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187,
No.
September
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
16, 1992
DESIGN,
COMMUNICATIONS Pages
SYNTHESIS AND CONFORMATIONAL ANALYSIS PEPTIDE MIMETICS OF BRADYKININ
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006
OF ?TURN
M. SatotTJ, Jessie Y.H. Lee*,§, Hiroshi Nakanish&§, Michael E. Johnson*,*, R. Alan Chruscielt~i and Michael KahntTBP +Department of Chemistry and $Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry & Pharmacognosy University of Illinois at Chicago, P.O. Box 6998, Chicago, IL 60680 Received
August
4, 1992
Summary.
Gamma-turnsare regular secondarystructure elements,found with somefrequency in small peptides, that have been implicated in the biologically active conformations of several systems.This report describesthe design,synthesisand conformational analysisof a non-peptide y-turn mimetic. Low energy conformations of the mimetic systemexhibit good conformational agreementwith an experimentally observedpeptide y-turn. The mimetics were incorporatedinto the nonapeptide bradykinin, for which a y-turn, formed by residuesSer 6 to Phe 8, has been hypothesized to be a bioactive conformation. The resultsindicate that a bioactive conformation of bradykinin may include a reverse turn at this position. 0 1992Acadrmlc Press,Inc.
Bradykinin is a nonapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) with potent vasodilative and bronchoconstictive properties(1). Despiteextensive investigation, the bioactive conformation of bradykinin remainsto be determined (2-7). Proposedbioactive conformations for bradykinin include a y-turn incorporating residues Ser 6 - Phe 8 (2,3), or a p-turn incorporating residues 6-9 (5-7). Additionally, bradykinin is rapidly inactivated in vivo via proteolytic cleavage of the amide bond between Pro 7 and Phe 8 (8) thus making this segment an attractive candidate for the development of mimetics. It hasalsobeen suggestedthat y-turns are presentin the solution conformations of several other peptides, including substanceP (9) cyclic somatostatinanalogs(lo), and cyclolinopeptide §PresentAddresses: M.S., Lion Akzo Co. Ltd., No. 13-12,7-Chome, Hirai, Edogawa-Ku, Tokyo 132, Japan; J.Y.-H.L., Abbott Laboratories, Abbott Park, IL 60064; H.N., Molecumetics Ltd., 2023 120th Ave. N.E., Suite 400, Bellevue, WA 98005; R.A.C., Searle Research and Development, St. Louis, MO; M.K., Department of Pathobiology, SC38, University of Washington, Seattle, WA 98195. *To whom correspondenceshould be addressed. 0006-291X/92
999
$4.00
Vol.
187,
No.
2, 1992
BIOCHEMICAL
RESEARCH
COMMUNICATIONS
F
R2
w
N-C,/
Hr;J
cap0
BIOPHYSICAL
0
RI
R"\
AND
N-b-O--Me // 0 0
.-Ii
I Peptide y - turn
Ph
Figure 1. Schematicrepresentation of a peptidey-turn andlactam(1) mimeticstructures.
A (11). Additionally, it has been proposedthat enkephalin assumesa ‘y-turn conformation in binding to membranesand to the b-opioid receptor (12-15). Gamma-turns are elements of peptide secondary structure characterized by a 3 + 1 hydrogen bond between the CO group of amino acid residuei and the NH group of amino acid residuei+2, as shown schematically in Fig. 1. Two types of y-turns exist -- the conventional or classic (16) y-turn with ($,I# values generally in the range, (70 to 95, -75 to -45). and inverse y-turns with ($,v) values generally in the range, (-95 to -70, 45 to 75) (16,17). The main chain conformations of the two y-turns are mirror imagesof each other. Although rare in proteins, a recent analysis of 54 proteins with high resolution X-ray crystal structures has shown the existence of approximately ten conventional y-turns, with all but one producing chain reversal at the endsof P-hairpins; inverse y-turns are about six times more common than conventional “Iturns in proteins, but produce chain kinks, and are not usually involved in chain reversal (16). However, y-turns are found more commonly in small cyclic peptides(lo-12,17-23). The use of conformationally constrained peptides or conformationally defined peptide mimetics has proven to be a very useful approach for defining the biologically active conformation of peptides(24-28). Thus, as an initial approach to further investigate the role of y-turns in defining the biologically active conformations of peptides, we have designeda cyclic mimetic, 1, of a peptide y-turn (Fig. 1) that provides for the facile developmentof hybrid peptideturn mimetic systems. To investigate the proposedbioactive conformation of bradykinin, and generate a degradation resistant analogue, we have synthesized‘y-turn mimetic 1 (Fig. I), and have incorporated it into the bradykinin sequence.
