Symposium on Molecular Architecture
Molecular Recognition Design and Synthesis of Artificial Receptors Employing Directed Hydrogen Bonding Interactions Andrew D. Hamilton University of Pittsburgh, Pittsburgh, PA 15260 An important goal in modern hioorganic chemistry concerns the design of synthetic molecules that mimic various aspects of enzyme chemistry ( I ) . Detailed study of such models can lead not only to insights into the nature of enzyme action but also to new chemical species with some of the specificity and speed normally associated only with enzymes. Central to the success of this endeavor is the development of molecular architecture in which different regions or functional groups are positioned in a well-defined array to provide a specific chemical microenvironment. In recent years particular progress has been madein the areaof molecular recognition. A number of artificial receptors have been constructed that show strong and selective binding to small organic substrates. In the main, these have been based on a complementary relationship between the host and its guest. This complementarity must include both the shape and the chemical characteristics of the suhstrate. Thus, for effective molecular recognition the receptor must provide a cavity that matches the shape and size of the substrate. The cavity must in addition he lined with hinding groups capable of interacting with complementary regions on the substrate. Nature provides many extraordinary examples of molecular recognition. The recent X-ray structure (2)of a D-glucose hinding protein shows in enormous detail the complementary relationship between the binding groups on the protein and the substrate (Fig. la). Thirteen hydrogen honds (Fig. l b ) are formed between peptide residues and the hydroxyl groups or pyranose oxygen of D-glucose to give a very high affinity for the sugar. In addition, two aromatic residues, phenylalanine-16 and tryptophan-183 are positioned above and below the plane of the pyranose ring forming two hydro-
Figure 1. X-ray
phobic caps to the hinding pocket for the strongly hydrophilic suhstrate. In seeking to mimic and extend the strategies of biological molecular recognition, we must develop synthetic microenvironments with similarly controlled binding groups. Artificial receptors should be prepared in which mutually separated hydrogen bonding, hydrophobic, and charged groups are directed to converge on a cavity of appropriate size. Progress toward the artificial recognition of important hiomolecules (peptides, nucleotides, drugs, etc.) may lead to the development of new pharmaceutical strategies, drug delivery systems or chemical sensor designs. Such &dies are direciedat rhe ground state structur& uf the target suhstralea. Extension of this molecular recogni. tion approach to the design of artificial receptors t h a t a r e complementary to the proposed transition state structure of a reaction will lead to selective stabilization of the transition state and acceleration of the reaction rate (3).
Early Work
An important base in the areaof artificialmolecularrecognition has come from three key classes of molecules, the cvclodextrins, cyclo~hanes,and crown ethers. Work in manv laboratories h i shown that these macrocyclic derivatives can form discrete complexes with a wide range of substrates. The naturally occurring cyclodextrins and'their synthetic counterparts, the cyclophanes, are cylindrical molecules with a hydrophobic interior and hydrophilic exterior. In aqueous, and in some cases organic, solutions these receptors hind small organic substrates into their central cavity. The
structure of D-glucose binding protein. A. Crystal structure 8. Schematic of sugar binding site. Volume 67
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recognition is based primarily on molecular shape and surfarecharacteristics rather thanany diatribution of functional groups. As a result, the potentidfor specifically orienting a substrate within the cavity is limited. However, recent elegant methods for functionalizing cyclodextrins and cyclophanes are overcoming this problem. Crown ethers (particularly 18-crown-6 derivatives) form strong complexes with primary ammonium ions. Binding involves three hydrogen bonds between the ammonium NH's and oxygen or nitrogen atoms on the crown ether and has been the basis of many functionalized, and even catalytic, receptor systems. Crown ethers are limited by their conformational flexibility and single functionalgroup selectivity. Thesyntheticand molecular recognition properties of the cyclodextrins ( 4 ) . cyclophanes ( 5 ) ,and crown ethers (6) have been the subject of many reviews and commentaries (7).
may provide a secondary recognition of the many different substituents in the 5,5-positions of barbiturates.
