VOL. 15, 1129-1141 (1976)
BIOPOLY MERS
Coenzyme Models. IV. Effect of Polymer Micelles on Rate and Equilibrium of Addition of Cyanide Ion to N-Substituted 3Carboxamidopyridinium Ions SEIJI SHINKAI and TOYOKI KUNITAKE, Department of Organic Synthesis, Faculty of Engineering, Kyushu University, Fukuoka 812, Japan
Synopsis The water-soluble poly( 1-vinyl-2-ethylimidazole) quaternized with ethyl bromide and lauryl bromide was prepared; lauryl group content, 8.8 mol% (L-9),28.9 mol% (L-29),and 40.9 mol% value of methyl orange near 460 nm shifted to shorter wavelengths (417433 (L-41). The A,, nm) in the aqueous solution of L-29 and L-41, and the intrinsic viscosity of L-29 was more than ten times smaller than that of L-9. The rate and equilibrium constants ( k f and K ) for addition of cyanide ion to the N-substituted 3-carboxamidopyridinium ions were studied a t 30°C, where N-substituents employed were n-propyl, n-hexyl, benzyl, 2,6-dichlorobenzyl, and n-lauryl. The kinetic parameters for n-lauryl-3-carboxamidopyridiniumwere markedly increased in the presence of L-29 and L-41 and with increasing polymer concentrations (84-fold fork/ and 7800-fold for K ) ,especially a t low ionic strength, whereas L-9 decelerated the addition reaction. These distinct behaviors mean that L-29 and L-41 are classified as micellelike polymers and L-9 as a polyelectrolytelike polymer. However, I,-29 depressed the rate of the forward reaction for benzyl-3-carboxamidopyridinium, acting like a simple polyelectrolyte. Therefore, the polymer micelle can provide both the microenvironments characteristic of polyelectrolytes and micelles, depending on the hydrophobicity of substrates.
INTRODUCTION The reaction environments produced by microheterogeneous systems have been of much interest in relation t o enzymic catalyses. T h e most expeditious methods for such studies are the use of micelles and polyelectrolytes. Both of them are known to be effective catalysts for organic rePolymeric Schiff bases, such as polyvinylpyridine, when quaternized by long aliphatic groups, can solubilize many kinds of water-insoluble compounds, and are well known as polymer micelles or p o l y ~ o a p s . ~ - ~ These polymers may possess properties characteristic of micelles, polyelectrolytes, or both, depending on the content and length of the alkyl chain. In fact, partially laurylated polyvinylpyridine gives rise to a drastic decrease in intrinsic viscosity and an increase in solubilization ability a t ca. 10 mol% of the lauryl g r o ~ p . ~This - ~ change is attributable t o the transition from a polyelectrolyte to a polymer micelle. 1129
0 1976 by John Wiley & Sons, Inc.
SHINKAI AND KUNITAKE
1130
The catalytic effect of polymer micelles has been reported by Cordes and co-workers8and Okubo and Isegfor the alkaline hydrolysis of several phenyl esters. In order to assess the characteristics of polymer micelles more clearly, we chose the addition reaction of the cyanide ion toward N-substituted 3-carboxamidopyridinium ions [Eq. (l)].This reaction is considered to be one of the most highly promoted reactions by micelles10and is known, in contrast, to be decelerated in the presence of polyelectrolytes.ll
This system may be also useful for understanding the influence of apoenzymes on the reactivity of NAD coenzyme. We employed the following polymers and N-substituted 3-carboxamidopyridinium ions: substrates:
Pr: -(CH&CH3 Hx: -(CH&,CH,
I
R
CI,Bzl: wdich lorobenzyl
po1y mers:
--CHGH-/
I CH,CH,
EXPEkIMENTAL Materials N-substituted 3-carboxamidopyridinium ions were prepared from appropriate alkyl halides and nicotinamide. Mixtures of alkyl halides and nicotinamide (1:2 molar ratio) were refluxed in minimum volumes of ethanol for 24 hr. The precipitates were collected and recrystallized from methanol: N-propyl-3-carboxamidopyridinium iodide[NPr(Pyr+)CONH2X-1, mp 181°-1830C (lit.12 182°C); N-benzyl-3-carboxamidopyridinium chloride[NBzl(Pyr+)CONH2X-], mp 235O-236OC (lit.13 235'236OC); N-2,6-dichlorobenzyl-3-carboxamidopyridinium chloride [NC12Bzl(Pyr+)CONH2X-], mp 2380-240°C (lit.13 239O-242OC); N hexyl-3-carboxamidopyridinium bromide[NHx(Pyr+)CONH2X-], mp 215O-218' c.
COENZYME MODELS. IV
1131
TABLE I Preparation of Polymers
Polymer
Ratio in Feed ([lauryl bromide]/ [polymer unit])
Lauryl Group Content (mol %)
Residual Free Imidazole (mol YO)
L-9 L-29 L-41
0.10 0.30 0.40
8.8 28.9 40.9
1.8 3.9 10
Found: C, 50.51; H, 6.64; N, 9.68%. Calcd for C12H19N20Br: C, 50.19; H, 6.68; N, 9.75%.
T h e preparation of N-lauryl-3-carboxamidopyridiniumbromide "La(Pyr+)CONHzX-] was described previ0us1y.l~ 1-vinyl-2-ethylimidazole was kindly provided by Toho Rayon Company and distilled prior to use, bp 108'C/35 mmHg.
Polymerization and Quaternization Polymerization of 1-vinyl-2-ethylimidazole was conducted using azobisisobutyronitrile as initiator a t 70°C in methanol solvent, and the polymer was reprecipitated from methanol and ether. 4 g of poly(1-vinyl-2-ethylimidazole)and aliquots of lauryl bromide in 50 ml of dimethylsulfoxide were heated a t l0O'C for 24 hr. 1 ml of the reaction mixture withdrawn was poured into ether, and the recovered polymer was subjected to elemental analysis after reprecipitation from methanol and ether, in order to determine the content of the lauryl group incorporated. Excess ethyl bromide was then added and heated further a t 5OoC for 72 hr. Polymers were recovered by precipitation in acetone, purified as aqueous solutions by extensive dialysis, and subjected to elemental analysis after reprecipitation. The results are summarized in Table I.
Kinetics Kinetic measurements were carried out spectrophotometrically with the aid of a Hitachi 124 spectrophotometer equipped with a thermostated cell holder (30' f 0.l'C). The reaction was initiated by mixing a polymer solution containing KCN with a pyridinium solution. T h e reaction rates were followed by observing the appearance of an absorption near 340 nm, which is characteristic of 1,d-adducts of the pyridinium ions.15 In all cases an excess of cyanide ion was present, so that the pseudo-first order behavior was observed. From the pseudo-first order rate constant hobs thus obtained was calculated the second order rate constant k f according to Eq. (2):
kf =
kobs
[CN-]
+ K-l
SHINKAI AND KUNITAKE
1132
where K denotes the equilibrium constant for Eq. (1)(i.e., K = k f / k , = [4-adduct]/[substrate][CN-I).
