Reaction of Acetaldehyde with !i,!i-DimethylGEORGE W. GAFFNEY
and
WILLIAM A. WILLIAMS,
and
HERBERT MCKENNIS, J R . ~ ,
Surgical Research Laboratory, M e d i c a l College o f Virginia, Richmond, V a . , and Department o f Physiological Chemistry, The Johns Hopkins University, Baltimore,
Md.
IIb. Ilcc.eiit ~ v o r kof Gustnfwoii (.i 1 h:s shuivii that this dei,irativc undergoes further reartion with dimedon under alkaline conditions t o give a compound of the general type represented by 111, with loss of chlorine. Yoe and Reid ( 1 6 ) in their .studies of thc form:rtion of the Ilkdimedon derivative of acetald(~hyc1efound that in aqueous Iiuffer solution, the maximum yield of insoluble protlurt \v:t.s ohtained a t pH 4.02. MacFadyen (10) followed the rcaction of formadehgde with dimedon by noting the disnppearnncsc of free formaldehyde as measured by the chroiiiotropic arid (4.5-dihydroxy-2,7-n~phthalenedisulfonic :wid) color reaction. The pH region 7 to 8 favored a fast rcwtion in his studies. In :id& tion, mildly acidic conditions h a r r l ) ~ ( , nsugge3ted for thc. formation of aldeh>-de-diniedon dpiiy:itiv(,3 (1;).
s THE course of the investigation of certain biological mixtures,
1 need arose for a procedure for the quantitative separation and
identification of acetaldehyde present in small amounts. After other means had been tested, the practicability of separating various volatile carbonyl-containing substances 1)y aeration procedures was investigated. By taking advantage of the known specificity of dimedon (5,5-dimethyldihydroresorcinol; 5,5dimethyl-1,3-cyclohesanedione) for aldehydes, as opposed t o ketones, it was sought to absorb acetaldehyde quantitatively in aqueous solutions of dimedon and to absorb acetone finally in sodium bisulfite solution. During these studies the desirability and necessity of rvorking out some of the elements of the reaction mechanism for dimedon and aldehydes hecamc :tpparent.
I1
I
TI1 IIa R = I I a It =
3
4
5
6
7
8
Initial p H o f Dimedon Solution
Figure 1. Effect of pH on Efficiency of Retention of Aerated Acetaldehyde by Dimedon Solutions I . 0.4% dimedon with sodium 11)drolidc 11. 0.4% dimedon with diethanolaniine 111. 0.4% dimedon with piperidine
Prior to this investigation, Shinii and Sicolet ( 1 3 j had found that v,-ell-cooled saturated aqueous solutions of dimedon did not absorb acetaldehyde quantitatively. Experiments of the authors indicated that at room temperature only about one third of the aerated acetaldehyde was absorbed. These difficulties focused attention upon the possible mechanisms of the reaction of dimedon with acetaldehyde. The reaction represented as the most likely one occurs in two separate stages ( H a , IIIa). The postulated intermediate 11s is presented not only because of the unlikelihood of a trimolecular reaction t o give directly ~ - 2 ( 5 ' , 5 ' dimethyl-1 ',3'-dioxocyclohexyl)ethyl-5,5-dimethyl-3hydrosy-3cyclohexene, but also because Desai ( 2 ) and others (3) have obtained from trichloroacetaldehy(3e and dimedon the compound 1
Present address, Medical College of Virginia, Richmond, Va.