Dlrected Hydrogen Bondlng Interactions
The most widespread binding interaction in biological recognition (as in Fig. 1) involves a hydrogen bond directed from protein to substrate. Thus, a cleft or cavity containing several directed hydrogen bonding groups should lead to binding and potential orientation of a substrate with complementary groups. Our first receptors based on this strategy involved as substrates the barbiturate family of drugs (e.g., 1). These are attractive targets for molecular recogni-
tion studies due to their widespread use as sedatives and anticonrmlsants and their simple and rigid arrangement of hvdroeen bondine-,. erouns. , . Indeed. we recoenized that all six olthe acresdihle hydrogen bonding sites (four CO lone pairs and t w o imide A'H's) would be comolexed br, two 2.6-diaminopyridine units i&orporated into-a macro&clic cavity of appropriate size (Fig. 2). An important design question concerns not only the positioning of the hydrogen honding groups but also the rigidity of the supporting framework. If the receptor is too flexible then intramolecular hydrogen bonds may occur (e.g., between the two diaminopyridine groups in Fig. 2) causing a collapse of the cavity. Molecular modelling studies suggested that an isophthalate group (as X in Fie. .. 2). would nrovide the correct soacine and rieiditv - . for l~arhituratecompl~xation. Thesecond spacergroup IYin ~ hydrogen bonding region but Fig. 2) is further from t h main
-
The first barbiturate receptors (e.g., 2 and 3) were prepared by a simple two-step sequence from 2,6-diaminopyridine (8).Reaction of the diamine with isophthalolyl dichloride gave diamidodiamine 4, which was coupled under high dilution conditions with the appropriate diacid chloride to form macrocycles 2 and 3 in 12%and 7% yield, respectively. The integrity of the binding cavity in 3 was confirmed by Xray crystallography (9).This shows (Fig. 3) an open conformation for the macrocycle with all six hydragen honding groups directed toward its center. The receptor is not fully preorganized (i.e., with a planar array of t h e six binding groups) but has a twisted conformation with a 44" angle between the pyridine rings. In addition, a T H F molecule of crystallization is occupying the cavity and forms a single hydrogen bond to an amide NH. The barbiturate binding properties of 2 and 3 can he readily followed by 'H NMR. Addition of one equivalent of barbital l a to a CDC13 solution of 2 or 3 results in larae downfield shifts of the host amide and guest imide proton resonances ( 1 .G6,1.63, and 1.38 ppm, respectively, for 2). Such large shifts are strongly indicative of a hexahydrogen bonded c k p l e x of the typeshown in 5. Continuing the addition of 1 into 2 or 3 leads to a titration curve that shows clear saturation with a sharp knee at a gueskhost ratio of 1:l.Further analysis of the binding curve using either a Scatchard or nonlinear regression approach (10)yields an association constant ( K J for the interaction. Association constant values for the binding of 2 or 3 to a number of barbiturate substrates are collected in the table. These show that for a complementary substrate such as barbital large K. values (1.37 X 106 M-' and 1.35 X 105 M-') Association Constants for the Receptor-Barbiturate interaction
Figure 2. Schematic of an artificial binding site for barbiturates.
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Receptor
Barbiturate
K,, M-' (25 ' C . CDCld
2 3 2 3
Mephobarbital 1c Phenobarbital lb Phenobarbital lb Barbital l a Barbital la
6.80 X lo2 2.80 X lo5 1.97 X lo5
2
1.35 X lo5
1.37 X lo6
I
Figure 3. X-ray structure of 3.
Figure 4. X-ray structure of complex 3:la.
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are possible. Enforced removal of two or three hydrogen bonds from the interaction, as with mephobarbital, results in a 103-fold drop in binding. Precise structural details of the recognition process have been obtained by X-ray crystallography. The structure of the complex between l a and 3 (9) confirms the hexahydrogen bonded nature of the interaction between receptor and substrate (Fig. 4). The bond lengths of the six hydro en bonds fall into three pairs corresponding to N(4)-0(41), N(3)-O(43 ), medium (-3.0 A; short (-2.9 N(5)-N(7), N(2)-N(8) and long (-3.2 N(6,l)-O(42)) hydrogen bonds (11). In addition, the macrocycle bas undergone a conformational change (compared to Fig. 3) to bring all six hydrogen bonding groups into the same plane. The barbiturateitself is not coplanar to themacrocycle but lies a t a 27' angle relative to the pyridine rings. The skewed position of the guest within the cavity is most likely due to unfavorable steric interactions between the barbital-2carbonyl group and the isophthaloyl-2-proton. Nonplanar hydrogen bonded complexes of this type are common in nucleic acid chemistry where propeller twists between interacting rings of up to 30° are often encountered (12). Thus, in 2 and 3 we have constructed strong and highly selective receptors for a pharmaceutically important class of substrates. A number of other approaches to the design and synthesis of oriented hydrogen bonding receptors have been reported (13-15). Of particular note is the work of Kelly (14) who has constructed receptors for uric acid derivatives. Linking two benzoquinoline units as 6 creates a cavity with four converging hydrogen bonding sites in a rigid arrangement. 6 forms strong complexes with functionalized uric acid derivatives (as in 7) with a K. of 1.0 X lo6M-'. A very different approach to the synthesis of receptors has been exploited hy Rebek and his group (15). Using Kemp's triacid 8 as a key binding and linking component they have constructed a large number of nonmacrocyclic derivatives that force two or more carboxylic acid or other functional groups to converge on a central cavity. With an appropriate spacer, the resulting receptor defines a rigid and convergent hydrogen bonding site. For example, acridine receptor 9 forms strong complexes with substrates containing two basic centers. Association constants of greater than lo6 M-I have been measured for such difunctionalized substrates as diazabicyclooctane (9) and pyrazine (10).
