IONIZATION Yu.
I.
CONSTANTS Khurgin
and
OF I.
N-ACYL
DERIVATIVES
OF GLYCINE
V. V i k h a
UDC541.124.7+547.466o22
Automation of the p r o c e s s of peptide synthesis on a r a t h e r large scale is based not only on the use of solid-phase p o l y m e r c a r r i e r s , but also on the i m p r o v e m e n t of c l a s s i c a l methods of condensation and p r o t e c tion of the t e r m i n a l g r o u p s . T h e r e f o r e , one of the important p r o b l e m s of peptide c h e m i s t r y is the quantitative study of the r e a c t i o n s of N - a c y l a t e d amino acids and t h e i r derivatives - n e c e s s a r y components of peptide synthesis - and the establishment of the relationship of their s t r u c t u r e to their r e a c t i v i t y . The relative r e a c t i v i t y in the s e r i e s of N - a c y l a m i n o acids XCONHCHCOOH should be d e t e r m i n e d
i
R
basically by s t r u c t u r a l (electronic) f a c t o r s of the side group of the amino acid residue R and the substituent X in the acyl group. We had studied e a r l i e r the dependence of the ionization constants of carbobenzoxyamino acids X = C6I~CH2O - upon the s t r u c t u r e of the side group of the amino acid residue R [1] and the kinetics of a m i n o l y s i s of the c o r r e s p o n d i n g p-nitrophenyl e s t e r s [2]. The p u r p o s e of this work was to study the influence of the substituent X in the acyl group on the i o n i z a tion constants of N - a c y l - ( ~ - a m i n o acids. Usually for the ionization constants of carboxylic acids with one v a r i a b l e substituent, fulfillment of a c o r r e l a t i o n dependence of the type of the H a m m e t t - T a f t equation is obs e r v e d [3, 4]: Ig k //co = Z_~_p~ ~ (i) where p* is the r e a c t i o n constant; or* is the induction constant of the variable substituent; and Z_M_ is the t r a n s m i s s i o n constant, which c o n s i d e r s the weakening of the inductive effect of the substituent when it is t r a n s m i t t e d through the bridge group - - M - . F o r carboxylic acids of the aliphatic s e r i e s , the r e a c t i o n constant of ionization p* in aqueous medium, a c c o r d i n g to the data of v a r i o u s authors [3, 5], fluctuates in c o m p a r a t i v e l y n a r r o w limits f r o m 1.7 to 1.8. A value p* = 1.6 has been obtained for carbobenzoxyamino acids in 20% aqueous dioxane m e d i u m [1, 2]. The c h e m i c a l nature of the substituent in the acyl group of N - a c y l a m i n o acids influences the values of their ionization constants [6]. However, the available data are insufficient for a quantitative evaluation of the inductive effect. Various authors have used nonstandard conditions for the m e a s u r e m e n t of pKa, while the n e c e s s a r y values of the inductive constants of groups of the type of X C O - or XCONH- are unavailable for the analysis of these data with the aid of the c o r r e l a t i o n Eq. (1) [7]~ T h e r e f o r e , thus far there has been no evaluation of the bridge effect of the amide group - C O N H - . The c o m p a r a t i v e l y high t r a n s m i s s i o n of the inductive effect through the nitro gen atom in substituted N-phenylglycines is well known [8]. In the r e c e n t work [9] the bridge effect of the carbonyl carbon atom Z_CO_ = 0.5 was determined. To evaluate the bridge effect of the amide group, in this work we m e a s u r e d the values of the ionization constants of a n u m b e r of acylated d e r i v a t i v e s of glycine (R = H) and analyzed the data obtained according to Eq. (1). In addition, we m e a s u r e d the values of pK a for d e r i v a t i v e s of glycine with the substituents usually r e q u i r e d in peptide synthesis. Since we had e a r l i e r d e m o n s t r a t e d a c o r r e l a t i o n of the ionization constants of c a r b o b e n z oxyamino acids and the rate constants of aminolysis of the derivatives with an active carboxyl group [2], these data can be used to e s t i m a t e the expected reaction rate constants of peptide bond formation. The fulfillment of the c o r r e l a t i o n E q . (1) for earboxylic acids is a reliably established fact; in a n u m b e r of c a s e s the ionization reaction is used as a standard reaction to determine the values of the inductive constants of substituents [5]. F o r an analysis of the data obtained, we subsequently used the scale of inductive constants ~I, obtained on the basis of a separation of the inductive and r e s o n a n c e effects in u n s a t u r a t e d s y s t e m s [3]. The inductive constants used were obtained with the aid of the well-tmown e x p r e s s i o n o~ ~- 6.2~i
{2)
N. D. Zelinskii Institute of Organic C h e m i s t r y , A c a d e m y of Sciences of the USSR. T r a n s l a t e d f r o m I z v e s t i y a Akademii Nauk SSSR, Seriya Khimicheskaya, No. 6, pp. 1245-1250, June, 1968. Original article submitted O c t o b e r 4, 1967.
