IY.E. MAYBERRY, J E. KXLL,A
3308
~
D. D BERTOLI
L'ol. hfi
[ C O S T R I B C T I U N FROM T H E S A T I O S A L ISSTITCTE O F .ARTHRITIS AND METABOLIC DISEASES, S A T I O S A L I S S T I T U T E S O F H E A L T H ,
BETHESDA, hfARYLASD]
Kinetics of Iodination. I. A Comparison of the Kinetics of Iodination of N-Acetyl-L-Tyrosine and N-Acetyl-3-iodo-~-Tyrosine BY u'.E. ~ I A U B E R R J.YE., ' R A I L ,AXD D. BERTOLI RECEIVEDJUSE 29, 1963 The kinetics of iodination of S-acetyl-i,-tyrosine and S-acetyl-3-iodo-~-tyrosine has been studied. The biInolecular second-order w t e equation d.c/dt = [.XI[ B],where :I and B are the stoichiometric concentrations of tyrosyl derivative and iodine, fits both reactions when iodide and hydrogen ion concentrations are held conitant \.dues for the rate constants have been calculated with the aid of a high speed digital computer. The data for both reactions are compatible with the concept of phenoxide ion iodination by either molecular iodine or hypoiodous acidinium ion. I n keeping with the Hatnmett meta u-constant of +0.352, the rates of iodination of acetyltyrosine exceed those of acetylinonoiorlotyro~iiieby a factor of 30 over the p H range 5.40 to 9.80.
The chemical kinetics of the iodination of tyrosine was studied by Li' in 1912. The data were interpreted to show that monoiodination was the rate-limiting step in diiodination and further that once the phenolic ring had been tnonoiodinated, the second iodine atom entered the ring instantaneously. Biologic and chemical evidence;' later raised questions regarding the tenability of those conclusions. Kecent preliminary studies have shown t h a t S-acetyl-L-tyrosine iodinates more rapidly than X-acetyl-3-iodo-L-tyrosine over the pH range 5.90 to 10.57.4 Comparisons of the kinetics of monoiodination and diiodination have been made to gain some clue as to the mechanistic detail of phenolic iodination. Such comparisons may have significance in their biologic analogies to in vivo thyroxine formation. A computer program has been employed in the present studies. In addition to increasing speed and facility of rate constant calculations, the statistical fit of the data to the rate equation has been greatly improved by increasing the number of observations per kinetic run. Ai comparable analysis of the observed data would be precluded by the usual mathematical methods.
Experimental Equilibria.-The iodination reactions were performed in aqueous solution and were designed with consideration for the equilibria involving the reactants as: 1,
I]-
I?
+ I--, K~ = 1.3 x
10-3 at 25"
(1)5
k - 1~
k?
IzClk-
I? + C1 -, K z
=
0 60 a t 25"
(2)fi
7
( 1 1 Aided by a g r a n t for n Postdoctoral Fellowship f r o m t h e American Cancel Society Addres- correspondance t o t h e SlayClinic Rnchester, Slinn 82: C . H 1.i J A m C h v m Soc , 6 4 , 11-i7 (I!>%: B i \$' I.udwig a n d P Llutzenbecher. Z . Pn of Chi,m., 268, 195 ( i Y 3 9 ) . K 11 Heirirltt. .I Gcii P h ? s ~ o i ,26, 18.5 11941 1942). C F t o m a g e u t . AI. J u t i s r , SI I a f o n , antl J Roche, Comoi. r r i i d . S o c . bioi , 1 4 3 , 785 IlW82, R SI. Fink a n d K F i n k . .5'ci?ncr, 108, .is5 '1!+481, J Roche S . I.issitzky, 0 Alichrl and I< S l i c h e l . Riochiin n i r , p h y \ . A ilo. I , 4 3 9 (19511, C I , Gemmill, T v d u u / t o >I'i.,,r ~ , 14,t342 ' I # , A I \v I: A1aybett-y- a n d J . rs R ~ I I !. b i d aa, 821 t l u t n i ) ; \\' E. h f a y h e r i y a n d J K. K a l l . Erpost.s A n n B i o r h i m h f d , 2 6 , 2 1 (19641. ( 5 ) R1 Ilavies a n d E. G w y n n e . J . A m . Chum. Soc , 74, 2748 (1952) 801 1). I. Cason a n d H . R I . S e u m n n n . i h i d , 83, 1822 (1961). (71 I1 P Bell a n d E . Gelles, J . C h e n i . .S,x , 2734 (1951)
HaOI'
+ HzO
kt
HOI k-
31,
+ 3H20
ks
103.. k-
+ HtO-,
K,
=
3 X lo-'
(41'
4
+ 51.- + 6 H + ,
Kj
=
4 X 10 - I i
6
a t 25' ka
K-C&OH
K-CsHjO-
+H
l,
Kg
!5)y
(6)
k- R
