GEORGEDE STEVENS A N D F. F.NORD
Vol. 74
(3-Hydroxy-2-pyridylniethyl)-diisopropylamine.-A mixture of 24.7 g . of (3-hydroxy-2-pyridylmethyl)-trimethyl'I he first three of the follotving syntheses are representa- ammonium bromide and 60 g. of diisopropylamine was stirred tiye of the preparation of compounds liqted in Tables I and and refluxed. There did not seem t o be any reaction a t ir. the reflux temperature. On heating the above mixture in a Dimethyl Carbamate of (3-Hydroxy-2-pyridylmethy1)- rocking autoclave a t 12.5' for 2 hours, reaction took place. dimethylamine Dihydrochloride. - A solution containing 4:; After boiling off excess diisopropylaminr, the residue was g . of (~-hydroxy-2-pyridylm~thyl~-dimcthylatnine in SO cc. extracted lvith benzene and the benzene layer then washed of pyridine and 32 cc. of dirnethylcarbarnyl chloride was with water. The residue after removal of benzene, was disheated on a steam-bath for 2 hours. X o s t of the pyridine tilled. This gave 13.7 g. of (3-hydroxy-2-pyridylmethy1)\ v i i s then removed by distillation in V U C I I O . The residue was diisopropylamine, b.p. 98-101' (1.2 mm.); yield 66%. dissolved in cold water, made alkaline with sodium hydrox(3-Hydroxy-2-pyridylmethyl)-diisopropylmethylammoide, arid extracted with ether. The ether layer was washed nium Bromide.--A cold solution of (3-hydroxy-2-pyridylwith water and dried over anhydrous sodium sulfate. After methyl)-diisopropylamine in acetone containing methyl removal of the ether by distillation, the residue was heated bromide gave crystals of the quaternary salt. After two on a steam-bath under a vacuum of 1 mni. to remove traces crystallizations from a mixture of methanol and ether, it of pyridine. The residue was dissolved in ether and satu- melted a t 171--172' dec.; yield 82%. rated with anhydrous hydrogen chloride. A light brown Anal. Calcd. for C13H230N2Br: C, 51.49; H, 7.64; K, crystalline solid separated. This mas dissolved in hot eth- 9.24. Found: C, 51.19; H, 7.49; N, 8.96. anol, decolorized with charcoal, and crystallized on addition 2-Benzylaminomethyl-3-pyridolDihydrochloride .-On adof anhydrous ether. After recrystallization from a mixture dition of 60 g. of benzylamine t o 24.7 g. of (3-hydroxy-2of ethanol arid ether or from acetonitrile, the dihydrochlopyridylmethy1)-trimethylammonium bromide, the temperaride melted a t 163-167"; yield 47%. ture rose to 33". The reaction mixture was then stirred and Dimethyl Carbamate of (3-Hydroxy-2-pyridylmethy1)heated a t 50" for 1.5 hours, during which time the origintrimethylammonium Bromide.-The esterification product ally milky mixture became clear. After removal of excess obtained in the above example, after removal of traces of benzylamine by distillation in vacuo, the residue was dispyridine i n zlacuo, was dissolved in a cold acetone solution of methyl bromide. Crystals of the quaternary ammonium solved in 10% sodium hydroxide. The insoluble oil that separated was extracted with ether. On neutralization of salt began t o separate in a short time. After recrystallizathe alkaline layer with hydrochloric acid to about PH 8, an tion from a mixture of ethanol and ether, the product melted oil separated that was extracted with benzene. The residue at 175-177" dec.; yield 60%. after removal of benzene was dissolved in ethanol and alcoPhenylmethyl Carbamate of (3-Hydroxy-2-pyridylmethholic hydrogen chloride added. The crystalline dihydroy1)-diethylamine Monohydrochloride. -To a solution of 10 chloride separated in 71 yoyield. After two crystallizations g . of (3-hydroxy-2-pyridylmethyl)-diethylamine in 10 cc. of from methanol, it melted a t 236-241'. pyridine, there was added 10 g. of phenylmethylcarhamyl Acknowledgments.-The authors are indebted chloride. In about two minutes, the acid chloride dissolved with considerable evolution of heat and bubbling. The to the late Dr. G. Lehmann and Dr. L. Randall of solution was cooled in water and kept for about 16 hours at the Pharmacology Dept., Hoffmann-La Roche, room temperature. The large crystals of the monohydroh e . , for the pharmacological data presented. LVe chloride which formed were filtered and washed with pyri- wish to thank Dr. A. Steyermark and his staff for dine and anhydrous ether. After recrystallizatio~i froin a the microanalyses reported. mixture of isopropyl alcohol and ether, the product melted it 142-144'; yield 52%. S [ . T I E Y I O , S.J