Equilibrium Constants ( K ) The equilibrium constants in the absence of polymers were evaluated using the method of Behme and Cordes,16 from the slope of the plots of (ODo-OD)/[CN-] against OD, where OD0 denotes the absorbance of the substrate, OD the absorbance of the reaction mixture a t infinite time. Since the reaction product of NLa(Pyr+)CONH2X- with CN- precipitated in the absence of polymers a t ca. 80%reaction, OD values for NLa(Pyr+)CONH2X- were evaluated as follows. At first the pseudo-first order rate constants k,bs was determined from theGuggenheim procedure and the corresponding half-life (t1/2) from Eq. (3).17 Subsequently, OD,/, (absorbance at the half-life) was read from the reaction chart, and OD was determined according to Eq. (4): 0.693 t1/2 = k obs OD = 2 X OD,/,
(3)
(4)
The molar absorption coefficient for each substrate can be determined according to the method of Cordes and co-workers.1° The equilibrium constants in the presence of polymers were evaluated using these t values [Eq. (511:
K=
-
OD/t 1 [CN-]([substrate] - OD/e. 1)
where 1 is the cell width.
Viscosity Measurements Viscosities of the polymers were measured a t 30°C, using a modified Ubbelhode viscometer. Linear plots of q,,/C versus C were observed for all the polymer samples in 0.02 M KBr. This rather low ionic strength is sufficient to suppress the electroviscous effect even for highly charged polymers.
RESULTS Viscosity of Polymers and Visible Spectra of Methyl Orange Table I1 gives viscosities of the quaternized polymers and absorption spectra of methyl orange in the presence of these polymers. The intrinsic viscosity of L-9 is more than ten times larger than that of L-29. The L-41 polymer gives a viscosity somewhat larger than that of L-29. These viscosity variations supposedly reflect the conformation change of the polymers, since the same starting polymer was used for quaternization.
COENZYME MODELS. IV
1133
TABLE I1 Viscosity and Absorption Spectrum of Methyl Orange
a
Polymer
Lauryl Group Content (mol %)
[VI" (dug)
h m a x b (nm)
L -9 L-29 L-41 none
8.8 28.9 40.9 -
0.496 0.037 0.048 -
463 417 433 464
30"C, 1-1
=
0.02 with KBr.
M , [L-91 = 0.091 M , [L-291 = 0.075 M ,
b 30"C, [methyl orange] = 2.67 X
[L-41] = 0.101 M . TABLE I11 Rate and Equilibrium Constants for Addition of the Cyanide Ion t o N-Substituted 3-Carboxamidopyridinium Ions in the absence of Polymera Substrate NPr(Pyr+)CONH,XNHx(Pyr+)CONH,XNLa(Pyr+ )CONH,XNBzl(Pyr+)CONH,XNCI,Bzl(Pyr+)CONH,X-
€,axb
7000 6880 6750 6630 6700
k f (M-I sec-' )
K (M-I)
0.0217 0.0223 0.0800 0.0895 0.146
0.11 0.15 25 1.99 4.36
lo-'
a 30°C, 1-1 = 0.1 with KCl, [KOH] = M , [KCN] = 0.02-0.10 M . b Molar absorption coefficient of 1,4-adducts.
The aqueous solution of L-9 hardly affected the visible spectrum of methyl orange, whereas ,A, of methyl orange appreciably shifted to shorter wavelengths in the presence of L-29 and L-41. The largest hypsochromic shift was seen for L-29, consistent with the viscosity data.
Kinetic Parameters in the Absence of the Polymer The results of kinetic measurements in the absence of polymers are recorded in Table 111. The reaction temperature in this study is higher by 5°C than that employed by Lindquist and Corded5 and k f values for NPr(Pyr+)CONHZX- and NBzl(Pyr+)CONH2X- are a little larger and the K values a little smaller, compared with their results. The reaction parameters for NHx(Pyr+)CONH2X- are almost identical with those of NPr(Pyr+)CONH2X-. The rate constants k f of the cyanide addition toward NLa(Pyr+)CONH2X- is greater by fourfold and the corresponding K value by 230-fold than those of NPr(Pyr+)CONH2X-.
Kinetic Studies in the Presence of Polymer Micelles In Figures 1 and 2, the rate and equilibrium constants for the addition of the cyanide ion to NLa(Pyr+)CONH2X- a t 3O0C and pH 11 are shown
SHINKAI AND KUNITAKE
1134
as a function of the concentration of polymers. Typical reaction parameters are collected in Table IV. Both reaction parameters, kf and K , are remarkably increased with increasing concentrations of L-29 and L-41; and L-29 showed larger acceleration effects for both parameters. For example, these constants for NLa(Pyr+)CONH2X- in the presence of L-29 are enhanced by factors of 23 for k f and 34 for K compared with those in the absence of the polymers. If NPr(Pyr+)CONHzX- is regarded as a typical nonmicellar substrate and used as reference,'O L-29 increases these constan by 84-fold and 7800-fold7respectively. Interestingly, the addition reaction is completely inhibited when the unit concentration of the polymers is much larger than [CN-] (Figs. 1 and 2). The inhibitory phenomenon will be described later in detail. The increases in rate and equilibrium constants observed at the optimal condition in the presence of L-29 are comparable to those in conventional cationic micellar systems.1°
2
I
I
0.10 0.08
L-Ll
/ J
0.06
0.04 0,02
0
-,.
I
I
0.02
0.04
(PNYVER)
I
*
I
0
0.08
0.06 M
Fig. 1. Effect of polymers on the k f of NLa(Pyr+)CONHzX- = CN- reaction: "LaM. (Pyr+)CONHzX-] = 5.00 X M ; [KCN] = 0.01 M ;[KOH] = 1.0 X
-"""I 800
30
-
20
1 -
-. 10
, &
0
0.02
0.04
I
0.08 (PIILYMEP)
0
1:
Fig. 2. Effect of polymers on the K of NLa(Pyr+)CONHzX- = CN- reaction. The reaction conditions employed are the same as in Fig. 1.
COENZYME MODELS. IV
1135
T A B L E IV R a t e a n d Equilibrium Constants for NLa(Pyr+)CONH,Xin t h e Presence of Polymera
Polvmer x l o 2M k f M-' sec-' KM-' __ L -9 1.81 0.0106 5.4 L-29 6.00 1.82 862 L-41 8.08 1.36 316 - ~ _ _ _ __ __ a 30"C, [ K C N ] = 0.01 M , [ K O H ] = 1.0 x low3M . N o inorganic salt was a d d e d e x c e p t KCN a n d K O H . ~
In contrast, the presence of L-9 decelerated the addition reaction (Figs. 1 and a), and the decrease was similar for kf and K . When NBzl(Pyr+)CONH2X- was used as the substrate, the forward reaction (kfterm) was decelerated by both L-29 and L-9 (Fig. 3a). This is in marked contrast to the above-mentioned data obtained with NLa-
?-I
oa14
L x - 0.12
?-I
0.10
+u L-29
O
O
0.02
0.04
(POLYMER]
M
(a)
3.k :
0
0
0.02 [POLYMER]
0.04 M
(b) Fig. 3. (a) Effect of polymers on the h, of NBzl(Pyr+)CONHZX- = CN- reaction. (b) Effect of polymers on the K of NBzl(Pyr+)CONHZX--CN- reaction. [NBzl(Pyr+) CONHzX-] = 5.00 X M ; [KCN] = 0.01 M ; [KOH] = 1.0 X M.
1136
SHINKAI A N D KUNITAKE
(Pyr+)CONH2X- as substrate. This different behavior observed for the same polymer, L-29, would be caused by the unfavorable partitioning of NBzl(Pyr+)CONH2X- in the polymer phase compared with NLa(Pyr+)CONH2X-. The association constants K increased gradually with the increase of the L-29 concentration, in spite of the decrease in the forward reaction rate (Fig. 3b). Therefore, the rate of the reverse reaction k, is much more depressed by the L-29 polymer than that of the forward reaction.