MATERIALS
AND
METHODS
IWR spectra were measuredin CDCl, or in D,O at either 24 or 70°C; conditions are noted where appropriate. Resonanceswere assignedusing double quantum filtered COSY and 1000
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heteronuclear correlation pulse sequences on Nicolet NMC360 or Bruker AM400 spectrometers. IR spectra were recorded on a Pet&n-Elmer Model 137 infrared spectrophotometer. Low resolution mass spectra were recorded on an HP5985 system. All reactions were monitored by thin-layer chromatography carried out on 0.25 mm E. Merck silica gel plates (60F-254) with UV light and 7% ethanolic phosphomolybdic acid and heat as the developing agent. E. Merck silica gel 60 (particle size 0.040-0.063 mm) was used for flash chromatography. All reactions were carried out under an argon or nitrogen atmosphere with dry, freshly distilled solvents under anhydrous conditions unless otherwise noted. Yields refer to chromatographically and spectroscopically (lH NMR) homogeneous materials, unless otherwise noted. Bradylcinin binding assays were performed by Dr. Jan Rosenbaum at Procter & Gamble following a published protocol (29). Structures of the y-turn mimetics studied here were built using the MacroModel molecular modeling program (30), employing the MM2 force field. Conformational searches for low energy ring conformers utilized the MacroModel Batchmin program, retaining only methyl groups at positions of chain extension from the ring. Each conformation was minimized using the MacroModel-MM2 force field by the block diagonal Newton-Raphson method, and compared with idealized y-turns. Solvent effects were roughly taken into account by employing a distancedependent dielectric constant with a cutoff distance of 20 A.
RESULTS
AND DISCUSSION
Conformational
Analysis.
In comparing mimetic structures with that of a peptide y-turn,
we have concentrated on three parameters that particularly reflect the positioning of the peptide chain as it extends away from the turn (or the mimetic): carbons; the torsional angle between the C,‘C’(=O) bonds in the mimetics);
the distance between the C,’ and CoLi+2
and Ni+2Cai+2 bonds (and the equivalent
and the 4-atom root-mean-square
distance between the peptide C&,
C’(=O), Ni+2, and Cai+2 atoms and the equivalent y-turn mimetic atoms. For comparison, we have used the ‘y-turn observed in the crystal structure of a cyclic pentapeptide, cycle-(Gly-ProGly-D-Ala-Pro)
(31). In calculating energy minimized mimetic ring conformations,
chain extension was truncated with single methyl groups corresponding
the peptide
to the C,’ and C&+’
carbons. Each isomer conformations,
with
of the lactam mimetic, the ring exhibiting
1, can exist in three relatively
various puckering
low energy
patterns that position the chain
extension bonds in differing orientations. Table 1 lists the conformational energies, the CaiCi(=O)Ni+2Cai+2 torsional angles, the CaiC,i+2 distances, and the 4-atom C&Ci(=O)Ni+2Coi+2 rms differences between the y-turn peptide crystal structure and the four lactam conformers for each of the two isomers. From the values listed in Table 1, it can be seen that the C,‘C’(=O)Ni+2Ca’+2 torsional angle and C,iCai+2 distances for the first and third conformers of the S-isomer are closer to those observed in the peptide y-turn than are those for the other conformers.
The rms deviation from the peptide crystal structure ‘y-turn for these conformers is
also smaller than for the other conformers.
An overlay of the low energy lactam conformer with
the peptide y-turn region is shown in Fig. 2. The energy separation between conformers 1001
1,2 and
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Table 1. Conformr+tipns of lactam y-turn mimetics, as calculated with MM2 force fields. For comparison, the Ca’C’(=O)Ni+2(H)C,i’2 torsional angle and C,‘-C, i+2 distances are also listed for the y-turn in the crystal structure of cyclo-(Gly-Pro-Gly-D-Ala-Pro).29 System/ Conforme?