Experimental
[ COXrRIBUTIOS
?\TO
255
F R O X 1 H E L)EPARTXEVT O F
ORGANIC CHEMISTRY
AND
ENZYMOLOGY, FORDHAM UNIVERSITY]
Investigations on Lignin and Lignification. IX. The Relationship Between the Action of Brown Rot Fungi, Cellulose Degradation and Lignin Composition in Bagasse BY GEORGEDE STEVENS AND F. F. NORD RECEIVED FEBRUARY 28. 1952
A comparative study oi the effect of four wood-destroying fungi of the "brown rot" type on the dissimilation of cellulose i n bagasse has been carried out. The lignins liberated by the cellulolvtic action of each of these molds have been characterized. Their identity with bagasse native lignin is discussed.
'The importance of wood-destroying fungi of the "brown rot" type as agents of cellulose degradation for the isolation of additional amounts of unaltered lignin from woody tissues has been well established.',' However, these molds reveal a certain amount of specificity with respect to the type of wood attacked and their rate of growth when cultivated on a wood species. That is, certain "brown rots" thrive better when softwoods are used as substrates, whereas others show optimum activity with hardwood^.^ I n a recent report froni this Laboratory,4 the isolation and characterization of the 113. \
(1) W. J. Scfuiliert : i r i i l 1'. 1'. riortl. T r i r s J U I . K S . I I . , 72, 977, 3885 1950). 12) S. 1'. Kudzin and F 1'. Xord, i b i d . , 73, ICjliJ r l 9 5 l r . l:3) M. K. h'obles, Cmr. J. Kcscai.ih. 26C, 281 i l Y 4 8 ) . (4) .(; d e Stevens a n d I; 1: S o r d . T ~ r r s~ O l i R S A l . 73,46?? 11951).
tive lignin and enzymatically liberated lignin from bagasse, the supporting fiber from the annual plant, sugar cane, was described. Since bagasse cannot be assigned to either the softwood or hardwood class, the problem of the choice of the "brown rot" fungus which would give rise to the highest rate of cellulose depletion necessarily presented itself. The resolution of this problem is, in part, reported in this paper. Experimental The wood specie5 investigated in this study was virgin bagasse, and the "brown rot" organisms employed t o effect the decay were the softwood molds, Poria uaillantii anti Lentznus lepideus, and the hardwood fungi, Daedalea quer cinia and Polyporus sulphureus. Sterilization and Inoculation of Bagasse Samples.-Ten gram samples of the virgin bagasse were weighed into each
July 5, 1952
DISSIMILATION O F CELLULOSE
of forty 500-ml. Fernbach-type culture flasks, and to each flask was added a 2.5-ml. portion of a nutrient medium consisting of: neopeptone, 1.0 g.; KHzPO,, 1.5 g.; MgSO,. 7H20,0.5 g.; thiamine hydrochloride, 2.0 mg.; tap water to, 1 liter. The flasks were plugged with cotton, and, t o avoid thermal destruction of the woody tissue, sterilized with streaming steam a t 100" for 60 minutes on three successive days. After cooling, each flask was inoculated with a 5-ml. sporemycelial suspension of a pure culture of one of the organisms referred to above. The inoculated flasks were incubated in the dark a t 27-28'. The decayed bagasse was analyzed periodically. Analytical Methods.-After separation of the fungal mycelia from the decayed bagasse,' the latter was collected and dried. Lignin was determined according to the standard method,6 and the percentage solubility of the decayed residue in 1% NaOH determined according to the method outlined.' All values were corrected for moisture content. Isolation of Native Lignin.-The Brauns method' of extraction was used. This consists essentially of first extracting the ground bagasse with water and with ether. It was then extracted thoroughly with 95% ethyl alcohol a t room temperature. Upon removal of the ethyl alcohol a t reduced pressure, the remaining resinous material was dried, dissolved in dioxane, centrifuged, filtered and precipitated into thirty times its volume of ice-cold distilled water. Hereupon, the precipitate was dried, redissolved in dioxane, centrifuged, filtered and precipitated now into thirty times its volume of ether. This procedure was repeated until a constant methoxyl value was obtained. This method of purification must be applied in order to obtain a uniform lignin of maximum purity. The enzymatically liberated lignin was extracted from the decayed bagasse and purified in the same manner. Needless to say, the fungal mycelia were separated from the decayed bagasse before extraction.
