC-Z centrifuge•. Melting points (uncorrected) were taken on aB U.chi melting point apparatus. An Aerograph autoprep model A-700was us ed for separation of extract'ed portions. Liquid nitrogen wasused as a coolant. during collection. Infrared spectra were baken ona Perkin-Elme rmodel 137 B Infracord sp~ctrophotometerand on aBeckman IR lZ. Mas s s pe ctral data was obtained on 'an A tlas model·CH-4 mass spectrometer operating at 70 eV voltage,' 10}J.A currentand an inlet temperature of 1800 , C. '
>~*Work of Dr. M. Taylor, Dept. of Biology, P. S.C. ,>:o:c>:cMatheson, Colema n and,B ell (Practical). Methyl abietate
was used as the nutrient only during bacterial isolation.
, , I
15
Isolation of bacteria was by streaking the liquid enrichme nt
cultures onto plates containing the above me dium with 2% agar. After
testing each strain of bacteria under various conditions, optimum
yields of degradation products were obtained from cultures of an un-
characterized species of A rthrobacter cultivated at room tempera-
ture (24-26 0 C).
Stock cultures were maintained on nutrient agar (Difco),
stored'at 40 C, and were transferred every two months.'
Purification of methyl abietate by chtomatography (gas
chromatography, coiut:nn chromatography) proved to be impractical,
the refore, the nutrient was changed to methyl dehydroabietate.