1;
A;
O.Y.~ H
Y 0
'7
CPh,
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Figure 5. Twoaite strategy for Mymine recognition.
Figure 6. X-ray structure of active site of ribonuclease T,.
U
CPh,
Figure 7. Schematic of synthetic, two site approach to nucleotide base recognition.
Directed T-Slacklng Interactions
The combination of directed hydrogen honding and sstacking interactions offers a powerful approach for the recognition of planar heterocyclic substrates such as the nucleotide bases. The hydrogen bonding groups a t the periphery of the heterocycle and the flat r surface on its top and bottom faces suggest a perpendicular convergence of binding interactions (Fig. 5). This recognition strategy is seen in the Xray structure of the active site of the guanine-binding protein, ribonuclease TI (16) (Fig. 6). Two hydrogen bonds are formed between the ~ e ~ t i backbone de and the N-1 and 0-6 of guanine. In additibn; a a-stacking interaction occurs between the guanine and a tyrosine residue (Tyr 45) held in a face-to-face orientation a t 3.4 A above the bound substrate. We have incorporated this two-site binding approach (hydrogen bonding and aromatic stacking) into a series of receptors for the nucleotide bases. Our strategy was to link within a macrocyclic framework, a group capable of stacking with the nucleotide base to one complementary to its hydrogen bonding periphery (Fig. 7).
Figure 8. X-rsy
strucrure of 11.
The first receptor 11for thymine combined a 2,7-dialkoxynaphthalene as s-stacking component with a 2,6-diamidopyridine unit as hydrogen honding component (17). This latter group is interesting as i t forms a triple hydrogen bonded complex with the imide gmup of thymine. Figure 8 shows the X-ray structure of 11 and confirms that the macrocycle takes up an open conformation with no intramolecular stacking between the two aromatic rings. A side view of this structure is seen in Figure 9a. Once again, the binding prop-
11 Figure 9. Side view d X-ray sbucture of (a) 11, (b) complex 11:12.
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erties of 11 could be followed by IH NMR. Treatment of 11 with one equivalent of 1-butylthymine 12 in CDCls results in large downfield shifts (-2 ppm) of the amide NH and imide NH resonances and small upfield shifts (-0.3 ppm) of the thymine CH3, ring H, and NCHz resonances. TheselH NMR changes are consistent with the formation of a complex between 11 and 12 that combines simultaneous aromaticstacking (with accompanying ring current effects) with three hydrogen bonding interactions. Association constant measurements also confirm the contribution of s stacking to binding and show a threefold increase (from 89 to 290 M-') for 11 when compared to a simple 2,6-dimidopyridine derivative that lacks the stacking group. A side view of the X-ray structure of the complex between 11 and 12 is shown in Figure 9b. The naphthalene ring is positioned a t a 14O angle above the thymine ring with a closest approach of 3.4 A. Comparison of the structures of uncomplexed (Fig. 9a) and complexed (Fig. 9b) forms of 11 shows that on substrate binding the naphthalene swings through a40° arc to its faceto-face stacking position.