A large ratio (66 to 1 a s a minimum) of iodide to iodine has been maintained. Ionic strength has been maintained constant by the addition of sodium chloride; therefore, equilibrium K. has significance in the system. This complicates the system somewhat; however, this salt was chosen because of its lack of absorption in the wave length range of the present studies. 111 addition, as will be subsequently shown, the correction factors necessary because of equilibrium K Z are not major. Hydrogcii ion concentration has been kept constant by the presence of buffer. Iodate formation may result in loss of iodine if iodide antl hydrogen ion concentrations are low. This possibility has been studied for each set of experimental conditions. Equilibrium Kfi has been determined for each phenolic compound used. Measurement of the rate constant of iodination requires that all of the equilibria involved be rapid with respect t o the actual iodination step. Materials.-Recrystallized S-acetyl-L-tyrosine ( S - a c T Y ) , Sacetyl-3-iodo-~-tyrosine(M-acMIT), and S-acetyl-3,5-diiodo-1,tyrosine ( S - a c D I T ) were the phenolic derivatives employed.1° Paper chromatography with a l-butanol-l,4-dioxane-2 .V ammonium hydroxide (30 : 20 : saturated) solvent system and high voltage electrophoresis in barbital buffer a t p H 8.68 revealed each compound to run as one spot as detected by the ferric chloride-potassium ferricyanide reagent for phenols.Il The acetyltyrosine had a melting point on a Kijfler stage of 119.5-151.0° (uncor.), while the literature value is given as 1R2154" (cor.).12 Anal. Calcd. for CIIHILSOI:C, 59.19; H , 5.87; S , 6.27. FouridI3: C, 59.35; H , 5.82, S ,6.31. The acetylmonoiodotyrosinehad a m . p . or 159-160.5" (uncor.), but no literature value was found for comparison. Ancll. Calcd. for C I I H 1 ~ I N O aC, : 3i.84; H , 3.36; I , 36.35; S ,4.01. Foundls: C,38.02; H , 3.47; I,36.52; S ,4.18. T h e m . p . of the acetyldiiodotyrosine.0.5Hz0 was 125-126' (uncor.) confirming the value previously reported for this compound.14 Anal. Calcd. for CllH111zX01~0.5H~O: C , 27.30; H , 2.50; (8) KI was estimated by Bell a n d Gelles (ref. 7 ) b y dividing t h e h y d r d y a i i constant of \I' C Bray a n d E. I, Connolly. J A m . ( ' h e m . .Coc 3 3 , 148.5 :1911J, by Ki. !1 B a r t o n , I l a s s . , 1968. p. 259. (9) H K . V . Arnstein a n d R I . E C l u h h , B i o c h e m . J . , 63, 528 (1958).
received a definitive test, and we were intrigue- with the possibility of effecting a transannular synthesis of the bicyclic system present in penicillin. The requirement that the configurations a t C-6 and C-3 of I1 be fixed before creation of the bicyclic ring system is implicit in the findings of ,Irnstein and his co-workers,j>'" so that, a t least in the biosynthetic conversion to penicillin, 3~-carboxy-2,2-dimethyl-5-oxo-6~-phenylacetamidoperhydro-1,4-thiazepine(I, R = C6H5CHs)would be the desired stereomer to test, with the added provisions that it could serve as a competitive precursor and that the cells would be permeable to the compound. The 1,4thiazepines related to the penicillins have received relatively little attention, and the stereochemistry of these compounds has not been investigated. Previous syntheses leading to 3-carboxy-2,d-dimethyl5-oxoperhydro-1,I-thiazepine (111)] ' - l a and its closely related derivativesg, have utilized open-chain starting materials and have generally employed a ring-closure step involving either addition of a thiol to an acrylate derivative or the formation of the amide C--K bond. Thus, 3-carbomethoxy-T-chloro-2,2-dimethyl-3-oxoperhydro-1.4-thiazepine (IV) and X-carboniethoxy2,2 - dimethyl - 5 -oxo - %,3,4,5 -tetrahydro - 1 .-lthiazepine (1711) were reported as products from condensations of (IO) H . K . V . Arnstein and J . C. Crawhall. i b z d . , 6 6 , 181' (10.57). 67, 180 (1957). (11) T. Wieland, G . Ohnackei and W Ziegler, Chem. B F ~ 90, . , 19.1 ilY.i7) (12) I . I,. K n u n y a n t s , 0.V Kil'disheva, and SI. G . Lin'kova Impst. Akad. .\-auk S . S . S . R , OfiieI Khzm. V o i d , 71 fl95.5). C'hrni. A b s f ? . 6 0 , 13!23 (1966). ( 1 3 ) I . L. K n u n y a n t s a n d R.1. G . L i n ' k o v a , ibid , 62 (19 60, 1692 (19.56). ( 1 4 ) I . L K n u n y a n t s , 0 . V, Kil'disheva, .\I. P..K r a s u s k a y a . h l . G . Lin'kova, V. V . Shokina. Z. V. Benevolenskaya, and L P. Kasteikene, ibid.. 1777 (1959), Bull. Acad. S c i . C . S . S . R . ,Dio Chem. S c t . , 1702 (1959); Chem. Abslr., 64, 8843 (1960)
