7258
J. Org. Chem. 1992,57,7258-7265
Synthesis of Chiral 1,lO-Phenanthroline Ligands and the Activity of Metal-Ion Complexes in the Enantioselective Hydrolysis of N-Protected Amino Acid Esters John G. J. Weijnen and Arie Koudijs Laboratory of Organic Chemistry, Wageningen Agricultural University, Dreijenplein 8, 6703 HE Wageningen, The Netherlands
Johan F. J. Engbersen* Laboratory of Organic Chemistry, Faculty of Chemical Technology, University of huente, 7500 AE Enschede, The Netherlands
P.0. Box 217,
Received June 23, 1992
The synthesis of seven new, chiral 1,lO-phenanthrolinea(1-7) containing a 2-pyrrolidinemethanol or ephedrine substitutent at the a-position is reported. The catalytic activity of Zn2+,Co2+,Cu2+,Ni2+,and Cd2+complexes of these ligands in the hydrolysis of p-nitrophenyl esters of picolinic acid (PNPP) and chiral N-protected amino acids was examined in water and micellar solution. The Zn2+complex of the chiral, asymmetrically disubstituted (S)-l-[[9-[(N-methyIdodecylamino)methyl]-l,l0-phenanthrolin-2-yl]methyl]-2-pyrrolidinemet~ol(l) exhibits the higheat activity toward PNF'P. The highest enantioselectivity in the stereoselectivehydrolysis of p-nitrophenyl N-dodecanoyl-D(L)-phenylalaninate (D(L)-CI2-Phe-PNP)is observed for 1-Co2+solubilized in Brij 35 micelles (kD,,b/kL ob = 15.3). In a mixed micellar system of 1-Zn2+with Brij 35 as the cosurfactant, hydrolysis of D-CI2-Phe-%"P predominates over that of the L-enantiomer (kDa,,b/khob = 2.41, whereas with CTABr as the cosurfactant an inversion of enantioselectivity is observed (kDa,ob/kLa,ob= 0.54).
The bidentate l,l0-phenanthroline nucleus is a strongly chelating agent for a variety of metal ions.' Therefore, it is an attractive building block for incorporation into host molecules where a ligated metal ion serves as a Lewis acid binding site and ~ a t a l y s t . ~ -Metal-ion ~ complexes of functionalized 1,lO-phenanthrolines have been used as catalysts in the oxidative cleavage of DNA" and in the enantioselective reduction of acetophenone?b*cThe complexing ability of the 1,lO-phenanthroline ring has also been beneficially used in the development of biomimetic models for metalloenzymea. In these models the metal ion is coordinated in a fixed position in the metallocleft of 2-substituted 1,lO-phenanthrolinesand is consequently in close proximity to the reaction site! These biomimetic models provide insight into the mechanism by which metalloenzymes may operate. The catalytic role of the metal ion in the active site of carboxypeptidase A (CPA) has been the subject of several This Zn2+-containingproteolytic enzyme model ~tudies.4~3 catalyzes the hydrolysis of the C-terminal amino acid residues of peptides and the hydrolfsis of corresponding esters. The function of the metal ion chelated at the active site of CPA is thought to be manifold: bringing the sub(1)Holyer, R. H.; Hubbard, C. D.; Kettle, s. F. A.; Wilkins, R. G. Inorg. Chem. 1965,4,929. (2)(a) Keipert, S.J.; Knobler, C. B.; Cram, D. J. Tetrahedron 1987, 43,4861. (b) Chandler, C.J.; Deady, L. W.; Reisa, J. A. J. Heterocycl. Chem. 1986,23,1327.(c) Newkome, G. R.; Kiefer, G. E.; Puckett, W. E.; Vreeland, T. J. Org. Chem. 1983,48, 5112. (d) Buhleier, E.; VBgtle, F. Liebigs Ann. Chem. 1977,1080. (3)(a) Sigman, D. S. Biochemistry 1990,29,9097. (b) Gladiali, S.; Ghelucci, G.; Soccolini, F. J. Organomet. Chem. 1989, 370, 285. ( c ) Gladiali, S.;Pinna, L.; Delogu, G.; De Martin, S.; Zasainovich, G.; Mestroni, G. Tetrahedron: Asymmetry 1990,1, 635. (4)(a) Engbersen, J. F. J.; Koudijs, A.; van der Plas, H. C. J. Org. Chem. 1990,55,3647.(b) Fife, T. H.; Przystas, T. J.; Squillacote, V. L. J. Am. Chem. SOC. 1979, 101, 3017. (c) Sigman, D. S.; Wahl, G. M.; Creighton, D. J. Biochemistry 1972, 11, 2236. (d) Breslow, R.;Fairweather, R.; Keana, J. J. Am. Chem. SOC.1967,89,2135. (5)(a) Groves, J. T.; Chambers, R. R. J. Am. Chem. SOC. 1984,106, 630. (b) Fife, T. H.; Przystas, T. J. J. Am. Chem. SOC.1982,104,2251. (c) Fife, T. H.; Pnystas, T. J. J. Am. Chem. SOC.1985,107,1041.(d) Suh, J. Bioorg. Chem. 1990,18,345.