Effect of Ionic Strength In the conventional micellar catalysis, the efficiency is generally depressed by the increase in salt concentrations. The largest decrease in the addition reaction of the cyanide ion was reported to be about sixfold.1° Figure 4 shows the effect of the KBr concentration on the rate and equilibrium constants for NLa(Pyr+)CONH2X- substrate in the presence of 0.015 M L-29. As expected, additions of KBr decreased both the rates and the association constants, with the largest rate decrease about threefold.
Inhibition of Addition Reaction by Polymer Micelles As shown in Figure 1, the cyanide addition to NLa(Pyr+)CONH2X- was completely inhibited by polymer micelles under the condition, [polymer] >> [CN-1. The pseudo-first order rate constants k,bs were measured as a function of KCN concentration, while maintaining the concentration of L-41 constant (Fig. 5). The absorption increase based on the 4-adduct formation was undetectable a t KCN concentrations below 0.012 and 0.003
- 200 4
SI
- 100 s
Imrl
M
Fig. 4. Effect of ionic strength on the k f and K of NLa(Pyr+)CONH*X- = CN- reaction M; [KCN] = 0.01 in the presence of the L-29 polymer: [NLa(Pyr+)CONHzX-] = 5.0 X M ; [L-291 = 0.015 M. M ;[KOH] = 1.0 X
COENZYME MODELS. IV
1137
M for the L-41 concentration of 0.101 and 0.0202 M , respectively. The absorbance near 340 nm increased rapidly a t higher KCN concentrations, and then reached saturation with further increases. The critical inhibition for the ratios of [polymer]/[KCN] are 7-8 for L-41 and ca. 6 for L-29. The absorption of the 4-adduct of NLa(Pyr+)CONH2X- has disappeared in the presence of 0.05 M L-9. However, this polymer only depressed the rate and equilibrium constants progressively with its increasing concentration, so that it is difficult to conclude whether a similar, critical inhibition is involved in this polyelectrolyte-type polymer.
DISCUSSION Selection of Polymer The polymer micelle can be usually prepared by quaternization of polymeric Schiff bases such as polyvinylpyridine.8J8 Unfortunately, these quaternized polymers may undergo addition of CN- and other reactions, which are characteristic of alkylpyridinium salts. Winters and co-workerslg found that N-laurylpyridinium bromide forms the laurylviologen cation radical with aqueous sodium cyanide a t room temperature. N-ethylpyridinium bromide is inert under the same condition,lg so that this radical formation should be possible only in a micellar system. In a separate study, we found that the polymer micelle of poly( 2-vinylpyridine) (laurylated units, 30 mol%) generated the weak absorption of the viologen cation radical
0.03
0.02 -l
u v) w m v)
s
0.01
0 0
0.01
0.02 (KCN)
0,03
M
Fig. 5. Inhibition of NLa(Pyr+)CONHZX- = CN- reaction by L-41 polymer: "La( 0 )[L-411 = 0.101 M ; (A)[L-41] (Pyr+)CONHzX-] = 5.0 X 10-4M; [KOH] = 1.0 X lo-"; = 0.0202 M .
1138
SHINKAI AND KUNITAKE
a t near 600 nm under anaerobic conditions (unpublished results). To obviate these complexities,we employed polymeric l-vinyl-2-ethylimdazole, the polymer micelle of which was found to be inert in aqueous sodium cyanide solutions.
Conformation of the Polymers The conformational characteristics of the polymers employed may be assessed from viscosities of the polymers and absorption spectra of methyl orange in the presence of the polymer. The intrinsic viscosity of the L-9 polymer was much larger than that of the L-29 and L-41 polymers. Presumably, the latter polymers possess globular, compact conformations, while the L-9 polymer assumes an expanded conformation (a typical polyelectrolyte). Strauss and c o - ~ o r k e r s previously ~-~ found for quaternized polyvinylpyridines that drastic changes of physical properties (viscosity decrease, solubilization of hydrocarbons) occurred at ca. 10-13 mol% of the lauryl group content. They proposed that the polymer formed intramolecular micelles at high lauryl group contents. Thus, it is concluded that the L-29 and L-41 polymers formed polymer micelles. This was also confirmed by absorption spectra of methyl orange. Visible spectra of methyl orange are frequently used to detect the hydrophobic region present in aqueous media, since the apolar environment elicits the hypsochromic shift of the ,A, value near 460 nm.20’21 That appreciable hypsochromic shifts were observed in the presence of L-29 and L-41 polymers suggests that these polymers possess hydrophobic regions. The expanded conformation of the L-9 polymer cannot provide a hyof methyl orange is not affected by this drophobic binding site; thus ,A,, polymer. According to Strauss and Gershfeld? the incorporation of long aliphatic chains into polymers results in two opposing factors affecting the polymer conformation. They are 1)hydrophobicity, which holds polymer molecules in compact conformations and 2) the chain stiffness, which causes expansion. At intermediate lauryl contents, the former factor overcomes the latter and gives rise to compact conformations, whereas the rodlike conformation is favored in highly laurylated polymers. The possibility that increased contents of the lauryl group facilitate intermolecular aggregation must be also taken into account. The transition occurs at the lauryl content between 29 and 38 mol% for polyvinylpyridine derivative^.^ Consistent with this proposition is the fact that the L-29(29%laurylated) polymer possesses more compact conformations and greater hydrophobicity than L-41(41%laurylated), as inferred from the data of Table 11.
Polymer Micellar Effects
It has been established by Cordes and co-workers1°that rate and equilibrium constants for the addition of cyanide ion to 3-carboxamidopyri-
COENZYME MODELS. IV
1139
dinium ions with long alkyl chains are pronouncedly increased in dilute aqueous solutions of cationic surfactants. For example, these constants for the cyanide addition to NLa(Pyr+)CONH2X- in the presence of 0.02 M hexadecyltrimethylammonium bromide a t 25°C are kf = 1.1-1.5 M-l sec-' and K = 600-1000 M-'. Typical nonmicellar values are kf = 0.014 M-' sec-l and K = 0.185 M-l for NPr(Pyrt)CONH2X-. On the other hand, Okubo and Isell reported that rate and equilibrium constants of a similar reaction system are decelerated by polyelectrolytes in the same proportion. This indicates that the reverse reaction (h, term) is not influenced by polyelectrolytes. Therefore, this reaction would be one of the most sensitive systems to study the transition between the micellar and polyelectrolyte-type states of the quarternized polymer. The hf and K values for NLa(Pyr+)CONH2X- were markedly increased in the presence of L-29 and L-41 polymers. The enhancements are almost comparable in magnitude to those observed in micellar systems.1° On the other hand, L-9 decelerated these parameters for NLa(Pyrf)CONH2Xin the same proportion (Table IV). Therefore, L-29 and L-41 polymers behave as typical micelles, and the L-9 polymer as a typical polyelectrolyte. T h e L-29 polymer showed an influence greater than t h a t of L-41. These results are in complete accord with the conclusion derived from the conformational study. Contrary to the data obtained for NLa(Pyr+)CONH2X-, the L-29 polymer showed a polyelectrolyte-type influence on the cyanide addition t o NBzl(Pyr+)CONH2X-. T h e NBzl(Pyr+)CONH2X- molecule is so soluble in aqueous media that it would be hardly partitioned in the micellar phase of L-29. Thus, this reaction, which mainly occurs in the bulk phase, would be influenced by the polyelectrolytelike portion of L-29, and the forward reaction rate would become depressed (Fig. 3). In contrast, the association constants gradually increased with the increase in the concentration of L-29, indicating the gradual decrease of rates of the reverse reaction (k, term). A simple polyelectrolyte should depress rates kf and association constants K in the same proportion, since the reverse reaction is unaffected." It is likely t h a t the 4-adduct of NBzl(Pyr+)CONHzX-, being neutral and less water soluble, is solubilized and stabilized in the hydrophobic region of L-29, resulting in the depressed rate of the reverse reaction. Evidently, the effect of L-29 on the reaction of NBzl(Pyr+)CONH2X- is polyelectrolytelike for the forward reaction and micellelike for the reverse reaction. Thus, the chemical influence of the polymer depends not only on the polymer structure, but also on the hydrophobicity of the substrate employed. The cyanide addition to NLa(Pyr+)CONH2X- was thoroughly inhibited by polymer micelles under the condition [polymer] > ca. 7[CN-]. This phenomenon is not found with conventional micelles so that this is probably related to the inherent property of polymer micelles. T h e possible explanation for this phenomenon is that the polymer micelle may constitute a number of small, local micelles. If so, the dilution effect would be more clearly observed for the polymer micelle than for the conventional micelle.