Energy (kcal/Mol)
C,iCi(=O)Ni+2C,i+2
c,i-cai+2
Torsional Angle
Distance (A)
CaiCi(,0)Ni+2Cai+2
RMS (A)
Lactam 1 S (R)
0.0
46” (-46’)
4.9
0.29 (0.3 1)
Lactam 2 S (R)
1.35
82” (-82’)
4.7
0.42 (0.44)
Lactam 3 S (R)
1.51
16” (-16”)
5.0
0.29 (0.29)
Lactam 4 S (R)
4.29
107” (-1070)
4.6
0.52 (0.54)
Pentapeptide Crystal29
--
+8”
5.2
-.
%alues for R-isomers are shown in parantheses. The S and R isomers form mirror images, thus the conformational energies and C&-C,i+2 distances are equal, and dihedral angles simply reverse sign.
3 is relatively
low, and the lactam ring is reasonably
flexible, thus it is probable that there will
be some conformational averaging between the three low energy conformers. Based on this analysis,the low energy conformers for the lactam mimetic 1 provide good agreementwith the observedy-turn conformation. The CaiCai+:!distance is only slightly shorter than that observed experimentally, the rms deviation is about 0.3 A, and the torsional angle is reasonably close to that observed experimentally. Synthesis. The synthesisof 1 is shown in Scheme1. The acid chloride of 5bromovaleric acid participates in a [2+2] cycloaddition via its derived ketene with the N-benzylimine of formaldehydeto provide the bromoazetidinone2 in 88%. Condensationwith glycine methyl ester proceededsmoothly to provide 1 in 45% yield, via the intermediacy of azetidinone 3.
,’
.’
Figure 2. Overlay of low energy lactam conformers with crystal structure y-turn.
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0
NH*.HCI (5 eq.) M&&C KHC03 (5 cq.) CH3CN + Hz0 (9: 1) * retlux, overnight
1) (COCI), CHzCllL reflux. 2 hr
OH
2) Phpy?IAph c
Br
COMMUNICATIONS
N ‘Ph BF3-OEt7 Et,N CH,Cl, -78 c r.t.
2
cc
NH
N
\
0
Ph
COzMe
CO?Me
1
3
Scheme 1. Synthesis of y-turn mimetic ring system.
We prepared
hybrid mimetic 4, shown in Fig. 3, by solid phasepeptide synthesison an
Advanced Chemtech200 synthesizerusingstandardprotocols(32) from Boc-Arg-(Tos)-Pam resin (Advanced Chemtech). The y-turn mimetic intermediate 5, preparedin analogousfashion to 2,
Arg -Pro --Pro -Gly
-Phe
4b
0
N
Boc-N I H
OH
$
0
‘-’ I Ph
H
5 Figure 3. Structures of y-turn mimetic intermediate, incorporating the y-turn mimetic.
1003
5, and hybrid bradykinin
mimetic, 4,
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Table 2. Displacement of [3H] BDK in NC10815 cell cultureassaya
Concentration(M)
% ControlSpecificBinding(* SEM)b
10-s
91 *3
10-6
54 f 0
10-s
28 * 1
lOA
8*1
10-5MBDK
0 fO.l
aReceptorbindingassaywasperformedin membranes preparedfrom NG108-15cells. The competitionbindingassaywasconductedat 25 “C for 20 min in the presenceof a protease inhibitorcocktail,followingpublishedprocedures (33). bControlSpecificBinding= 2998f 192 DPM, NSB: 10e5BDK = 82 f 4 DPM. All samplesweredonein triplicate. SEM represents standarderror of the mean.
was incorporated by preactivation (5 minutes, O-5” in DMF) with BOP reagent (33). Protected amino acids were incorporated by double couplings of their respective symmetrical anhydrides or, for Boc-Arg(Tos), as its HOBt ester. Couplingsjudged incomplete by qualitative ninhydrin assay were capped with acetic anhydride. The peptide was removed from the resin using anhydrousHF/anisole (9: 1) at O-S’, and purified by reverse-phaseflash chromatography (bonded phaseethyl (C,), Merck) followed by HPLC on a C,, column using a water/acetonitrile gradient in 0.1% trifluoroacetic acid to separatethe two diastereomersof 4. Retention time for the first diastereomer was 16 m, corresponding to an elution at 42% CH,CN, that for the second diastereomerwas 19.9 m, correspondingto elution at 48% CH&N.