1179
TABLE i. Ionization Constants of N-Acylglycines XCONHCH2COOH and Induction Constants of Substituents ~XCONH and a X ,
,
The m e a s u r e d values of the ionization constants of N - a c y l derivatives of glycines are p r e sented in Table 1.
,,,
N-Acy1 derivatives df glycine N-acetylglycine N- Formylglycine N-Chloroacetylglycine N-Trifluoroaeetylglycine GlycvlgIvcine lg-Afanylgly.eine N-Benzoy]glycine
X
PKa
~CONtI
CHa H CH~CI CF,,
3,90 3,78 3,65 3,47
1,80 1,96 2A6 2,40
+ NH~CII2-+ NH3CH~CH2 C,~l~
3,36 3,63 3,98
2,5 2;2
1,72
~
-0,5O
o
1,55
2,60 1,5O
0,56
0,60
The data of Table 1 p e r m i t an estimation of the induction constants for a n u m b e r of a c y l amino groups, for which it is n e c e s s a r y to know the values of the ionization constants of the ionization of carboxylic acids, the constants of t r a n s m i s s i o n through the methylene group Z_CI:I2_, and the ionization constant of the carboxylic acid with a standard substituent (PKa) 0.
In aqueous medium, the reaction constant of the ionization of carboxylic acids is equal to + p* = 1.75 [5]. On account of the poor solubility ~.CM z+ o of N - a c y l a m i n o acids in water, aqueous organic NH,CH,CHr.I -'~ ~ ~ CF~ medium (20% aqueous dioxane) was used to m e a sure their J ionization constants. The r e a c t i o n ] C6H~ J with the inductive 9 constant for the usual aliphatic carboxylic acid constant crX of the proved equal to 2.05 under these conditions, , , substituents in the 5 36• acyl group X. which is in good a g r e e m e n t with the value p* = 2.0 [7]. The t r a n s m i s s i o n constant of the m e t h y l ene group Z_CH 2_ = 0.36 [10]. The ionization constant of "standard" acetic acid in 20% aqueous dioxane is cited in Table 1 were calculated according to a f o r m u l a equal to (PKa) 0 = 5.25 [1]. The values of ~XCONH * obtained f r o m Eq. (2): F~a 3~ i L
Fig. 1. Correlation of the ionization constants of N - a c y l d e rivatives ofglycine
~. _
(pKa)0-- pKa = t.38(5.25 -- pKa) Z-CH~-P*
(3)
9 The value crCH3CON H = 1.83 obtained according to Eq. (3) is in good a g r e e m e n t with the table data - 1.74 [10, 11]. As can be seen f r o m Table 1, the ionization constants of n - a c y l a t e d derivatives o f glycine depend app r e c i a b l y upon the chemical nature of the substituent X, which indicates t r a n s m i s s i o n of the inductive effect of the substituent through a chain of t h r e e atoms C - N - C and is a qualitative sign of c o m p a r a t i v e l y high t r a n s m i s s i o n of the inductive effect through the amide bond. All the acylamino groups studied are e x t r e m e l y strong electronegative substituents: their inductive constants are l a r g e positive values a~CON H > 1.5, while aliphatic amino groups do not have pronounced electronegative p r o p e r t i e s (usually (r~H ~ 0.6 [3, 10]). The strong shift of the f r e e pair of e l e c t r o n s