SHINKAI AND KUNITAKE
1140
It is interesting that the compact L-29 polymer inhibits the reaction more efficiently than L-41. Abnormal Affinity of N-lauryl-3-carboxamidopyridinium Bromide In the absence of polymer micelles, NLa(Pyr+)CONHzX- showed an unusually high affinity toward the cyanide ion. The rate constant kj is greater by only fourfold compared with that of NPr(Pyr+)CONHzX-, and, in contrast, the association constant increased by 230-fold; that is, the abnormality of NLa(Pyr+)CONHzX- is mainly associated with depression of the reverse reaction. Since the concentration of NLa(Pyr+)CONHZXM ) is definitely below the cmc of the analogous surfactants (ca. (5.0 X 1.5 X M),l the abnormally high reactivity of this substrate cannot be explained by the micelle formation. This peculiar finding is explicable by stabilization of the product, which is caused by the hydrophobic nature of the long alkyl substituent. It is already known that the nicotinamide mononucleotide and both the (Y and /3 isomers of NAD coenzyme have an abnormally great affinity for cyanide ion when compared with their model compound^.'^ This trend is particularly pronounced for the /3 isomer. Lindquist and Cordes15 pointed out that this observation accorded with the fact that the /3 isomers of NAD and NADH could exist in folded conformations with the adenine m~iety.~ A~ good * ~linear ~ relationship that is approximated by Eq. (6) is recognized between log K and log kj determined by Lindquist and Corded5 a t 25OC for the cyanide addition to six N-substituted 3-carboxamidopyridinium ions (Fig. 6). Our data for NPr(Pyr+)CONHzX-, NHx0
22-
"3
0 0 4
-1
-2
-1
0
2
1
log K data cited horn Ref. 15, I-CY-NAD, 2-nicotinFig. 6. Log K - log k/ relationship: (0) amide mononucleotide, 3-0-NAD; ( 0 ) data from the present study, 4-NPr(Pyr+) CONHzX-, 5--NHx(Pyr+)CONHzX-, 6-NBzl(Pyr+)CONHzX-, 7-NClzBzl(Pyr+)CONHzX-, 8--NLa(Pyr+)CONHzX-.
COENZYME MODELS. IV
1141
(Pyr+)CONH2X-, NBzl(Pyr+)CONHzX-, and NC12Bzl(Pyr+)CONH2Xa t 30°C are also located close to this linear relationship [Eq. (7)]: log hj = 0.55 log K - 1.45, r = 0.999
(6)
log hf = 0.53 log K - 1.19, r = 0.998
(7)
Interestingly, plots for the above-mentioned biological cofactors deviate from the linearity to the lower area in the log K - log hf map, due to the much too large association constants. A plot for NLa(Pyr+)CONH2Xalso deviates downward from the linearity. These results mean that the abnormally high affinity of the cyanide ion toward biological cofactors and NLa(Pyr+)CONHzX- is commonly caused by the increased stability of the CN adducts. We consider that the 4-adduct of NLa(Pyr+)CONHzXis stabilized by the apolar lauryl group and that of 0-NAD by the stacking with the adenine ring. The authors thank Miss Reiko Ando for her capable technical assistance
References 1. Fendler, E. J. & Fender, J. H. (1970) Aduan. Phys. Org. Chem. 8,271-406. 2. Cordes, E. H. & Dunlap, R. B. (1969) Acc. Chem. Res. 2,329-337. 3. Ise, N. (1973) Reaction on Polymers, ed. by Mooe, J. A,, Ed., Reidel, The Netherlands, p. 27. 4. Strauss, U. P. & Gershfeld, N. L. (1954) J . Phys. Chem. 58,747-753. 5. Strauss, U. P., Gershfeld, N. L. & Crook, E. H. (1956) J . Phys. Chem. 60,577-584. 6. Strauss, U. P. & Williams, B. L. (1961) J . Phys. Chem. 65, 1390-1395. 7. Inoue, H. (1964) Kolloid-Z. Z . Polym. 196, 1-7. 8. Rodulfo, T., Hamilton, J. A. & Cordes, E. H. (1974) J . Org. Chem. 39, 2281-2284. 9. Okubo, T. & Ise, N. (1973) J. Org. Chem. 38,3120-3122. 10. Baumrucker, J., Calzadilla, M., Centeno, M., Lehrmann, G., Urdaneta, M., Lindquist, P., Dunham, D., Price, M., Sears, B. & Cordes, E. H. (1972) J . Amer. Chem. Soc. 94,8164-8172. 11. Okubo, T . & Ise, N. (1973) J . Amer. Chem. Soc. 95,4031-4036. 12. Kosower, E. M. & Bauer, S. W. (1960) J . Amer. Chem. SOC.82,2191-2194. 13. Kim, C. S. & Chaykin, S. (1968) Biochemistry 7,2339-2350. Jap. 48,1914-1917. 14. Shinkai, S., Ando, R. & Kunitake, T. (1975) Bull. Chem. SOC. 15. Lindquist, R. N. & Cordes, E. H. (1968) J . Amer. Chem. SOC.90,1269-1274. 16. Behme, M. T. A. & Cordes, E. H. (1965) Riochim. Biophys. Acta 108,312-313. 17. Jencks, W. P. (1969) Catalysis in Chemistry and Enzymology, McGraw-Hill, New York, N.Y., p 558. 18. Kunitake, T., Shinkai, S. & Hirotsu, S.(1975) J . Polym. Sci., Polym. Lett. Ed. 13, 377-381. 19. Winters, L. J., Borror, A. L. & Smith, N. (1967) Tetrahedron Lett. 2313-2315. 20. Klotz, I. M. & Shikama, K. (1968) Arch. Biochem. Biophys. 123,551-557. 21. Takagishi, T., Nakata, Y. & Kuroki, N. (1974) J . Polym. Sci., Polym. Chem. Ed. 12, 807-816. 22. Weber, G. (1957) Nature 180,1409. 23. Shifrin, S. & Kaplan, N. 0. (1959) Nature 183, 1529.
