NOTES
Oct. 20, 1933
5438
scissa intercept of -Ks. It will be seen that this plot is similar to the second plot of Lineweaver and BurkIPcf., Table I , but is superior to this plot in t!([S]o - [SI,)= ( K u - K s ) / k i [ E ] K p (Ka (KP that for t = 0 the ordinate t/(ln([S]o/[S]t)) de[SIo)/k~[ElKp)((ln ([Sld[SIt))/([Slo- [Sit)) ( 8 ) scribes the initial velocities in terms of [S]O/VO. (In( [ S ] O /[SIt))/([SIo- [SIt) there will be obtained Also Kp may be simultaneously evaluated by equatfor various values of [SIoa family of lines of slope ing the observed slope of the parallel lines to the &(KP [SIo)/k3[E]Kpwith a common ordinate quantity (KP - Ks)/Ra[E]Kp. ] . ~each of the intercept of (1 - K ~ / K P ) / ~ ~ [ EFor While it appears that all three of the graphical lines of slope &(Kp [S]o)/k3[E]Kp there is a procedures based upon equation 6 will be found to point corresponding to t = 0 and as before' this be superior to those based upon equations 2 and 3, point may be located by examination of the limits because i t is very probable that a large number of of the two parameters t / ( [SI0 - [SI,) and (In( [S]O/ enzyme-catalyzed reactions are competitively in[SIt))/([ S ] O- [SIt) as t -P 0. Since the limit of t/ hibited by one or more of their reaction products, it ( [ S I 0 - [SI,) as f + 0 is 1/-d[S]/dt = l/ao and is difficult to assert that any one of the procedures that of (In( [SIo/[Slt))/([SI0 - [SIt) as t -F 0 is 1/ which are based upon equation 6 is superior to the [SIoit is evident that the points a t which the lines of other two for all cases that may be encountered. slope Ks(Kp [S]o)/K3[E]Kp possess abscissa Therefore, for any particular situation it is sugvalues equal to 1/ [SIowill be the points where t = 0. gested that all three be given consideration. In Therefore, as the parameters of the points corre- conclusion we wish to note that the procedures desponding to t = 0 for various values of [SI0 are, re- scribed in this communication are capable of extenspectively, 1/ - d [S]/dt = l/vo for the ordinate and sion to systems more complicated than those rep1/' [SI for the abscissa it follows2 that a line drawn resented by equations 1 , 4and 5 . However, we wish through these points will have a slope of Ks/k3[E] to defer discussion of such systems a t this time. and an ordinate intercept of l/Rs[E]. I t will be CONTRIBUTION h-0. 1989 FROM THE recognized that this plot is similar to the first of the GATESAND CRELLINLABORATORIES OF CHEMISTRY two plots proposed by Lineweaver and Burk,* cf., CALIFORNIA INSTITUTEOF TECHNOLOGY 4, CALIFORNIA Table I, but that in the present case the initial PASADENA velocities need not be separately evaluated since they are given, in the form of their reciprocals, in terms of the ordinate t / ( [SI0 - [SI,) for the condi- Nitration in the Presence of Polyphosphoric Acid tion that t = 0. Furthermore, Kp can be simulBY J. P. KISPERSKY AND K . KLACER taneously determined either from the intercept (1 RECEIVED MAY20, 1955 Ks/Kp)/ks [EI or the slope Ks(Kp [SIo)lka[ElKP. Steinkopf and Supan previously reported that TABLE I the nitration of the alkylmalonates required the use GRAPHICAL PROCEDURES FOR THE EVALUATION OF K RAND k3 of fuming nitric acid-acetic anhydride mixture a t Ordinate 40 to 5 O O . l The authors cautioned against allowing Ref. Ordinate Abscissa intercept Slope the temperature of the reaction to rise over 50' pre2 1/00 l/[SIo sumably because an uncontrollable reaction sets in . . t/([S]O - [sit) (h([S]O/[S]t)/([SlO- I / k s [ E ] KS/k8[E] [Sit) due to the exothermic nature of the nitration. I t 2 [SlO/VC 1sIo has been found that polyphosphoric acid may re. . l/(ln([Slo/[Slt)) ( K i l o - lSlt)/(lnKs/ka[EIa I/ka[El place the acetic anhydride in the nitration mixture (Islo/[Sit)) giving good yields of alkylnitromalonates and re3 Z'O WO/[s]O k3[EIb ducing the potential hazard of the reaction medium. -KS 4 ([SI@- ISlt)/t (ln([Slo/[Slt))/L The compounds thus prepared are listed in Table a Abscissa intercept = --lis. Abscissa intercept = I along with the yield, b.p., refractive index and kr [El/ K s . analysis. On the basis of the data presented it is Equation 7 may also be rearranged to give equa- evident that branching on the carbon atom attion 9 and for this situation a plot of t/(ln([S]~/ tached to the malonic ester moiety reduces the yield on nitration. t / ( l n ( [ S l d [ S l t ) ) = Ks(KP [ S l o ) l ~ s [ W+K{~( K PWe are indebted to the Office of Naval Research Ks)/kr[EIKpl ( ( [ S I 0 - [Slt)/(ln( [Slo/[Slt))J (9) for the funds which supported this work. [ S l t ) ) v s . ([SI0 - [Slt)/(ln(ISIo/[Slt))will give, for various values of [SIo,a series of parallel lines of Experimental slope (Kp - Ks)/ka[E]Kp.lo I t can be shown that Inasmuch as the same experimental procedure was used the points corresponding to t = 0 for the lines of in the preparation of all of the compounds listed in Table I slope (Kp - K s ) / k s [ EJKp are the points where the a general method will be described. Fifty grams of technical 100'% nitric acid was added to lines of slope (Kp - K s ) / k 3 [ E ] K ppossess abscissa g. of polyphosphoric acid* contained in a 200-1111. threevalues of [SIoand that a line drawn through these 80 necked flask equipped with stirrer, thermometer and droppoints, whose coordinates are [S]o/-d [S]/dt = ping funnel. The mixture was heated to 60" and, when [ S ] O / Vand O [SI0 when t = 0, will have a slope of 1/ homogeneous, 0.14 mole of diethyl alkylmalonate was added ka[E],an ordinate intercept of K s / k 3 [ E ]and an ab- dropwise over a period of 15 to 30 minutes. After a small Equation 7 may be rearranged to give equation 8 and it follows that in a plot of t / ( [SI0 - [ S ] t ) OS.