("5
Ill) Ii = CC1,
C"1
Thcw reports, together with a consideration of aldehyde solu bility and the possibility that a pH optimum for formation of the hypothetical intermediate I I a would bc the p H most favorable for retention of acetaldehyde, led t o the exploration of thc effect of hydrogcn ion conrentration on the retention of acetal~lc~hytle in dimedon solution. The p H v d u e s of OAy0 wlutioiis of dimc~;lonwere adjusted from pH 3.4 to p H 8 with sodium hydroxide prior to aeration. I n the regions pH 5 to 6 essentially quantitative recovery was obtained (Figure 1). By acidification of the rosulting solutions t o 1)H 3.9- high yields of acetaldch?-dc-dimedon derivative were obt :lined. Horning and Horiiing (6),following observ:itious by Desai ( I ) , suggested t,he use of piperidine as a catalyst in a general procedure for thc preparation of aldehyde-dimedon derivat,ives. It ap])eared that piperidine might excvt its catalytic action through the int,ermediate S-or-hydros)-eth?-lpiperitliuc. Accordingly, in some rsperiments piperidine was used instend of sodiumhydroxide to adjust the pH of the dimedon solutions. lcetaldehyde \vas :ierated into these solutions under the same conditions as previously employed for the sodium hydroside experiments. No superiority of piperidine as a catalyst, was disclosed and, furthermore, acetaldehyde retention v a s at a masimum in the Pame pH regions as iii the sodium hydroxide csperinients. These result? indicate that it is not necessary to invoke the intcrmedi:ition of
588
589
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4 the .\--a-hyclroxyethyl compound, although participation of the latter has not been excluded. I n additional experimentsj diethanolamine \v:is employed to adjust the p H of the dimedon solutions, and maximum retention was again obtained in the same region, giving further support to thc concept of a general hydrogen ion effect. The fact that the shapes of the curves are so similar and independent of the type of alkalinizing agent employed indicates that the primary reaction is not dependent upon reaction of acetaldehydc with added base. I n early experiments it was notod that follon+ng :ter:ition with acetaldehyde the pH of dimedon solution7 1i:itl shifted slightly to the alkaline. I n an effort t o increase acetaldehyde retention t.hc c8oric:entration of diniedon was increased. This enabled the aldehyde rengent to serve as its own buffer and consider:tbly reduced previously encountered pH shifts. The efficiency of this system m:tkes possible passage of the effluent gas into 1)isulfite solution for assay of acetone. EXPERIII ENTA L
.lqueous solutions of acetone and acetaldeliycle \\-ere aerated with nitrogen through a Van Slyke-Cullen apparatus ( 5 ) using tubes approximately 28 X 200 mm. The first tube contained carbonyl compound in 10 to 25 ml. of water. This was followed by an empty tube (trap) and two tubes, each of Jvhich contained 50 ml. of dimedon solution. Carbonyl compound which passed through these tubes was absorbed for assay in a pair of tubes, each of which contained 25 ml. of sodium bisulfite solution. Two hours' aeration a t 1000 cc. per minute was found sufficient for quantitative passage of acetaldehyde into the diniedon, and longer aeration (6 hours) produced no differenccs in results. Acetone does not pass through tho dimedon into bisulfite quantitativclly in less th:m 6 hours. The aeration time does not rc.sult from properties of dimedon sincc water d o n e delays passagr for an equivalent length of time. The insertion of :In empty tube (trap) b e t w e n dimedon and the bisulfite solutions \vas found to be particularly important since any contamination of the bisulfite solution by dimedon interferes with the estimation of bisulfite binding substance. For increased retention of acetaldehyde, the second dimedon tube was replaced with a 1-liter cylinder containing 250 ml. of dimedon solution and a gas dispersion tube constructed n i t h a 20-mm. coarse glass frit (Corning 39525). Estimation of Acetaldehyde. ,llclcliyde solutions were prepared from freshly distilled acetaldehyde. These were t'hen titrated ti>- the previously described method ( 11 ) for &ndardization. .lldrliyde which escaped the dimedon solutions and subsequently was caught in 0.33% sodium bisulfite solutions was titrated by the same procedure. I n some esperiments it was found equally convenient and reliable to use 2% bisulfite instead of 0.33% bisulfite and to titrate I)isulfite-l)ound acetaldehyde by the method used for acetone as describd belon-. JVhen smaller amounts of acetaldehyde \yere present in bisulfite together with acetone, the colorimetric method developed bj, Stotz ( 1 4 ) was employed. Isolation of a-2( 5',5'-Dimethyl-l',3'-dioxocyclohexyl jethyl5,5-dimethyl-3-hydroxy-3-cyclohexene. 1)imedon solution which had becn initially adjusted to pH 6 \vi(h sodium hydroxide and into which acetaldehyde had been aet,:ttcd as dwcribed above, was adjusted to pH 3.9 with acetic acid. Prrcipitation of crystals began almost a t once. After standing 1.5 hours, the precipitate was collected and dried at 50" C , I-ield, uncorrected for mechanical losses, was 727, of the calculated value: nielting point (capillary) was 137" to 139' 0. Adniisture with an authentic sample prepared by the procedure of Horning and Horning ( 6 ) did not depress the melting point. Conversion of Acetaldehyde Dimedon Derivative to 2,2,7,7,9The a l d ~ h y d c Pentamethyl-4,5 - dioxo-octahydroxanthene. dimedon drrivative obtained aftcxr aeration (itliove) \vas trentetl according to the general procedure of Horning and Horning ( 6 ) . Tho product was air dried; melting point (capillary) was 174.5' to 175' C. The melting point was not depressed upon admixture with :in authentic sample. Estimation of Acetone. Iledistilled acetone was used to prepare aqueous solutions which iver(~ standardized acneording to thc, procedure used by Hubbard (7). The Ihulfite solutions of acetone were titrated with iodine after the method of Klein (8). I t was found convenient to decompose the acetone-sodium bisulfite addition compound b)- addition of borax in excess instead of the customary disodium Preparation of Dimedon Diniedoii from t,he Eastman Kotlnli Po. v a s employed in most of the experiments. Be-
&$?:?