Received September 9,1975 Accepted December 8,1975
BIOPOLY MERS
Coenzyme Models. IV. Effect of Polymer Micelles on Rate and Equilibrium of Addition of Cyanide Ion to N-Substituted 3Carboxamidopyridinium Ions SEIJI SHINKAI and TOYOKI KUNITAKE, Department of Organic Synthesis, Faculty of Engineering, Kyushu University, Fukuoka 812, Japan
Synopsis The water-soluble poly( 1-vinyl-2-ethylimidazole) quaternized with ethyl bromide and lauryl bromide was prepared; lauryl group content, 8.8 mol% (L-9),28.9 mol% (L-29),and 40.9 mol% value of methyl orange near 460 nm shifted to shorter wavelengths (417433 (L-41). The A,, nm) in the aqueous solution of L-29 and L-41, and the intrinsic viscosity of L-29 was more than ten times smaller than that of L-9. The rate and equilibrium constants ( k f and K ) for addition of cyanide ion to the N-substituted 3-carboxamidopyridinium ions were studied a t 30°C, where N-substituents employed were n-propyl, n-hexyl, benzyl, 2,6-dichlorobenzyl, and n-lauryl. The kinetic parameters for n-lauryl-3-carboxamidopyridiniumwere markedly increased in the presence of L-29 and L-41 and with increasing polymer concentrations (84-fold fork/ and 7800-fold for K ) ,especially a t low ionic strength, whereas L-9 decelerated the addition reaction. These distinct behaviors mean that L-29 and L-41 are classified as micellelike polymers and L-9 as a polyelectrolytelike polymer. However, I,-29 depressed the rate of the forward reaction for benzyl-3-carboxamidopyridinium, acting like a simple polyelectrolyte. Therefore, the polymer micelle can provide both the microenvironments characteristic of polyelectrolytes and micelles, depending on the hydrophobicity of substrates.
INTRODUCTION The reaction environments produced by microheterogeneous systems have been of much interest in relation t o enzymic catalyses. T h e most expeditious methods for such studies are the use of micelles and polyelectrolytes. Both of them are known to be effective catalysts for organic rePolymeric Schiff bases, such as polyvinylpyridine, when quaternized by long aliphatic groups, can solubilize many kinds of water-insoluble compounds, and are well known as polymer micelles or p o l y ~ o a p s . ~ - ~ These polymers may possess properties characteristic of micelles, polyelectrolytes, or both, depending on the content and length of the alkyl chain. In fact, partially laurylated polyvinylpyridine gives rise to a drastic decrease in intrinsic viscosity and an increase in solubilization ability a t ca. 10 mol% of the lauryl g r o ~ p . ~This - ~ change is attributable t o the transition from a polyelectrolyte to a polymer micelle. 1129
0 1976 by John Wiley & Sons, Inc.
SHINKAI AND KUNITAKE
1130
The catalytic effect of polymer micelles has been reported by Cordes and co-workers8and Okubo and Isegfor the alkaline hydrolysis of several phenyl esters. In order to assess the characteristics of polymer micelles more clearly, we chose the addition reaction of the cyanide ion toward N-substituted 3-carboxamidopyridinium ions [Eq. (l)].This reaction is considered to be one of the most highly promoted reactions by micelles10and is known, in contrast, to be decelerated in the presence of polyelectrolytes.ll
This system may be also useful for understanding the influence of apoenzymes on the reactivity of NAD coenzyme. We employed the following polymers and N-substituted 3-carboxamidopyridinium ions: substrates:
Pr: -(CH&CH3 Hx: -(CH&,CH,
I
R
CI,Bzl: wdich lorobenzyl
po1y mers:
--CHGH-/
I CH,CH,
EXPEkIMENTAL Materials N-substituted 3-carboxamidopyridinium ions were prepared from appropriate alkyl halides and nicotinamide. Mixtures of alkyl halides and nicotinamide (1:2 molar ratio) were refluxed in minimum volumes of ethanol for 24 hr. The precipitates were collected and recrystallized from methanol: N-propyl-3-carboxamidopyridinium iodide[NPr(Pyr+)CONH2X-1, mp 181°-1830C (lit.12 182°C); N-benzyl-3-carboxamidopyridinium chloride[NBzl(Pyr+)CONH2X-], mp 235O-236OC (lit.13 235'236OC); N-2,6-dichlorobenzyl-3-carboxamidopyridinium chloride [NC12Bzl(Pyr+)CONH2X-], mp 2380-240°C (lit.13 239O-242OC); N hexyl-3-carboxamidopyridinium bromide[NHx(Pyr+)CONH2X-], mp 215O-218' c.
COENZYME MODELS. IV
1131
TABLE I Preparation of Polymers
Polymer
Ratio in Feed ([lauryl bromide]/ [polymer unit])
Lauryl Group Content (mol %)
Residual Free Imidazole (mol YO)
L-9 L-29 L-41
0.10 0.30 0.40
8.8 28.9 40.9
1.8 3.9 10
Found: C, 50.51; H, 6.64; N, 9.68%. Calcd for C12H19N20Br: C, 50.19; H, 6.68; N, 9.75%.
T h e preparation of N-lauryl-3-carboxamidopyridiniumbromide "La(Pyr+)CONHzX-] was described previ0us1y.l~ 1-vinyl-2-ethylimidazole was kindly provided by Toho Rayon Company and distilled prior to use, bp 108'C/35 mmHg.
Polymerization and Quaternization Polymerization of 1-vinyl-2-ethylimidazole was conducted using azobisisobutyronitrile as initiator a t 70°C in methanol solvent, and the polymer was reprecipitated from methanol and ether. 4 g of poly(1-vinyl-2-ethylimidazole)and aliquots of lauryl bromide in 50 ml of dimethylsulfoxide were heated a t l0O'C for 24 hr. 1 ml of the reaction mixture withdrawn was poured into ether, and the recovered polymer was subjected to elemental analysis after reprecipitation from methanol and ether, in order to determine the content of the lauryl group incorporated. Excess ethyl bromide was then added and heated further a t 5OoC for 72 hr. Polymers were recovered by precipitation in acetone, purified as aqueous solutions by extensive dialysis, and subjected to elemental analysis after reprecipitation. The results are summarized in Table I.
Kinetics Kinetic measurements were carried out spectrophotometrically with the aid of a Hitachi 124 spectrophotometer equipped with a thermostated cell holder (30' f 0.l'C). The reaction was initiated by mixing a polymer solution containing KCN with a pyridinium solution. T h e reaction rates were followed by observing the appearance of an absorption near 340 nm, which is characteristic of 1,d-adducts of the pyridinium ions.15 In all cases an excess of cyanide ion was present, so that the pseudo-first order behavior was observed. From the pseudo-first order rate constant hobs thus obtained was calculated the second order rate constant k f according to Eq. (2):
kf =
kobs
[CN-]
+ K-l
SHINKAI AND KUNITAKE
1132
where K denotes the equilibrium constant for Eq. (1)(i.e., K = k f / k , = [4-adduct]/[substrate][CN-I).