+
+
+
+
+
+
t t1
+
(9) It will be noted that this intercept is the reciprocal of that obtained in the case of a ([Slo [Slc)/t os. (In([Slo/[Slt))/t plot. (10) It is seen from this relation that when KP > Ks the dope will be positive, when K P = Ks the lines will parallelthe abscissa and when K u < Ka the slope will be negative.
-
quantity of ester had been added the mixture required slight cooling to maintain a temperature of 60'. After the addition of the ester the reaction was stirred for one (1) W.Stdnkopf and A. Supan, Ber., 43, 3239 (1910). (2) Obtained from the Victor Chemical Works, Chicago 4, Illinols.
NOTITS
TABLE I
1-02
SITRATIOX OF SUBSTITUTED MALONIC ESTERS,R-C( ~
R
a
Yield
Isopropyl Isobutyl n-Butyl Cyclohexyl n-Decyl -411alysisby the
BO
cc.
83-84 88-88 93---94 110-120
Pressure
0 . 3 mm. 0 . 5 mm. iJ.,?mm,
n25n
1.4337 1.4351 1.4340 1.4597
c
FI
COpC2Hj)y ..
alyqe. N
5.67 7.29 5.37 I i) 50.6 7.29 ,?.87 15 ,5p 54,4 7.31 4.88 8Th l4p 1.4450 30.2 905 4.06 120-130 Elek Microanalytical Laboratories, Los Angeles, Calif. Yield based on
i8 -r
hour a t 60” then cooled and poured onto 200 g. nf chipped ice. After the ice had melted the oily layer was extracted with ether, and washed with 5% sodium bicarbonate solution until neutral, then with water. After drying, the ether was removed by distillation and the remaining liquid distilled through a small Vigreuu column. The diethyl alkylnitromalonates were obtained as colorless oil>- liquids. AEROJET-GENERAL CORPORATIOX AZUSA,CALIFORSIA
Reaction of Tyrosine with Formaldehyde in Acid Solution BY HAROLD S. OLCOTT RECEIVED M A Y31, 1955
In a study of protein reactions,‘ we wished to have available, as one type of model, a non-dialyzable substance containing a number of free amino groups. I t occurred to us that tyrosine should react with formaldehyde in acid solution? to form such a polymer. The authors of the only previous studies on this ~ y s t e r nbelieved ~.~ that they had obtained isoquinoline derivatives. We have prepared products by the methods they described and also under other reaction conditions and find that they are not isoquinoline derivatives but that instead they display the properties to be expected of acidcatalyzed tyrosine-formaldehyde polymers. The preparations were soluble in dilute acid and alkali but insoluble in the neutral range. Depending upon the conditions used in the polymerization, 40-7070 was retained in dialyzing bags (Visking tubing) after extensive dialysis. Free amino nitrogen (by Van Slyke) was equivalent to 75-93% of the total nitrogen. These observations, together with the known reactions of other substituted phenols with f ~ r m a l d e h y d eindicate ,~ that the polymer is composed for the most part of units with the structure /
__
_..-- ~. -. ...Calcd.
R.p.
OH
The low total nitrogen and amino nitrogen analyses in all likelihood reflect the presence of additional methylol groups and methylene cross-links involving amino nitrogen in parts of the polymer. (1) A. Mohammad, H. Fraenkel-Conrat a n d H. S . Olcott, Arch. Biochem., 24, 157 (1949). ( 2 ) T h e polymer formed from tyrosine a n d formaldehyde in alkaline solution has been described b y A. E. Brown, THISJOURNAL, 68, 1011 (1946). It contains no free amino nitrogen. (3) A . Pictet and T. Spengler, Ber., 44, 2030 (1911). (4) J. Wellisch, Biochem. Z., 49, 173 (1913). f.5) Reviewed in J. F. Walker, “Formaldehyde,” 2nd Ed., Reinhold Publ. Corp., New York, N. Y.,19.53.
.48.B 50.6
6.89
~~. .
I‘oiind-- H
~
s 5.