strate and nucleophile together in a ternary complex, polarizing the substrate carbonyl bond, activating the nucleophile, and stabilizing the tetrahedral intermediate.6~~ Although the protein matrix in which the metal ion is embedded will be of influence on substrate specificity and orientation, the coordination geometry of the metal ion is considered to be a major factor for the catalytic activity of the en~yme.4~1~ Several of the above-mentioned metal-ion features have been investigated in artificial hydrolytic metalloenzymes. Metallomicelles have been developed in order to improve the substrate binding properties of synzymes."ll The adsorption of apolar substrata into or onto these molecular assemblies and their subsequent reaction resemble enzymatic reactions>2and large rate accelerations are observed for the hydrolysis of carboxylic,@Jland phosphoric eatersl0 in these systems. High stereoselectivitycould be attained in the hydrolysis of p-nitrophenyl esters of N-protected amino acids catalyzed by histidine containing di- and tripeptide8 in surfactant aggregat4~4.l~However, until now only a few model (6)(a) Dugas, H. Bioorganic Chemistry; Springer-Verlag: New York, 1988. (b) Breslow, R.; Schepartz, A. Chem. Lett. 1987,l. (7)Hanzlik, R. P. Inorganic Aspects of Biological and Organic Chemistry; Academic Press: New York, 1976. (8)(a) Tagaki, W.; Ogino, K. Top. Curr. Chem. 1985,128,143 and references cited therein. (b) Ogino, K.; Kashihara, N.; Fujita, T.; Ueda, T.; Isaka, T.; Tagaki, W. Chem. Lett. 1987,1303.(c) Tagaki, W.; Ogino, K.; Tanaka, 0.; Machiya, K.; Kashihara, N.; Ycehida, T.; Bull. Chem. Soc. Jpn. 1991,64, 74. (9)(a) Fornasier, R.; Scrimin, P.; Tecilla, P.; Tonellato, U. J. Am. Chem. SOC.1989,111,224.(b) De Santi, G.; Ssrimin, P.; Tonellato, U. Tetrahedron Lett. 1990,31,4791.(c) Scrimin, P.; Tecilla, P.; Tonellato, U. J. Org. Chem. 1991,56,161. (10)(a) Gutsche, C. D.; Mei, G. C. J. Am. Chem. SOC. 1985,107,7964. (b) Gellman, S.H.; Petter, R.; Breslow, R. J. Am. Chem. SOC.1986,108, 2388. (c) Menger, F. M.; Gan, L. H.; Johnson, E.; Durst, D. H. J. Am. Chem. SOC.1987,109,2800. (11)(a) Weijnen, J. G. J.; Koudijs, A.; Engbersen, J. F. J. J. Chem. SOC.,Perkin Trans. 2 1991,1121. (b) Weijnen, J. G. J.; Koudijs, A.; Schellekens, G. A.; Engbersen, J. F. J. J. Chem. SOC.,Perkin Tram. 2 1992,829. (12)Fendler, J. H.;Fendler, E. J. Catalysis in Micellar and Macromolecular Systems; Academic Press: New York, 1975.
0022-3263/92/1957-7258~03.00/0 0 1992 American Chemical Society
J. Org. Chem., Vol. 57, No.26, 1992 7269
Synthesis of Chiral 1,lO-Phenanthroline Ligands Scheme I
'
H3C,
Scheme I1
NH
R'
C.0 R
a
C,2-Phe-PNP : R = (CH2)&H3
$IO R
CI2-Leu-PNP : R = (CH,),oCH,
Z-Phe-PNP : R = OCH,C,H,
'
HC .
QLc,o+o2
s
BryC
PNPP
8
determines largely the catalytic efficiency." In the present work the factors controlling the magnitude and direction of the stereoelective hydrolysis of enantiomeric substrata in the presence of chiral l,l0-phenanthroline ligands coordinated with bivalent metal ions was Seven new chiral 1,lO-phenanthrolines were synthesized, which are all functionalized with a hydroxymethyl group in close proximity to the metallocleft and the catalytic activity and enantioselectivity in the cleavage of p-nitrophenyl esters of N-protected amino acids were studied.