Equilibrium Constants ( K ) The equilibrium constants in the absence of polymers were evaluated using the method of Behme and Cordes,16 from the slope of the plots of (ODo-OD)/[CN-] against OD, where OD0 denotes the absorbance of the substrate, OD the absorbance of the reaction mixture a t infinite time. Since the reaction product of NLa(Pyr+)CONH2X- with CN- precipitated in the absence of polymers a t ca. 80%reaction, OD values for NLa(Pyr+)CONH2X- were evaluated as follows. At first the pseudo-first order rate constants k,bs was determined from theGuggenheim procedure and the corresponding half-life (t1/2) from Eq. (3).17 Subsequently, OD,/, (absorbance at the half-life) was read from the reaction chart, and OD was determined according to Eq. (4): 0.693 t1/2 = k obs OD = 2 X OD,/,
(3)
(4)
The molar absorption coefficient for each substrate can be determined according to the method of Cordes and co-workers.1° The equilibrium constants in the presence of polymers were evaluated using these t values [Eq. (511:
K=
-
OD/t 1 [CN-]([substrate] - OD/e. 1)
where 1 is the cell width.
Viscosity Measurements Viscosities of the polymers were measured a t 30°C, using a modified Ubbelhode viscometer. Linear plots of q,,/C versus C were observed for all the polymer samples in 0.02 M KBr. This rather low ionic strength is sufficient to suppress the electroviscous effect even for highly charged polymers.
RESULTS Viscosity of Polymers and Visible Spectra of Methyl Orange Table I1 gives viscosities of the quaternized polymers and absorption spectra of methyl orange in the presence of these polymers. The intrinsic viscosity of L-9 is more than ten times larger than that of L-29. The L-41 polymer gives a viscosity somewhat larger than that of L-29. These viscosity variations supposedly reflect the conformation change of the polymers, since the same starting polymer was used for quaternization.
COENZYME MODELS. IV
1133
TABLE I1 Viscosity and Absorption Spectrum of Methyl Orange
a
Polymer
Lauryl Group Content (mol %)
[VI" (dug)
h m a x b (nm)
L -9 L-29 L-41 none
8.8 28.9 40.9 -
0.496 0.037 0.048 -
463 417 433 464
30"C, 1-1
=
0.02 with KBr.
M , [L-91 = 0.091 M , [L-291 = 0.075 M ,
b 30"C, [methyl orange] = 2.67 X
[L-41] = 0.101 M . TABLE I11 Rate and Equilibrium Constants for Addition of the Cyanide Ion t o N-Substituted 3-Carboxamidopyridinium Ions in the absence of Polymera Substrate NPr(Pyr+)CONH,XNHx(Pyr+)CONH,XNLa(Pyr+ )CONH,XNBzl(Pyr+)CONH,XNCI,Bzl(Pyr+)CONH,X-
€,axb
7000 6880 6750 6630 6700
k f (M-I sec-' )
K (M-I)
0.0217 0.0223 0.0800 0.0895 0.146
0.11 0.15 25 1.99 4.36
lo-'
a 30°C, 1-1 = 0.1 with KCl, [KOH] = M , [KCN] = 0.02-0.10 M . b Molar absorption coefficient of 1,4-adducts.
The aqueous solution of L-9 hardly affected the visible spectrum of methyl orange, whereas ,A, of methyl orange appreciably shifted to shorter wavelengths in the presence of L-29 and L-41. The largest hypsochromic shift was seen for L-29, consistent with the viscosity data.
Kinetic Parameters in the Absence of the Polymer The results of kinetic measurements in the absence of polymers are recorded in Table 111. The reaction temperature in this study is higher by 5°C than that employed by Lindquist and Corded5 and k f values for NPr(Pyr+)CONHZX- and NBzl(Pyr+)CONH2X- are a little larger and the K values a little smaller, compared with their results. The reaction parameters for NHx(Pyr+)CONH2X- are almost identical with those of NPr(Pyr+)CONH2X-. The rate constants k f of the cyanide addition toward NLa(Pyr+)CONH2X- is greater by fourfold and the corresponding K value by 230-fold than those of NPr(Pyr+)CONH2X-.
Kinetic Studies in the Presence of Polymer Micelles In Figures 1 and 2, the rate and equilibrium constants for the addition of the cyanide ion to NLa(Pyr+)CONH2X- a t 3O0C and pH 11 are shown
SHINKAI AND KUNITAKE
1134
as a function of the concentration of polymers. Typical reaction parameters are collected in Table IV. Both reaction parameters, kf and K , are remarkably increased with increasing concentrations of L-29 and L-41; and L-29 showed larger acceleration effects for both parameters. For example, these constants for NLa(Pyr+)CONH2X- in the presence of L-29 are enhanced by factors of 23 for k f and 34 for K compared with those in the absence of the polymers. If NPr(Pyr+)CONHzX- is regarded as a typical nonmicellar substrate and used as reference,'O L-29 increases these constan by 84-fold and 7800-fold7respectively. Interestingly, the addition reaction is completely inhibited when the unit concentration of the polymers is much larger than [CN-] (Figs. 1 and 2). The inhibitory phenomenon will be described later in detail. The increases in rate and equilibrium constants observed at the optimal condition in the presence of L-29 are comparable to those in conventional cationic micellar systems.1°
2
I
I
0.10 0.08
L-Ll
/ J
0.06
0.04 0,02
0
-,.
I
I
0.02
0.04
(PNYVER)
I
*
I
0
0.08
0.06 M
Fig. 1. Effect of polymers on the k f of NLa(Pyr+)CONHzX- = CN- reaction: "LaM. (Pyr+)CONHzX-] = 5.00 X M ; [KCN] = 0.01 M ;[KOH] = 1.0 X
-"""I 800
30
-
20
1 -
-. 10
, &
0
0.02
0.04
I
0.08 (PIILYMEP)
0
1:
Fig. 2. Effect of polymers on the K of NLa(Pyr+)CONHzX- = CN- reaction. The reaction conditions employed are the same as in Fig. 1.
COENZYME MODELS. IV
1135
T A B L E IV R a t e a n d Equilibrium Constants for NLa(Pyr+)CONH,Xin t h e Presence of Polymera
Polvmer x l o 2M k f M-' sec-' KM-' __ L -9 1.81 0.0106 5.4 L-29 6.00 1.82 862 L-41 8.08 1.36 316 - ~ _ _ _ __ __ a 30"C, [ K C N ] = 0.01 M , [ K O H ] = 1.0 x low3M . N o inorganic salt was a d d e d e x c e p t KCN a n d K O H . ~
In contrast, the presence of L-9 decelerated the addition reaction (Figs. 1 and a), and the decrease was similar for kf and K . When NBzl(Pyr+)CONH2X- was used as the substrate, the forward reaction (kfterm) was decelerated by both L-29 and L-9 (Fig. 3a). This is in marked contrast to the above-mentioned data obtained with NLa-
?-I
oa14
L x - 0.12
?-I
0.10
+u L-29
O
O
0.02
0.04
(POLYMER]
M
(a)
3.k :
0
0
0.02 [POLYMER]
0.04 M
(b) Fig. 3. (a) Effect of polymers on the h, of NBzl(Pyr+)CONHZX- = CN- reaction. (b) Effect of polymers on the K of NBzl(Pyr+)CONHZX--CN- reaction. [NBzl(Pyr+) CONHzX-] = 5.00 X M ; [KCN] = 0.01 M ; [KOH] = 1.0 X M.
1136
SHINKAI A N D KUNITAKE
(Pyr+)CONH2X- as substrate. This different behavior observed for the same polymer, L-29, would be caused by the unfavorable partitioning of NBzl(Pyr+)CONH2X- in the polymer phase compared with NLa(Pyr+)CONH2X-. The association constants K increased gradually with the increase of the L-29 concentration, in spite of the decrease in the forward reaction rate (Fig. 3b). Therefore, the rate of the reverse reaction k, is much more depressed by the L-29 polymer than that of the forward reaction.