in the amide bond leads to a d e c r e a s e in the f o r m a l negative charge on the nitrogen atom, as a result of which the e l e c t r o n e g a t i v i t y of the acylamino group i n c r e a s e s greatly. Substituents in the acyl group X- a r e of second a r y importance, inducing a s u p p l e m e n t a r y shift of the free pair of e l e c t r o n s of the nitrogen atom, and this has its own effect upon the individual values of the c o r r e s p o n d i n g inductive constants. Let us c o n s i d e r the dependence of t h e v a l u e s of PKa of N - a c y l g l y c i n e upon the p r o p e r t i e s of the substituent X. F r o m the data of Table 1 it is evident that with i n c r e a s i n g electronegativity of X, the values of the ionization constants i n c r e a s e . F o r a quantitative evaluation of the inductive effect of X, a graphical dependence of the values of pK a upon (r~ for the substituents~X, in which the carbonyl atom of the amide bond is bonded to a carbon atom, is c o n s t r u c t e d in Fig. 1. This dependence is not simple; the manifestation of the inductive effect is influenced by the chemical nature of the substituents X, which can probably i n t e r a c t in v a r i o u s ways with the amide group. Thus, N-benzoylglycine has a reduced ionization constant, while substituents with charged groups give r e l a t i v e l y higher values in c o m p a r i s o n with substituents in which the amide group is bonded to the substituent X through a t e t r a h e d r a l carbon atom. In the latter c a s e , the dependence is quantitatively e x p r e s s e d as pKa = 3,8i ___0.05 -- a x "(0.13 + 0 . 0 i 5 )
(4)
A c o m p a r i s o n of the coefficient at a ~ in Eq. (4) with the ionization constant of substituted acetic acids (XCH2COOH), p* = 0.735, shows that the amide group weakens the inductive effect by no l e s s than fivefold. The value Z_CONH_ = 0.18 was obtained, which is half the t r a n s m i s s i o n of the methylene bridge. Assuming
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TABLE 2. IonizationConstants of N-Aeylglycines XCONHCH2COOH PKa
Acyl derivatives of glycine
o*XCONH ~*X
m
t;H~O N-Carbomethoxyglyc[ne N-Carbo-tert-butoxy(.CH~,)3C0 glycine C~HnO N-Carbocyclohexyloxyglycine N-Carbobenzoxyglyeine CsHsCH20 N- Phthalylglycine N-Tosylglycine p-C/-I~CsH4S02 TABLE 3. Melting Points of N-Acyl Derivatives of Glycine Melting point, N-Acyl derivatives of g l y c i n e
N-Aeetylglycine N-Formylglycine N-Chtoroacetylglycine N-Trifluoroaeerylglyeine N-Benzoylglycine N-Carbomethoxygtycine N-Carbo-tert-butoxyglycine N-Carbocyelohexyloxycarbonylglyeine N- Carbobenzoxyglycine N- Phthalylglyeine N-Tosylg-!ycine
~
found
in lit. data 20~
I
1,88
4,05 3,60 3,87
1,62
1,45
1,6b 1,34
fulfillment of the additivity rule in the t r a n s m i s s i o n of the inductive effect through an amide group [4], i.e., Z-co.~H~ = Z - c o - Z _ ~ _
(5)
referLit.