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The structural and synthetic simplicity of 11 means that many changes can be made t o the receptor. Varying the hydrogen bonding region will lead to differences in the binding specificity for other nucleotide bases. 2-Amino-1,snaphthyridine 13 possesses a triple hydrogen bonding complementarity with the periphery of guanine (18). We have prepared a macrocyclic receptor 14 containing both an aminonaphthyridine and a r-stacking unit and shown that i t binds to guanine derivatives by a combination of aromatic stacking and hydrogen bonding interactions as in 15. The participation of the T-stacking group is confirmed by an increase in K. (126 to 502 M-l) on going from a simple acyclic amino naphthyridine to 14. A similar strategy can be applied to the recognition of adenine. Bis-(2-aminopyridine) derivatives 16 can form four hydrogen bonds to the periphery of adenine (combining both Watson-Crick and Hoogsteen interactions) (19). We have prepared a series of macrocyclic receptors 17 combining both a naphthalene a-stacking unit and a 1,2-bis-(2-amino-6-pyridy1)-ethaneunit and shown that they form strong complexes with 9-alkyladenine 18, (for example, K. value for 17:18 is 3400 M-I). The hinding conformation of 19 requires all four hydrogen bonding groups of the his-(2-aminopyridine) group to be directed toward the center of the cavity. This orientation is supported by an X-ray structure on uncomplexed 17 (n = 2) (Fig. 10). The pyridine N's are placed a t 5.04 A and the amide NH's at 7.33 A from each other in good binding complementarity to the amino group and purine N's of adenine 18. The second aspect of these two-site receptors that can be chanced - is the r-stacking component. This is particularly relevant since in a recent survey of protein crystal structures Petsko (20) has identified two important geometries for aromatic-aromatic interactions, face-to-face or edge-to-edge geometries (Fig. 11). In an effort to probe the origins of these two arrangements we have investigated the importance of the electronic characteristics of the stacking group on its orientation. Macrocycle 20, containing two electron withdrawing ester groups on the naphthalene ring, forms stronger complexes (K. = 570 M-I) than unsubstituted 11 (K. = 290 M-I) with I-butylthymine 12. The X-ray structure of complex 20:12 (Fig. 12) shows a parallel, face-to-face interaction with an interplanar distance of 3.5 A.An insight into the special stabilization involved in stacking comes from MNDO calculations on thymine and 2,7-dimethoxynaphthalene-3,6-dicarboxylate.The resulting charge distributions are superimposed (sign only) on a downward view of structure 20:12 (Fig. 13). This shows five points of contact where partially positive charged atoms on the naphthalene precisely align themselves with partially negative regions on the thymine (21). From this we can conclude that electrostatic interactions between regions of complementary charge distribution on the rings play an important role i n s stacking. If this analysis is correct, it should be possible to change the geometry of aromatic-aromatic interactions by varying the electronic characteristics of one ring. Replacing the diester
W
groups in 20 by two ether groups leads to macrocyclic 21, which shows substantially weaker binding (K. = 138 M-') to 12 than does 20. MNDO calculations on 2,3,6,7-tetramethoxynaphthalene show a reversal of sign on carbons-4 and -5. This would lead to a repulsive electrostatic interaction between receptor and substrate if a face-to-face geometry were to form (Fig. 14). The X-ray structure of complex 21:12 confirms that the faceto-face geometry is avoided and that the naphthalene takes up an almost perpendicular, edge-to-face orientation with respect to the substrate (Fig. 15). ThesolutionlH NMR data on complex 21:12 is consistent with this conformation. Interestingly, the naphthalene-1,8-protons in 21 project toward a region of partial negative charge formed by the tbymine imide group. The possible electrostatic stabilization between these regions may account for thesmall stabilization of 21:12 compared to the simple complex lacking a naphthalene (as discussed above). Thus, within a simple series of thymine receptors, we have shown that the geometry of aromatic-aromatic interactions can be controlled by modifying the electronic properties of one component. In particular, an electrostatic complementarity between partial charges on the rings can lead to strong face-to-face stacking, while in the absence of such effects a weaker edge-to-face interaction is preferred. In a further development of his Kemp's triacid strategy, Rehek (22) has elegantly shown that receptor 22 can form two site complexes with adenine. The convergence of a-stacking and hydrogen bonding interactions in 22 leads to association constant values of 120 M-'. Incorporation of two
Figure 10. X-ray structure of 17 (n = 2).
FACE-TO-FACE
EDGE-TO-FACE
Figure 11. Aromatic-ammatic interactions.
"-"-.," Figure 13. Charge disbibutionssuperimposedon structure 20:12.
. Figure 12. X-ray Bucture of complex 20:12.
Figure 14. Charge distribution of 21 pmduced by replacing lhe diester groups in 20: with two elher groups.
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Kemp's triacid units into receptor 23 allows combined Watson-Crick and Hoogsteen hydrogen bonding to occur to adenine and leads to a substantial increase in the association constant ( K . = 11,000 M - I ) . This approach to designing recognition interactions has led not only to effective binding but also to the transport of adenine across membrane systems (23).
Figure 15. X-ray structure of complex 21:12. 8. Chang,S.K.;Hamilton,A. 0. J. Am. Cham.Soc. 1988.110,1318. 9. Chang, S. K.; Van Engen, D.: Hamilton, A. D. J. Am. Chem. Sor., in prear.
lo. Foster,R.;Fife, C. A. Pro#. Nucl. Mogn. Reaon. Spertmsc. 1363,4,1.