Effect of Ionic Strength In the conventional micellar catalysis, the efficiency is generally depressed by the increase in salt concentrations. The largest decrease in the addition reaction of the cyanide ion was reported to be about sixfold.1° Figure 4 shows the effect of the KBr concentration on the rate and equilibrium constants for NLa(Pyr+)CONH2X- substrate in the presence of 0.015 M L-29. As expected, additions of KBr decreased both the rates and the association constants, with the largest rate decrease about threefold.
Inhibition of Addition Reaction by Polymer Micelles As shown in Figure 1, the cyanide addition to NLa(Pyr+)CONH2X- was completely inhibited by polymer micelles under the condition, [polymer] >> [CN-1. The pseudo-first order rate constants k,bs were measured as a function of KCN concentration, while maintaining the concentration of L-41 constant (Fig. 5). The absorption increase based on the 4-adduct formation was undetectable a t KCN concentrations below 0.012 and 0.003
- 200 4
SI
- 100 s
Imrl
M
Fig. 4. Effect of ionic strength on the k f and K of NLa(Pyr+)CONH*X- = CN- reaction M; [KCN] = 0.01 in the presence of the L-29 polymer: [NLa(Pyr+)CONHzX-] = 5.0 X M ; [L-291 = 0.015 M. M ;[KOH] = 1.0 X
COENZYME MODELS. IV
1137
M for the L-41 concentration of 0.101 and 0.0202 M , respectively. The absorbance near 340 nm increased rapidly a t higher KCN concentrations, and then reached saturation with further increases. The critical inhibition for the ratios of [polymer]/[KCN] are 7-8 for L-41 and ca. 6 for L-29. The absorption of the 4-adduct of NLa(Pyr+)CONH2X- has disappeared in the presence of 0.05 M L-9. However, this polymer only depressed the rate and equilibrium constants progressively with its increasing concentration, so that it is difficult to conclude whether a similar, critical inhibition is involved in this polyelectrolyte-type polymer.
DISCUSSION Selection of Polymer The polymer micelle can be usually prepared by quaternization of polymeric Schiff bases such as polyvinylpyridine.8J8 Unfortunately, these quaternized polymers may undergo addition of CN- and other reactions, which are characteristic of alkylpyridinium salts. Winters and co-workerslg found that N-laurylpyridinium bromide forms the laurylviologen cation radical with aqueous sodium cyanide a t room temperature. N-ethylpyridinium bromide is inert under the same condition,lg so that this radical formation should be possible only in a micellar system. In a separate study, we found that the polymer micelle of poly( 2-vinylpyridine) (laurylated units, 30 mol%) generated the weak absorption of the viologen cation radical
0.03
0.02 -l
u v) w m v)
s
0.01
0 0
0.01
0.02 (KCN)
0,03
M
Fig. 5. Inhibition of NLa(Pyr+)CONHZX- = CN- reaction by L-41 polymer: "La( 0 )[L-411 = 0.101 M ; (A)[L-41] (Pyr+)CONHzX-] = 5.0 X 10-4M; [KOH] = 1.0 X lo-"; = 0.0202 M .
1138
SHINKAI AND KUNITAKE
a t near 600 nm under anaerobic conditions (unpublished results). To obviate these complexities,we employed polymeric l-vinyl-2-ethylimdazole, the polymer micelle of which was found to be inert in aqueous sodium cyanide solutions.
Conformation of the Polymers The conformational characteristics of the polymers employed may be assessed from viscosities of the polymers and absorption spectra of methyl orange in the presence of the polymer. The intrinsic viscosity of the L-9 polymer was much larger than that of the L-29 and L-41 polymers. Presumably, the latter polymers possess globular, compact conformations, while the L-9 polymer assumes an expanded conformation (a typical polyelectrolyte). Strauss and c o - ~ o r k e r s previously ~-~ found for quaternized polyvinylpyridines that drastic changes of physical properties (viscosity decrease, solubilization of hydrocarbons) occurred at ca. 10-13 mol% of the lauryl group content. They proposed that the polymer formed intramolecular micelles at high lauryl group contents. Thus, it is concluded that the L-29 and L-41 polymers formed polymer micelles. This was also confirmed by absorption spectra of methyl orange. Visible spectra of methyl orange are frequently used to detect the hydrophobic region present in aqueous media, since the apolar environment elicits the hypsochromic shift of the ,A, value near 460 nm.20’21 That appreciable hypsochromic shifts were observed in the presence of L-29 and L-41 polymers suggests that these polymers possess hydrophobic regions. The expanded conformation of the L-9 polymer cannot provide a hyof methyl orange is not affected by this drophobic binding site; thus ,A,, polymer. According to Strauss and Gershfeld? the incorporation of long aliphatic chains into polymers results in two opposing factors affecting the polymer conformation. They are 1)hydrophobicity, which holds polymer molecules in compact conformations and 2) the chain stiffness, which causes expansion. At intermediate lauryl contents, the former factor overcomes the latter and gives rise to compact conformations, whereas the rodlike conformation is favored in highly laurylated polymers. The possibility that increased contents of the lauryl group facilitate intermolecular aggregation must be also taken into account. The transition occurs at the lauryl content between 29 and 38 mol% for polyvinylpyridine derivative^.^ Consistent with this proposition is the fact that the L-29(29%laurylated) polymer possesses more compact conformations and greater hydrophobicity than L-41(41%laurylated), as inferred from the data of Table 11.
Polymer Micellar Effects
It has been established by Cordes and co-workers1°that rate and equilibrium constants for the addition of cyanide ion to 3-carboxamidopyri-
COENZYME MODELS. IV
1139
dinium ions with long alkyl chains are pronouncedly increased in dilute aqueous solutions of cationic surfactants. For example, these constants for the cyanide addition to NLa(Pyr+)CONH2X- in the presence of 0.02 M hexadecyltrimethylammonium bromide a t 25°C are kf = 1.1-1.5 M-l sec-' and K = 600-1000 M-'. Typical nonmicellar values are kf = 0.014 M-' sec-l and K = 0.185 M-l for NPr(Pyrt)CONH2X-. On the other hand, Okubo and Isell reported that rate and equilibrium constants of a similar reaction system are decelerated by polyelectrolytes in the same proportion. This indicates that the reverse reaction (h, term) is not influenced by polyelectrolytes. Therefore, this reaction would be one of the most sensitive systems to study the transition between the micellar and polyelectrolyte-type states of the quarternized polymer. The hf and K values for NLa(Pyr+)CONH2X- were markedly increased in the presence of L-29 and L-41 polymers. The enhancements are almost comparable in magnitude to those observed in micellar systems.1° On the other hand, L-9 decelerated these parameters for NLa(Pyrf)CONH2Xin the same proportion (Table IV). Therefore, L-29 and L-41 polymers behave as typical micelles, and the L-9 polymer as a typical polyelectrolyte. T h e L-29 polymer showed an influence greater than t h a t of L-41. These results are in complete accord with the conclusion derived from the conformational study. Contrary to the data obtained for NLa(Pyr+)CONH2X-, the L-29 polymer showed a polyelectrolyte-type influence on the cyanide addition t o NBzl(Pyr+)CONH2X-. T h e NBzl(Pyr+)CONH2X- molecule is so soluble in aqueous media that it would be hardly partitioned in the micellar phase of L-29. Thus, this reaction, which mainly occurs in the bulk phase, would be influenced by the polyelectrolytelike portion of L-29, and the forward reaction rate would become depressed (Fig. 3). In contrast, the association constants gradually increased with the increase in the concentration of L-29, indicating the gradual decrease of rates of the reverse reaction (k, term). A simple polyelectrolyte should depress rates kf and association constants K in the same proportion, since the reverse reaction is unaffected." It is likely t h a t the 4-adduct of NBzl(Pyr+)CONHzX-, being neutral and less water soluble, is solubilized and stabilized in the hydrophobic region of L-29, resulting in the depressed rate of the reverse reaction. Evidently, the effect of L-29 on the reaction of NBzl(Pyr+)CONH2X- is polyelectrolytelike for the forward reaction and micellelike for the reverse reaction. Thus, the chemical influence of the polymer depends not only on the polymer structure, but also on the hydrophobicity of the substrate employed. The cyanide addition to NLa(Pyr+)CONH2X- was thoroughly inhibited by polymer micelles under the condition [polymer] > ca. 7[CN-]. This phenomenon is not found with conventional micelles so that this is probably related to the inherent property of polymer micelles. T h e possible explanation for this phenomenon is that the polymer micelle may constitute a number of small, local micelles. If so, the dilution effect would be more clearly observed for the polymer micelle than for the conventional micelle.