I ence
2(~6 153 100 121 188 ~5 89
:20--121 187,5 95--96 85,0.8~
98
97--99
[18]
120 194 150
120 192--19~ 149--15(
[i9]
53 54 IlO0'
3,86 4,03 4,26
[12] [i3] [14]
[151 [16] [17] [18]
[20]
we can estimate the t r a n s m i s s i o n of the nitrogen atom of the amide bond, if we use the value Z_CO_ = 0.5, obtained by T a l ' v i k [9]. F r o m this, f r o m Eq. (5), we obtain Z_NH_ = 0.36. Unfortunately, there a r e no other independent data for the quantitative estimation of Z_NH_ in aliphatic s y s t e m s , and this prevents our verifying the additivity of the t r a n s m i s s i o n of the inductive effect of the amide group, and consequently, the question of the principles of application of Eq. (5) still r e m a i n s open.
[21]
Another estimation of the t r a n s m i s s i o n of the nitrogen atom can be made f r o m data on the values of the ionization constants of substituted N-phenylglycines, obtained in [8]: Z_NH_ = 0.7, which is substantially higher than the constant that we calculated. This d i s c r e p a n c y may be due to the p r e s e n c e of a supplementary m e c h a n i s m of interaction of the free p a i r of e l e c t r o n s of the nitrogen atom with the a r o m a t i c ring. P r o b a bly for the s a m e r e a s o n the value of pK a of N-benzoylglycine does not fit into the c o r r e l a t i o n between the values of the ionization constants of N - a c y l g l y c i n e s and the inductive constants o-~. An analogous deviation f r o m the c o r r e l a t i o n straight line is also o b s e r v e d for carbomethoxyglycine, in which the substituent X is bonded to the amide group through an oxygen atom (Table 2). In this c a s e , the p r e s e n c e of a supplementary m e c h a n i s m of interaction of the substituent with the amide bond is e x p r e s s e d in the fact that the effective t r a n s m i s s i o n of the inductive effect for substituents of this type is reduced. This indicates that the effective value of the inductive constant of the acylamino group X C O N H - probably is d e t e r m i n e d chiefly by the state of the outer e l e c t r o n s of the nitrogen atom (prim a r i l y p e r h a p s by its free pair of e l e c t r o n s ) , while the double-bonded nature of the bridge amide group does not have this significance. Among the compounds e n u m e r a t e d in Table 1, there are two d e r i v a t i v e s of glycine with substituents having a net positive charge - glycylglycine and fi-alanylglycine. F r o m Fig. 1 it is evident that the c o r r e sponding values of pK a deviate f r o m the c o r r e l a t i o n . However, it should be mentioned that the deviations f r o m the c o r r e l a t i o n straight line for fl-alanylglycine a r e almost three t i m e s s m a l l e r than for g l y c y l g l y cine. This a g r e e s with the value of the constant of t r a n s m i s s i o n through the methylene group Z CH = 0.36. Consequently, the o b s e r v e d weakening of the inductive effect does not r e q u i r e the a s s u m p t i o n of t h ~ e x i s tence of s u p p l e m e n t a r y m e c h a n i s m s of interaction of the substituent with the r e a c t i o n c e n t e r in acylated amino acids, other than t r a n s m i s s i o n of the inductive effect of the aeylamino group through the a - c a r b o n atom of the amino acid r e s i d u e . The deviation f r o m the c o r r e l a t i o n straight line for aeyl derivatives of glycine with c h a r g e d substituents in the acyl group is an example of inconstancy of the inductive effect of the a m m o n i u m group. P r o b a b l y in this c a s e , together with the t r a n s m i s s i o n of the inductive effect along the chain of valence bonds, there is also a strong effect of the field of the charged group. The values of the inductive constants of the charged groups used, taking both these effects into consideration, were obtained on the basis of data of a m e a s u r e ment of the constants in aqueous m e d i u m [11]. However, the d e c r e a s e in the d i e l e c t r i c p e r m e a b i l i t y in aqueous dioxane medium m a y have an influence upon the field effects, intensifying it, while the t r a n s m i s s i o n of the inductive effect along the chain of valence bonds should not be so sensitive to the change in the medium.