8
0 6
io
HOH2& : 7
studies have been undertaken with respect to the enantioselectivity of hydrolytic metal lo enzyme^.'^ In previous studies on the catalytic effecta of metal-ion complexes of functionalized 1,lO-phenanthrolines in the hydrolysis of carboxylic and phosphoric esters, we have demonstrated that the nature of the ligated metal ion (13) (a) Ueoka, R.; Mataumoto, Y.; Moes, R. A.; Swarup, S.; Sugii, A.; Harada, K.; Kikuchi, J.; Murakami, Y. J. Am. Chem. SOC.1988,110,1588 and references cited therein. (b) Cleij, M. C.; Drenth, W.; Nolte, R. J. M. J. Org. Chem. 1991,66,3883. (c) Ueoka,R.; Cho, M.; Mataumoto, Y.; Goto, K.; Kato, Y.; Harada, K.; Sugii, A. Tetrahedron Lett. 1990,31,5335. (14) (a) Yarrmkov, I. A,; Berezin, B. B.; Belchich, L. A.; Davankov, V. A. Mukromol. Chem. 1979,180,799. (b) Yamekov, I. A,; Berezin, B. B.; Belchich, L. A.; Davankov, V. A. Eur. Polym. J. 1979,15,1067. (c) Ogino, K.; Tomita, I.; Machiya, K.; Tagaki, W. Chem. Lett. 1982, 1875. (d) Sakaki, S.;N h o , Y.; Ohkubo,K. Chem. Lett. 1983,413. (e) Fomasier, R.; Scrimii,P.: Tonellato. U.: Uta.N. J. Chem. SOC..Chem. Commun. 1988,716.
Results Ligands. The chiral 1,lO-phenanthroline ligands 1-7 were prepared according to the procedures outlined in Scheme I. The asymmetrically disubstituted ligands 1-4 were obtained by coupling of 2,9-bis(bromomethyl)-l,lOphenanthroline16 to 1equiv of N-methyldodecylamine," and without isolation of the unstable intermediate, the desired chiral amino alcohol was added to the reaction mixture. Compounds 5-7 were prepared by coupling of (S)-2-pyrrolidinemethanol t o the required 1 , l O phenanthroline halide. Ligands 1-7 contain a strongly metal-ion chelating moiety, composed of the l,l0-phenanthroline nucleus and one or two tertiary amino substituents at the 2 and 9 positions. Chirality in the ligan& is introduced by the rigid 2-pyrrolidinemethanol group with one chiral center, or by the more flexible ephedrine group with two chiral centers. In all ligands a nucleophilic hydroxyl group is at the same distance to the metallocenter (except the hydroxymethyl group at C9 of 6). Since 1,3, and 4 are only slightly soluble in water, their catalytic activity was studied in mixed micellar systems, composed of chemically inert surfactant molecules and metal-ion complexes of the lipophilic ligands. The water-soluble ligands 2,5, 6, and 7 were studied in pure buffer or, in case the substrate was not sufficient soluble in water, in Brij 35 micelles. Binding of metal ions to the l,l@phenanthrohenucleus can be monitored spectrophotometrically.' Addition of Zn2+to 1-7 shifta the absorbance maximum from 272 to 276 nm, and a shoulder in the region of 295-300 nm appears. From the changes in the W spectra it is shown that in the presence of 1equiv of Zn2+complexation of 1-7 is complete, indicating the high metal-ion affinity of these ligands. (15) Preliminary communication concerning part of this work Weijnen, J. G. J.; Koudijs, A.; Engbersen, J. F. J. J.Mol. Cutul. 1992, 73, L5. (16) Chandler, C. J.; Deady, L. W.; Reiss, J. A. J. Heterocycl. Chem. 1981,18,599. (17) W t o n , A. W.; Reck, R. A.; Harwood, H.J.; Dubrow, P. L. J. Org. Chenr. 1948,13, 186.