IN
BAGASSE BY
BROWN
ROTFUNGI
3327
TABLE I1 LIGNINCONTENTS OF DECAYED BAGASSE Organism
Period of decay Klason lignin." ( (mo.1 ioI
Dacdaeln qucrcina Polyporus su fphureus
Lentinus lepideirs Poria vaillantiL Control Average of two determinations.
10
22.8 93.9 26.3 32.2
3 I0
30.7 35.3
:3
46.0
8 0
50.1 21 ::
3 8
3
as compared to the pentosan content of some softwoods, such as spruce, having l2.l%.9,l1 Regell suggested that the ability of a wood to undergo decomposition is directly proportional to its pentosan content, since it may be regarded as an energy source. A recent report of Erdtman, et a1.,12purports to the non-identity of the native and enzymatically liberated lignins from spruce wood, the argument being based, in part, on the lowering of the methoxyl content of the Klason lignins and more so on the increase of reducing ability of the enzymatically liberated lignin with respect to the native lignin. These authors determined the copper numbers on the total, i.e., unpurified benzene-alcohol extract Results and Discussion from the decayed spruce wood. However, this resiAccording to Bray,8 an increase in alkali solubil- due contains contaminants and cellulose degradaity serves as a definite index of cellulose degrada- tion products, and this fact must be kept in mind tion. The percentage solubility in lY0 sodium hy- when these results are considered since it is known droxide of the decayed bagasse samples is listed in that the benzene-alcohol mixture is a suitable deresinifying agent. l 3 Table 1. On the other hand, we have purified our ethyl alTABLE I cohol extract from decayed bagasse according t o ALKALI SOLUBILITYOF BAGASSEDECAYEDFOR THREE Brauns. In Table 111 are recorded the chemical MONTHS compositions of the purified enzymatically liberated Solubility in 1 % NaOH,a Organism 70 lignins obtained from bagasse. Their identity atUninoculated control 23.72 tests to the necessity of using pure preparations for Daedalea puercina 24.6 comparative studies. Furthermore, if the methPolyporus sulphureus 28.1 oxyl content of the Klason lignins decreases with Lcntinus lepideus 38.6 the time of decay, then one would expect the methPoria vaillantii 69.6 oxyl contents of the enzymatically liberated fractions.to show the same trend. We have found no Average of three determinations. such change. Moreover, since the bagasse lignins Thus, with the exception of the case of Daedalea liberated by three different molds underwent no quercina, the selected organisms demonstrated a fundamental change in chemical composition, their marked efficiency for cellulolytic decay within a three-month period. This was verified by periodic TABLE I11 analyses of the resulting bagasse residues. The COMPARISOX OF BAGASSENATIVELIGNINWITH BAGASSE data in Table I1 express this change with respect to B Y IT^^^^^^ FUNGI LIGNINSLIBERATED the increase in the relative lignin content of the Polrporus Native Poria suiLcnliiius samples. lignin vaillanlii phureus icpidcus It appears, therefore, that the softwood as well as C 61.5 61.6 61.1 61.8 the hardwood "brown rot" molds will decay ba- Analyses, yo H 5.7 5.9 5.5 5.6 gasse, although Poria vaillantii causes the most OCHj 15.3 154 15.0 15.2 rapid dissimilation of cellulose. A possible under- (9) J. D.Reid, G. H. Nelson a n d S . I. Aronovsky, Ind. Enc. Chc?n., standing of this observation can be derived from A n a l . E d . , 18, 255 (1940). the high pentosan content of bagasse, e.g., 31.3% (10) A. G. N o r m a n a n d W. H. Fuller, A d v . i n Enaynrology, 2, 239 (5) E. C. Sherrard a n d E. E. Harris, I n d . Eiig. Chcm., 24, 103 (1923). (6) "Methods for t h e Chemical Analysis of Pulps a n d Pulpwoods," Forest Products Laboratory, Madison, Wis., 1939. (7) F. E. Brauns, THISJOURNAL, 61, 2120 (1939). (8) M .W, Bray, Papcr Tradc J . , 1 8 , No. 23, 58 (1924).