1181
Table 2 p r e s e n t s the values of the ionization constants of derivatives of glycine in which the amino groups are r e p l a c e d by N - p r o t e c t i v e groups, typical for peptide synthesis, including four with ureide p r o tection of the type X = R ' O - . In this c a s e , t r a n s m i s s i o n of the inductive effect of the substituent X = R ' O is complicated by the interaction of the oxygen atom with the amide bond. Unfortunately, the only value of . the inductive constant is that for the methoxy group a R ' O - = 1.45 [10], and this does not p e r m i t the use of Eq. (1). Nor are there any inductive constants for substituents of the type R ' O C O - . We should mention that the values of pK a of acetylglycine and carbomethoxyglycine differ v e r y little (see Table 1). This m a y be int e r p r e t e d with the aid of the assumption of v e r y high t r a n s m i s s i o n of the oxygen bridge. However, it is m o r e probable to a s s u m e that this coincidence is m o r e likely random, since in this case there is a supplementary m e c h a n i s m of interaction (conjugation) of the substituent with the amide group. This is confirmed both by the large difference of the inductive constants of the methyl and methoxyl groups and by deviation of pK a of carbomethoxyglycine f r o m the basic c o r r e l a t i o n straight line in Fig. 1. The available data do not p e r m i t a c o r r e l a t i o n of the values of pK a of glyeine with protective groups of the ureide type with the inductive con, stants of R g r o u p s . The effective values of the inductive constants ~XCONH in this case retain their high v a l u e s , although they a r e somewhat lower than the other inductive constants. EXPERIMENTAL N - A c y l a m i n o acids were produced according to the general methods of synthesis d e s c r i b e d in the lite r a t u r e , with c e r t a i n modifications in the p r o c e s s of synthesis, isolation, and purification of the product. The absence of free amino acids in the p r e p a r a t i o n s was verified by the method of p a p e r c h r o m a t o g r a p h y . All the substances w e r e c h a r a c t e r i z e d by their melting points, a determination of the equivalentby the method of potentiometric titration, and by elemental analysis. The synthesized acylamino acids and their melting points are p r e s e n t e d in Table 3. Glycylglycine and fi-alanylglycine were p r e p a r a t i o n s of the "Reanal" Company (Hungarian P e o p l e ' s Republic), purified by two r e c r y s t a l l i z a t i o n s f r o m water. The absence of free amino acids was d e m o n strated chromatographically. The ionization constants were d e t e r m i n e d by potentiometrie titration on the T T T - l c "Radiometer" a u t o t i t r a t o r with a K-401 calomel electrode and a G-202c glass e l e c t r o d e . F o r the titration we used 10 ml of 3.2 9 1 0 - 2 M solution of the N - a e y l a m i n o acid, containing 20% dioxane. The ionic strength of the solution # = 0.8 was maintained by introducing the c o r r e s p o n d i n g amount of KC1. As the titrant we used 1 N KOH, containing 20% dioxane. The exact concentration of the titrant was established by titration with HC1 with exact concentration on an a u t o t i t r a t o r . The titration was conducted in a t h e r m o s t a t i c a l l y controlled cell at the t e m p e r a t u r e 25=~0.1~ in a s t r e a m of nitrogen. The value of pK a of the mixed constant was calculated according to the f o r m u l a [22]: 9 [HA]-- {H+} pKo =
pH -- ,g
:+