Acknowledgment
We thank the National Institutes for Health for their support of the work described in this article. Literature CHed 1. Dugas, H. Bioorgonic Chamirtry; 2nd ed.; Springer New York, 1989. 2. Vyas,N. K.:Vyss,M.N.:Quiocbo, F. A.SeienrelWo~hington,DC),1988,242,1291. 3. Kraut. J. Science (Wahingfan,DC) 1388,242,533. a. Bender, M.: Komiyama, M. Cyelodertrin Chemlalry; Sprinpen Near York, 1978. 5. Diedorich, F. Ang. Chim. Int. Ed. 1388.27, 362. 6. Gokcl, G. W.; Korseniamki, S. H. MnerocyclicPolyether Syntheao8:Springer: B d i n , 1982. 7. Lehn, J. M. An#. Chem. Int. Ed. 1388,27,89: Cram, D. A w Cham. Int. Ed.
11. Taylor, R.:Kennsrd,O.; Versichel. W. J.Am. Chom.Soe. I383,105,57S1. 12. Saenger, W. Prineiploa of Nueieic Acidstructure; Springer: New York, 1984: p 26. 13. Shoridan.R.E.: Whit1ock.H. W.J.Arn.Chem.Sac. 1986,108,7120:Aarts.V.M.L.J.; vanstaveron, C. J ; Groofenhui.. P. 0. J.;"a" Eerd.". J: Kruise, L.;Harkemu, S.; Roinhoudt.0.N. J.Am. Chem.Soe. 1986,108,503S;Foibu~h,B.;Saha,M.:Onan,K.; Kargsr,B.;Gcise,R. J . Am.Chem. Sac. 1387,109,753L: Kilhurn, J . D.;Mackenzie, A.R.:Still,W.C.J.Am.Chem.Soe. 1988.110,1307;Beil.T. W.:Liu,J. J.Am.Chom. Soc. 1388.110,3673. 14. Kel1y.T. R.;Maguire,M. P. J.Am. Chsm Soc. 1387,109,6549. 15. Rebek. J., J r . J Molac. Roeog. 1388.1, I. 16. Heinemann, U.;Ssenger, W. Nolure (Landon) 1982,299.27. 17. Hamilton, A. D.; van Engen, D. II Am. Chem. Sor. 1987,109,5035. 1s. Hamilton,A.D.: Pant. N. J. Chem.Sac., Chem. Commun. 1588,765. 19. Goswami. S.: van Engen, D.; Hamilton, A. D. J. Am. Cham. So?., in prcea. 20. Budoy, S. K.:P&ka, G.A.Seienre lWoshinglon,DC), 1985,229.23. 21. Muehldorf, A. V.: van Engen, D.; Warner. J. C.: Hamilton, A. D.J. Am. Chom. Sor. 1988,110,6561. 22. Robek,J., Jr.;Askew,B.:Ballester,P.Buhr.C.; Janea,S.;~imsth.D.,Williama,K. J . Am. Chem. Soc. 1387.109,5033. 23. Bsnzing, T. Tjiuikua. T.; Wolfe, J.;Rebek, Jr. Science lWa&h#lon. DCI. 1988,242, 266.
Call for Papers-Semon National Undergraduate Research Symposium The 13th Annual Waldo Semon Chemistry Symposium, will be held at Kent State U n i v e r s i t y on Monday, April 8, 1991. A major focus o f this event, which is co-sponsored by Kent State University and BFGaodrich, w i l l he the Semon National Undergraduate Research Symposium, held for the f o u r t h consecutive y e a r . Students at colleges and universities anywhere in the United States are invited tosubmit a paper describing their undergraduate research work. This should be limited to 10 double-spaced, typed pages with any number of additional figures, tables, and r e f e r e n c e s . The paper should be written entirely by the student and should he structured in a f o r m a t similar to that of a full page published in an American C h e m i c a l Society journal (i.e., abstract, introduction, results and d i s c u s s i o n , conclusion, experimental section, and references). A panel o f judges will select six finalists, who will he invited to present short (20-25 minute) seminars describing their research work st the symposium. The student presenting the best paper will receive a $ 2 , 0 0 0 prize, with $200 going to each of the f i v e remaining speakers. All travel arrangements will he made at no expense to the participants. Students who are interested in participating in the 1991Semon National Undergraduate Research Symposium should write for further details to: Paul Sampson, Chair, Semon Chemistry Symposium Committee, Department of Chemistry, Kent State University, Kent, OH 4 4 2 4 2 , or call (216) 672-2032. Letters of intent tosubmit papers should a r r i v e in Kent no later than December 1 9 , 1 9 9 0 . The deadline f o r receipt of papers is March 1,1991.
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