SHINKAI AND KUNITAKE
1140
It is interesting that the compact L-29 polymer inhibits the reaction more efficiently than L-41. Abnormal Affinity of N-lauryl-3-carboxamidopyridinium Bromide In the absence of polymer micelles, NLa(Pyr+)CONHzX- showed an unusually high affinity toward the cyanide ion. The rate constant kj is greater by only fourfold compared with that of NPr(Pyr+)CONHzX-, and, in contrast, the association constant increased by 230-fold; that is, the abnormality of NLa(Pyr+)CONHzX- is mainly associated with depression of the reverse reaction. Since the concentration of NLa(Pyr+)CONHZXM ) is definitely below the cmc of the analogous surfactants (ca. (5.0 X 1.5 X M),l the abnormally high reactivity of this substrate cannot be explained by the micelle formation. This peculiar finding is explicable by stabilization of the product, which is caused by the hydrophobic nature of the long alkyl substituent. It is already known that the nicotinamide mononucleotide and both the (Y and /3 isomers of NAD coenzyme have an abnormally great affinity for cyanide ion when compared with their model compound^.'^ This trend is particularly pronounced for the /3 isomer. Lindquist and Cordes15 pointed out that this observation accorded with the fact that the /3 isomers of NAD and NADH could exist in folded conformations with the adenine m~iety.~ A~ good * ~linear ~ relationship that is approximated by Eq. (6) is recognized between log K and log kj determined by Lindquist and Corded5 a t 25OC for the cyanide addition to six N-substituted 3-carboxamidopyridinium ions (Fig. 6). Our data for NPr(Pyr+)CONHzX-, NHx0
22-
"3
0 0 4
-1
-2
-1
0
2
1
log K data cited horn Ref. 15, I-CY-NAD, 2-nicotinFig. 6. Log K - log k/ relationship: (0) amide mononucleotide, 3-0-NAD; ( 0 ) data from the present study, 4-NPr(Pyr+) CONHzX-, 5--NHx(Pyr+)CONHzX-, 6-NBzl(Pyr+)CONHzX-, 7-NClzBzl(Pyr+)CONHzX-, 8--NLa(Pyr+)CONHzX-.
COENZYME MODELS. IV
1141
(Pyr+)CONH2X-, NBzl(Pyr+)CONHzX-, and NC12Bzl(Pyr+)CONH2Xa t 30°C are also located close to this linear relationship [Eq. (7)]: log hj = 0.55 log K - 1.45, r = 0.999
(6)
log hf = 0.53 log K - 1.19, r = 0.998
(7)
Interestingly, plots for the above-mentioned biological cofactors deviate from the linearity to the lower area in the log K - log hf map, due to the much too large association constants. A plot for NLa(Pyr+)CONH2Xalso deviates downward from the linearity. These results mean that the abnormally high affinity of the cyanide ion toward biological cofactors and NLa(Pyr+)CONHzX- is commonly caused by the increased stability of the CN adducts. We consider that the 4-adduct of NLa(Pyr+)CONHzXis stabilized by the apolar lauryl group and that of 0-NAD by the stacking with the adenine ring. The authors thank Miss Reiko Ando for her capable technical assistance
References 1. Fendler, E. J. & Fender, J. H. (1970) Aduan. Phys. Org. Chem. 8,271-406. 2. Cordes, E. H. & Dunlap, R. B. (1969) Acc. Chem. Res. 2,329-337. 3. Ise, N. (1973) Reaction on Polymers, ed. by Mooe, J. A,, Ed., Reidel, The Netherlands, p. 27. 4. Strauss, U. P. & Gershfeld, N. L. (1954) J . Phys. Chem. 58,747-753. 5. Strauss, U. P., Gershfeld, N. L. & Crook, E. H. (1956) J . Phys. Chem. 60,577-584. 6. Strauss, U. P. & Williams, B. L. (1961) J . Phys. Chem. 65, 1390-1395. 7. Inoue, H. (1964) Kolloid-Z. Z . Polym. 196, 1-7. 8. Rodulfo, T., Hamilton, J. A. & Cordes, E. H. (1974) J . Org. Chem. 39, 2281-2284. 9. Okubo, T. & Ise, N. (1973) J. Org. Chem. 38,3120-3122. 10. Baumrucker, J., Calzadilla, M., Centeno, M., Lehrmann, G., Urdaneta, M., Lindquist, P., Dunham, D., Price, M., Sears, B. & Cordes, E. H. (1972) J . Amer. Chem. Soc. 94,8164-8172. 11. Okubo, T . & Ise, N. (1973) J . Amer. Chem. Soc. 95,4031-4036. 12. Kosower, E. M. & Bauer, S. W. (1960) J . Amer. Chem. SOC.82,2191-2194. 13. Kim, C. S. & Chaykin, S. (1968) Biochemistry 7,2339-2350. Jap. 48,1914-1917. 14. Shinkai, S., Ando, R. & Kunitake, T. (1975) Bull. Chem. SOC. 15. Lindquist, R. N. & Cordes, E. H. (1968) J . Amer. Chem. SOC.90,1269-1274. 16. Behme, M. T. A. & Cordes, E. H. (1965) Riochim. Biophys. Acta 108,312-313. 17. Jencks, W. P. (1969) Catalysis in Chemistry and Enzymology, McGraw-Hill, New York, N.Y., p 558. 18. Kunitake, T., Shinkai, S. & Hirotsu, S.(1975) J . Polym. Sci., Polym. Lett. Ed. 13, 377-381. 19. Winters, L. J., Borror, A. L. & Smith, N. (1967) Tetrahedron Lett. 2313-2315. 20. Klotz, I. M. & Shikama, K. (1968) Arch. Biochem. Biophys. 123,551-557. 21. Takagishi, T., Nakata, Y. & Kuroki, N. (1974) J . Polym. Sci., Polym. Chem. Ed. 12, 807-816. 22. Weber, G. (1957) Nature 180,1409. 23. Shifrin, S. & Kaplan, N. 0. (1959) Nature 183, 1529.
Received September 9,1975 Accepted December 8,1975