where the pH c o r r e s p o n d s to a definite point on the titration curve; {H+} = 10-PH; [HA] and [A-] are the s t o i c h i o m e t r i c concentrations of the nondissociated acid and its anion at a given pH. The value of PKa was calculated for no l e s s than ten pH values. The a c c u r a c y of the determination of PKa was no lower than 0.03-0.04 l o g a r i t h m i c units. The average values of pK a cited in Tables 1 and 2 for the c o r r e s p o n d i n g N - a c y l a m i n o acids and peptides w e r e obtained as a r e s u l t of t r e a t m e n t of no less than three titration c u r v e s . The d i s c r e p a n c y between individual determinations of pK a did not exceed 0.05 pHunit. CONCLUSIONS 1. The ionization constant of a n u m b e r of N - a c y l a t e d d e r i v a t i v e s of glycine with acyl substituents of different types were m e a s u r e d by the method of potentiometric titration. 2. A quantitative explanation of the inductive effect of the substituent in the acyl group was p e r f o r m e d with the aid of the Taft c o r r e l a t i o n equation. The r e a c t i o n constant of ionization p* was m e a s u r e d . 3. The constants of t r a n s m i s s i o n of the inductive effect through the amide group were estimated: an e s t i m a t e of the t r a n s m i s s i o n through the nitrogen atom Z_NH- =:0.36 was given within the f r a m e w o r k of the additive s c h e m e :
1182
4o F o r substituents bonded with an amide bond to an atom with i n c r e a s e d e l e c t r o n density, and for charged s u b s t i t u e n t s , a deviation f r o m the c o r r e l a t i o n dependence is o b s e r v e d . A discussion of the m e c h a nism of the i n t e r a c t i o n of the substituents with the r e a c t i o n c e n t e r or with the amide bond, which causes the deviations, is cited. 5. The data obtained can be used for a p r e l i m i n a r y calculation of the r e l a t i v e reaction r a t e s of amino acids with v a r i o u s p r o t e c t i v e groups in r e a c t i o n s of peptide synthesis. LITERATURE
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18~ 19. 20. 21. 22.
CITED
Yu. I. Khurgin, M. G. D m i t r i e v a , and E. V. Tripolitova, Reakts. Sposobnost' Organ. Soed., 1, No. 2, 35, T a r t u (1964). Yu. I. Khurgin and M. G. D m i t r i e v a , T e t r a h e d r o n , 21, 2305 (1965). R . W . Taft, In. Steric Effects in Organic C h e m i s t r y [Russian translation], IL (1962), p. 562. V . A . P a l ' m , Correlation Equations in Organic C h e m i s t r y [in Russian], Vol. 1, Tartu (1967), p. 7. M. Charton, J. Organ. Chem., 2__9_9,1222 (1964). C. Cohn and J. I. Edsall, The P r o t e i n s , Amino Acids, and Peptides as Dipolar Ions, Reinhold Publ., New York (1943), p. 85. The C h e m i s t ' s Handbook [in Russian], "Khimiya, "Vol. 3, M o s c o w - L e n i n g r a d (1964), p. 935. A. Bryson, N. R. Davies, and E. P. Serjeant, J. A m e r . Chem. Soc., 85, 1933 (1963). A . I . Tal'vik, Reakts. Sposobnost' Organ. Soed., 3, No. 2, 11, Tartu (1966). V . A . P a l ' m , Uspekhi Khimii, 30, 1069 (1961). A. Riche and U. F. Sendger, Modern P r o b l e m s of Physical Organic C h e m i s t r y [Russian translation], "Mir" (1967), p. 498. T. Curtius, Ber~ 17, 1665 (1884). E. F i s c h e r and O. Warburg, B e r . , 38, 3997 (1905)o E~ Rouwin, J. Organ. Chem., 18, 127, 1546 (1953). F. Weygand and E. Leising, Chem. B e r . , 87,248 (1954). T. Curtius and L. Levy, J. P r a k t . Chem., 7.~0, 89 (1954). H. Leuchs, B e r . , 3 9 , 8 5 7 (1906). F . C . McKay and N. F. Alberton, J. A m e r . Chem. Soc., 79, 4686 (1957). M~ Bergmann, L. Z e r v a s , and V. du Vigneand, Chem. B e r . , 65_, 1192 (1932). E . W . McChesney and W. K. Swarm, J. A m e r . Chem. Soc., 59, 1116 (1937). I . H . Billmaa and W. F. Hatting, J. A m e r . Chem. Soc., 70, 1473 (1948). D. Albert and E. P. Sergeant, Ionization Constants of Acids and Bases [Russian translation], *'Khimiya" (1964).
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