(1942) (11) R.D.Rege, A n n . A p p l i e d B i d , 14, 1 (1927). (12) A. Apenitis, H. E r d t m a n a n d B. Leopold, Sucnsk. Kcifrisk Tidskuift, 63, no. 9, 195 (1951). (13) S. A. Mahood a n d D. E. Cable, I n d . E n & . Chcm., 14, 933 (1922).
identity with bagasse native lignin is well founded. This identity rnay be further recognized in thrir infrared and ultraviolet absorption spectra4 l n a forthcoming communication this conclusion will be further supported by means of oxidation studies with nitrobenzene and alkali and also by identiiying their lignosulfonic acids. Acknowledgments.-The bagasse used in these experiments was obtained through the courtesy
\CoVTRrnVTIOS FROM
of the Godchaux Sugars, Inc., New Orleans, La. The inold cultures used in this investigation were obtained through the courtesy of Dr. W. J. Robbins of the Kew York Botanical Garden. The analyses reported in this paper were carried out by Mr. A. -4. Sirotenko of this Department. This work was carried out under the auspices of the Office of Naval Research. SET?' YORK, F.Y.
'I'IIE nEPARTMES1' OF CfIERIISTRY A Y D THE r)EFEXSE RESEARClf I,ABORATORY, T E As
Allylic Chlorides.
x 1
THEI!NIVERSITP
OF
XVIII. Preparation and Properties of 1,1,3-Trichloro-2-fluoro-lpropene and 1,1,2,3-Tetrachloro-1-propene' BY LEWISF.HATCH AND DAVID W. MCDONALD? RECEIVED DECEMBER 22, 1951
The following compounds have been prepared and characterized for the first time : 3-bromo-l,l-dichloro-2-fluoro-lpropene, 3,3-dichloro-2-fluoro-~-~~ro~~e1i-l-ol, 1,1,3-trichloro-2-fluoro-l-propene, 2,8,X-trichloro-2-propen-l-ol and 1,1,2,3zind 1, I ,~,3-trtrachloro-l-prope11e with potassitim tetrachloro-I-propene. The reactions of 1,1,3-trichloro-~-fl~toro-l-properle iodide and with sodium ethoxide hnvc hwii studied
The study of the influence of various groups and pene was related to 1,1-dichloro-2-fluoro-~ -propene atoms on the reactivity of the allylic chlorine atom by replacement of the allylic chlorine atom by a of substituted allyl chloride has been extended to hydrogen atom using lithium aluminum hydride.6 include lI1,3-trichloro-2-fluoro-l-propene and 1,1,- The structure of 1,1,2,3-trichloro-l-propenewas 2,3-tetrachloro-l-propene. Both of these cum- also confirmed in the same manner by its conver' I sion to the known 1,1,2-trichloro-l-propene.7 pounds are of the type CC12=C-CH,CI, a type The relative reactivities of 1,1,3-trichloro-2which shows a marked difference in its reaction fluoro-1-propene and 1,1,2,3-tetrachloro-l-propene with potassium iodide in acetone from the similar with sodium ethoxide in ethanol (Table I) show the tvpe CR.