The ‘H and PDMS data for
the two diastereomerswere consistentwith their structures. Mimetic Activity.
The two diastereomersof 4 were assayedfor their ability to bind to
NG108-15 cells (29). The 19.9 min retention time diastereomerproduced a concentrationdependentdisplacementof 3H-labeledbradykinin (BDK) in the micromolar range (TabIe 2). The other diastereomerdid not bind to this preparation.
CONCLUSIONS In summary, we have described the synthesisof a y-turn mimetic. Incorporation of mimetic 5 into the bradykinin sequence,thereby mimicking a proposedbioactive conformation, leads to an analog that possesses micromolar binding affinity for the bradykinin receptor in NG108-15 nerve cells. Although the affinity of 4 for the BDK receptor is significantly lessthan bradykinin itself [Kd = 3 x 10“’ M (29)], this result lends further support to the presenceof a reverse turn (either y or, more probably, p (5-7)) at this site. Further investigations with second generation hybrid mimetics (26,28) are in progressto clarify the bioactive conformations of bradykinin, and will be reported in due course. 1004
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ACKNOWLEDGMENTS This research was supported in part by NIH grant GM38260. Additionally, MK wishes to thank the Camille and Henry Dreyfus Foundation, the Searle Scholars Program/The Chicago Community Trust, the NSF (Presidential Young Investigators Award), Monsanto, Procter and Gamble, Schering, Searle and Syntex for matching funds, and the American Cancer Society (Junior Faculty Fellowship) for generous financial support. The molecular modeling facilities were provided, in part, by a BRSG shared instrumentation grant. We thank Dr. Clark Still, Columbia University, for providing the MacroModel and Batchmin programs for our use, Dr. David O’Conner (Procter and Gamble) for obtaining the PDMS spectra for the two diasteromers of 7, Dr. Jan Rosenbaum (Procter and Gamble) for the results of the bradykinin assay, and Richard Shen for HPLC purification.
REFERENCES 1. 2. 3.
4.
5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Regoli, D. and Barabe, J. (1980) Pharmacol. Revs. 32, l-46. Cann, J.R., Steward, J.M. and Matsueda, G.R. (1973) Biochemistry 12, 3780-3788. Ivanov, V.T., Filatova, M.P., Reissman, Z., Reutova, T.O., Efremov, E.S., Pashkov, V.S., Galaktinov, S.G., Grigoryan, G.L. and Ovchinnikov, Yu.A. (1975) in Peptides: Chemistry, Structure and Biology (Walter, R. and Meinhofer, J., eds.) pp. 151-157. Ann Arbor Science Publishers, Ann Arbor, MI. Zabrocki, J., Smith, G.D., Dunbar, J.B., Marshall, K.W., Toth, M. and Marshall, G.R. (1988) in Peptides: Proc. 20th European Peptide Symp. (Jung, G. and Bayer, E., eds.) pp. 295-297. Walter de Gruyter, Berlin. Lee, S.C., Russell, A.F. and Laidig, W.D. (1990) Znt. J. Pept. Protein Res 35, 367-377. Kyle, D.J., Martin, J.A., Farmer, S.G. and Burch, R.M. (1991) J. Med. Chem. 34, 1230-1233. Kyle, D.J., Martin, J.A., Burch, R.M., Carter, J.P., Lu, S., Meeker, S., Prosser, J.C., Sullivan, J.P., Togo, J., Noronha-Blob, L., Sinsko, J.A., Walters, R.F., Whaley, L.W. and Hiner, R.N. (1991) J. Med. Chem. 34, 2649-2653. Gdya, C.E., Wilgis, F.P., Vavrek, R.J. and Stewart, J.M. (1983) Biochem. Pharmacof. 