cause acetone is suggested as a solvent for the recrystallization of crude dimedon, i t is well to test solutions of the compounds for volatile carbonyl compounds prior to use. Piperidine was freshly distilled; the fraction boiling a t 104.5' to 104.8" C. a t 744.8 mm. of mercury was employed. Diethanolanine (practical, Eastman Kodak Co.) was used without further purification. Typical results with the various reagents are shown in Figure I, while Table I characterizes many rsperiments a i t h the rclativcly simple apparatus. Addition of Acetaldehyde and Acetone with 2,4-Dinitrophenylhydrazine. In some experiments 2,4-dinitrophenylhydrazine solutions, prepared for water soluble compounds ( 9 ) , were used a t room or ice-bath temperature instead of dimedon with the procedure otherwise the same as for the dimedon experiments. Searly quantitative retention of acetaldehyde was achieved by increasing to five the number of hydrazine tuhcs, but quantitative retention of aretone \vas not achieved. Chromatographic Separation of Acetaldehyde 2,4-Dinitrophenylhydrazone and Acetone 2,4-Dinitrophenylhydrazone. Following evactly the chromatographic method of Rohrrts and Green ( l a ) , and employing the brand of silicic acid adsorbent used by them gave consistently unsatisfactory results. By heating the adsorbent to SO" C. in vacuo, however, it was .icstivntcd and fair separation was achieved. ACKNOWLEDGMENT
TIE authors are grateful to t,he John and Mttry 11. 1Iarkle Foundation and to the Office of the Surgeon General for support of this work. The interest of Everett I. Evans and the kindness of Sidney S. Segus in making available facilities in the Department of Biochemistry, Medical College of Tirginin, are also grc:ttly apprechted. 'l'ahle I. Effect of 4cetone on Retention of Acetaldehyde 11) Concentrated Dimedon (1.5%) in Sodium Hydroxide, pH 5.70" Aerated ~~ c r t , i l d e l ide j 7.5 7.5
Acetaldehyde In Bisulfite, 1Ig (Colorimetric)
0.2
0.4
7.5 7.5
i.5 i.5
a
hlg .icetono 0.2
~
0.05
0.4
0.1
1.o 1.0
0.1 0.1
2.0 2.0
0.06 0.04
3.8 2.0 7.5 2.0 Aeration, 6 hours at 1000 cc. p e r ininiltr. -
0.1 0.1
~.
0.01 0.02
~
--
LITERATURE CITED
Desai. It. D., J . I n d i a n Ciicm. SOC.,10, C,(i:3 (1933). Desai, R. D., and Wali, 11.d.,Ihid.. 13,735 (1936). Gustafsson, C., Suonicn Kwiistilehti, 18B, 30 (1945). I b i d . , 21B, 3 (1948). Hawk, P. B., Oser, R. L., sild Summcrson W. €I., "Practical
Physiological Chemistry," 12th ed., p. 503, Philadelphia, Blakiston Co., 1947. Horning, E. C., and IIorning, 11. G., 6.O w < * h < , t ? t . , 11, 95 (1946). --, Huhbard, R. S.,.J. Bid. Chcm., 43, 43 (1920). Klein, D., I b i d . , 135, 143 (1940). McElrain, S. >I,, "Chsracterizatioli of Organic: C'~,tlll,oun~is,'' Kew Tork, Nncmillan Co., 1945. NacFadyen, D. A , , ,J. B i d . U i w m . , 158, 107 (1945). Reinke, R. (',, and Luce, E. S , ,ISD. Esc,. (?HEM., .IsLL. ED.,
.-~
18,244 (1946).
Roberts, J. D., and Green, C., Ihid., 18, 336 (1946). Shinn, L. A, and Xcolet, R . Is., J . Biol. U t e m . , 138, 91 (1941). Stota, E., Ibid., 148,585 (1943). T'orlanrler, D., Z. ami. Chou., 77, 241 (1929). Y o e , J. H., and Reid, I,. C , , 1 s ~I