-C- --CI I,CI. same relationship between the electron-attracting ' 1 The 1,1,3-trichloro-2-fluoro1-propene was syn- ability of the substituent on the number two carbon thesized from l,l-dichloro-2-fluoro-l-propeneby atom and reactivity as do those compounds having the use of N-bromosuccinimide followed by hydrol- two hydrogen atoms on the number one carbon atom.* I n both series the replacement of the hyysis of the S-bromo-l,l-dichloro-2-fluoro-l-propene to 3,3-dichloro-2-fluoro-2-propen-l-ol and conver- drogen atom on the number two carbon atom by an sion of this allylic alcohol to the desired allylic electron attracting atom (Br, CI or F) causes a dechloride in a manner similar to that previously used crease in reactivity. This similarity does not to prepare the 1,3-dichloro-2-fluoropropenes from TABLE I the 1-chloro-Zfluoro-1-propenes.'The 1,1,2,3-tetRELATIVE REACTIVITY O F 1,1,3-TRICHLORO-2-FLUORO-1rachloro-1-propene was obtained by the dehydroPROPENE AND 1,1,2,3-TETRACHLOR0-1-PROPENEWITH S O chlorination of 1,1,2,2,3-pentachloropropane. DIUM ETHOXIDE I N ETHANOL AT 50" The pentachloropropane was prepared by the 1,1,3-Trichloro-2-fluoro-l-propene low temperature addition of chlorine to 1 ,2,3-trichl~ropropene.~Apparently no substitution ocTime, hr. 6.50 8.50 10.5 12.5 22.0 curred which conforms with the generalizations of Reacted, 7 5 8 , 6 63.3 67.4 70.7 81.9 Taftl on noti-activated, low temperature c1ilorin:tk , hr.-I mole-'I. 4 . 3 7 3.88 3.87 3.81 3.83 tion reactions. The pentachloride obtained by the Av. k 3 .95fO. 16 addition of chlorine t o l,Z,R-trichloropropene is Rrlutive reactivity' 3.3 considered to be I , 1,2,2,3-pentachlori~propnne,alI , 1,2,3-Tetrachloro-l-propene though the physical constants of this compound do Time, hr. 1.00 1.50 2.00 3.00 4.00 not agree very closely with those reported by StevReacted, % 30.1 43.5 50.7 60.7 67.9 ens,j which disagreement, however, is prohxblv due k, hr.-'rriole-l I. 1 1 . 3 10.2 10.3 10.4 10.6 i n part to the higher purity of our sample. Av. k 10.6f0.3 The structure of 1,1,3-trichloro-"-fluoro-l-pro(1) For number XVII of this series see I,. F. l r a t c h a n d D. W. McDonald. THISJ O U R N A L , 74, 2911 (19.52). (2) Research Corporation Fellow 1D4O-~IOJO,~ f o i i s a n t o Fellow, 1 0 30 - 19 5 I . (3) L. F. IIatch. J. j . U'Amico and E. V. Riihnke, Tiirs J O I I I I N \ L , 74, 123 (1Y.52). (4) R . W. T a f t , i b i d , T O , 33G4 (1948). ( 5 ) P. C,. Stevens, ibid., 68, 620 (1!14Ci)
Relative reactivitya 9.0 Allyl chloride as 1.00 with k = 1.18.
(6) L. 1'. Hatch and R. H. Perry, ibid., 71, 3262 (1949). (7) G . Bersche and R. Fittig, A n n . , 133, 117 (1865); W. Szenic and R.Taggesell, Ber., 28, 2GG8 (1885). CSj I. P. 1T:itc.h i l ~ i < lTI, 1'. Alexander, TJIISJOVRNAI., 71, 1037 (1949)