7260 J. Org. Chem., Vol. 57, No. 26, 1992 Table 1. Pseudo-First-OrderRate Constants (kob,) for the Hydrolysis of PNPP by Different Zn2+-LigandComplexesa catalyst comicellar additive lo3 knk. 8-l k,klkn none none 0.010 1 none CTABr 0.023 2.3 1-Zn2+ CTABr 68.7 6870 none 2-Zn2+ 2.52 252 CTABr 3-Zn2+ 3-Zn2+ 258 4-Zn2+ CTABr 3.22 322 none 5-Zn2+ 2.74 274 none 14.20 1420 6-Zn2+ 7-Zn2+ none 2.88 288 nConditions: 25 OC, pH = 7.00 (0.01M N-ethylmorpholine-HBr buffer), [CTABr] = 4 X M, [ligand] = 5 X lo-' M,[Zn2+]= 5 M. X lo-' M,and [PNPP] = 5 X
Kinetic Studies. Kinetic experiments were performed in pure N-ethylmorpholine-HBr buffer or in buffered micelles at pH = 7.00 and 25 OC under pseudo-first-order conditions. The release of p-nitrophenolate from the Substrate ester was followed spectrophotometricallyat 400 nm. First we investigated the catalytic activity of the synthesized ligands in the hydrolysis of the metallophilic substrate p-nitrophenyl picolinate (PNPP, Scheme 11). This ester has shown to be a useful substrate in studies of metal-ion-activated hydrolysis reactions, and catalytic data thus obtained could be used for comparison of catalytic efficiency. Pseudo-first-order rate constants for the hydrolysis of PNPP catalyzed by Zn2+complexes of 1-7 are given in Table I. Pure cationic CTABr micelles exhibit almost no rate accelerating effect. Large rate enhancements of 250-1400-fold are obtained in the presence of equimolar Zn2+-and ephedrine-containing ligands (3,4) or the water-soluble 2-pyrrolidinemethanol ligands (2,5, 6, and 7). The highest rate acceleration (6780X) is observed in the presence of 1-Zn2+. Next, we investigated the catalytic activities of metallocomplexes of 1-7toward chiral, nonmetallophilic substrates, Le., the N-protected p-nitrophenyl esters of D(L)-phenylalanine and D(L)-leucine (Scheme 11). Since these substrates are not sufficiently soluble in pure aqueous buffer, in experiments with the hydrophilic metal-ion complexes of 2,5,6, and 7 the substrate was dissolved in solutions of nonionic Brij 35 micelles. Cationic micelles are not appropriate in this case because of the electrostatic repulsion of these micelles with the positively charged metal-ion-ligand complex. b2+, Co2+,Ni2+,and Cd2+in the absence of ligand show no catalytic activity toward p-nitrophenyl esters of Nprotected amino acids, whereas addition of Cu2+even retards the rate of spontaneous hydrolysis. Rate enhancementa caused by nonmetalated ligands are relatively small and enantioselectivities are low. Metal-ion complexes of 1, however, are efficient catalysts in the hydrolysis of D(L)-Z-Phe-PNP, although this substrate has no strong metal-ion binding site as is present in PNPP. The metal-ion activation of 1 is in the order of Zn2+> Co2+> Cu2+ complexes hydrolysis of D-Z(Table 11). For all 1-M2+ Phe-PNP predominates over that of the L-enantiomer. The degree of enantioselectivity (kDa,ob/ kLa,obs)is dependent on the nature of the metal ion, and the highest value (4.2) is found for 1-Co2+.The ephedrine-containing metallosurfactants (3, 4) and the water-soluble 2pyrrolidinemethanol metallocatalysts (2,5, 6, and 7) are less reactive and stereoselective compared to 1-M2+. 5Co2+hydrolyzes the L-substrate 4.4 times faster than the D-substrate; however, the catalytic activity of this metallocomplex is low. The results indicate that both hydrophobic interactions between substrate and metallocatalyst and rigidity of the ligand are important factors for the
Weijnen et al. Table 11. Apparent Second-Order Rate Constants (k*ob., M-'8-l) and Enantioselectivities for the Hydrolysis of D(L)-Z-Phe-PNP,Catalyzed by Different Metal Complexes' comicellar kDa,obl catalyst additive kDa,ob kL,+,h kL.,ob CTABr 37.8 27.3 1-Zn2+ 1.4 1-c02+ 30.0 7.10 4.2 CTABr 19.2 CTABr 1cu2+ 6.44 3.0 2-Zn2+ 0.25 0.22 Brij 35 1.1 3.27 2.63 3-Zn2+ CTABr 1.2 3-Co2+ 1.03 0.81 1.3 CTABr 4-Zn2+ 2.81 5.12 CTABr 0.55 4-co2+ 0.80 1.24 0.65 CTABr 5-Zn2+ 0.31 0.67 Brij 35 0.46 5-c02+ Brij 35 0.07 0.31 0.23 6-Zn2+ 0.34 0.29 Brij 35 1.2 7-Zn2+ 0.35 0.36 Brij 35 0.97 "Conditions: 25 OC, pH = 7.00 (0.01M N-ethylmorpholineHBr M,[ligand] = buffer), [CTABr] = 4 X 10" M,[Brij 351 = 4 X 5 X lo4 M,[M2+] = 5 X lo-' M,and (D(L)-ZP~~-PNP] =5X M. The first-order rate conatanta measured in the absence of metallocatalyst (k,) are 7.99 X s-l and 7.56 X s-l for the hydrolysis of D- and L-Z-Phe-PNP, respectively. 1"
-
'*,
30
-
20
-
s
/i/-/
A" c1
2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
103[1c0*+], M
Figure 1. Pseudo-first-order rate constants for the hydrolysis of D- and L-Z-Phe-PNP as a function of 1-Co2+ concentration in CTABr micelles at pH = 7.00 and 25 OC: [CTABr] = 4 X M, [D(L)-Z-P~~-PNP] =5X M, and [1]:[Co2+] = 1.
activity and enantioselectivity. The effect of the l-Co2+concentration on the rate and enantioselectivity in the hydrolysis of D(L)-Z-Phe-PNP is depicted in Figure 1. The rate of hydrolysis of D- and L-Z-Phe-PNP linearly increases with [ 1-Co2+],and over the entire concentration range the D-enantiomer is hydrolyzed faster than the L-enantiomer in a ratio of 41. No saturation kinetics is observed, indicating that the binding constant of D(L)-Z-Phe-PNP to 1-Co2+ is low. Substitution of the N-(benxyloxycarbonyl) protecting group of the substrate into the more hydrophobic N-dodecanoyl group results in a substrate with a higher affinity for the micellar phase. Also for these substrates, the lipophilic ligand 1 is hardly active in the absence of metal ion and the enantioselectivity is relatively low (Table 111). In the presence of metal ion, however, the enantioselectivity of 1 toward D(L)-C12-Phe-PNPis higher compared to that of D(L)-%Phe-PNP. The most remarkable feature in the data of Table I11 is that the nature of the metal ion determines not only the degree of enantioselectivity but also the direction of enantioselectivity. 1-Co2+ and 1-Cu2+ hydrolyze D-C12-Phe-PNPmore rapidly than the L-enantiomer (8.0 and 4.4 times, respectively) whereas 1-Zn2+ gives an inversion of the enantioseleotivity. The dependence of the direction of enantioselectivity on the nature of the metal ion in 1-M2+is not limited to
J. Org. Chem., Vol. 5':: No. 26, 1992 7261
Synthesis of Chiral 1,lO-PhenanthrolineLigands Table 111. Apparent Second-Order Rate Constants M-' 8-l) and Enantioselectivity (kD.,ob/kL.,ob) for the Hydrolysis of D(L)-Cl2-Phe-PNPand D ( L ) - C , ~ - ~ U - P N P , Catalyzed by Mixed Micellar Systems Composed of l-MZ+ and CTABP Clz:Phe-PNP C,,-Leu-PNP ~~
1-Zn2+ 37.3 l-Cozt 74.0 1Cu2+ 15.8 0.70 1-Ni2+
68.8 9.26 3.60 1.40
0.54 8.0 4.4 0.50
21.1 45.9 10.6 0.74
40.1 7.26 3.08 0.52
0.53 6.3 3.4 1.4
"Conditions: 25 O C , pH = 7.00 (0.01M N-ethylmorpholine-HBr M, [l] = 5 X lo4 M, [M2'] = 5 X lo4 buffer); [CTABr] = 4 X =5X M, [ D ( L ) - C ~ ~ - P ~ ~ -=P5NXP ] M, [D(L)-C~,-L~U-PNP] M. The first-order rate constants measured in the absence of s-l (~-c,~-Phe-pNP), 10.7 X metallocatalyst (k,) are 11.1 X 8-l (DCl2-Leu-PNP), and 4.69 10" s-l (L-C,~-P~~-PNP), 4.59 X x 8-l (L-C,,-Leu-PNP). Table IV. Apparent Second-Order Rate Constants M-' e-,) and Enantioselectivity (kD.,ob/kL,,ob) for the and D(L)-C,2-LeU-PNP, Hydrolysis of D(L)-CI2-Phe-PNP Catalyzed by Mixed Micellar Systems Composed of 1-M2' and Brij 35" CI2-Leu-PNP C1,-Phe-PNE'.-
0.0
0.2
0.4
0.8
0.6
1.0
1.2
103[1c0~ ,+ M1
Figure 2. Pseudo-first.order rate constants for the hydrolysis of D- and L-CI2-Phe-PNPas a function of 1-Co2+ concentration in Brij 35 micelles at pH = 7.00 and 25 OC: [Brij 351 = 4 X M, [ D ( L ) - C ~ ~ - P ~ ~ -=P5NXP ] M and [l]:[Co2+]= 1.
20 c
m
-
15
2$
P 10
l