32, 3839-3847. Levian-Teitelbaum, D., Kolodny, N., Chorev, M., Selinger, Z. and Gilon, C. (1989) Biopolymers 28, 51-64. vander Elst, P., Gondol, D., Wynants, C., Tourwe, D. and van Binst, G. (1987) Znt. J. Peptide Protein Res. 29, 331-346. Raghothama, S., Ramakrishnan, C., Balasubramanian, D. and Balaram, P. (1989) Biopolymers 28, 573-588. Froimowitz, M. and Hruby, V.J. (1989) Int. J. Peptide Protein Res. 34, 88-96. Nikiforovich, G.V. and Balodis, J. (1988) FEBS Lett. 227, 127-130. Dhingra, M.M. and Saran, A. (1989) Biopolymers 28, 1271-1285. Milon, A., Miyazawa, T. and Higashijima, T. (1990) Biochemistry 29, 65-75. Milner-White, E.J., Ross, B.M., Ismail, R., Belhadj-Mostefa, K. and Poet, R. (1988) J. Mol. Biol. 204, 777-782. Smith, J.A. and Pease, L.G. (1980) CRC Crit. Rev. Biochem. 8, 315-399. Toniolo C. (1980) CRC Crit. Rev. Biochem. 9, l-44. Karle, I.L. (1981) The Peptides 4, l-53. Kessler, H. (1982) Angew. Chem. Int. Ed. Engl. 21, 512-523. Rose, G.D., Gierasch, L.M. and Smith, J.A. (1985) Adv. Protein Chem. 37, l-109. Spatola, A.F., Anwer, M., Rockwell, A.L. and Gierasch, L.M. (1986) J. Am. Chem. Sot. 108, 825-831. Stradley, S.J., Rizo, J., Bruch, M.D., Stroup, A.N. and Gierasch, L.M. (1990) Biopolymers 29, 263-287. Hruby, V.J., Alobeidi, F. and Kazmierski, W. (1990) Biochem. J. 268, 249-262. 1005
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25. Toniolo, C. (1990) Int. J. Peptide Protein Rex 35, 287-300. 26. Saragovi, H.U., Fitzpatrick, D., Raktabutr, A., Nakanishi, H. and Kahn, M., Greene, M.I. (1991) Science 253, 792-795. 27. Nakanishi, H., Chrusciel, R.A., Shen, R., Bertenshaw, S., Johnson, M.E., Rydel, T.J., Tulinsky, A. and Kahn, M., (1992) Proc. Natl. Acad. Sci. USA 89, 1705-1709. 28. Chen, S., Chrusciel, R.A., Nakanishi, H., Raktabutr, A., Johnson, M.E., Sato, A, Weiner, D., Hoxie, J., Saragovi, H.U., Greene, M.I. and Kahn, M., (1992) Proc. Natl. Acad. Sci. USA 89,
5872-5876. 29. Wolsing, D.H. and Rosenbaum, J.S. (1991) J. Pharmacol. Exper. Therapeut. 257, 621-633. 30. Still, W.C., Mohamadi, F., Richards, N.J.G., Guida, W.C., Liskamp, R., Lipton, M., Caufield, C., Chang, G. and Hendrickson, T. (1989) MACROMODEL V2.0 Department of Chemistry, Columbia University, New York, NY 10027. 31. Karle, I.L. (1978) J. Am. Chem. Sot. 100, 1286-1289. 32. Stewart, J.M. and Young, J.D. (1984) Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, IL. 33. Castro, B., Dormoy, J.R., Evin, G. and Selve, C. (1975) Tetrahedron Lett. 15, 1219-1222.
1006
187,
No.
September
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
16, 1992
DESIGN,
COMMUNICATIONS Pages
SYNTHESIS AND CONFORMATIONAL ANALYSIS PEPTIDE MIMETICS OF BRADYKININ
999-l
006
OF ?TURN
M. SatotTJ, Jessie Y.H. Lee*,§, Hiroshi Nakanish&§, Michael E. Johnson*,*, R. Alan Chruscielt~i and Michael KahntTBP +Department of Chemistry and $Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry & Pharmacognosy University of Illinois at Chicago, P.O. Box 6998, Chicago, IL 60680 Received
August
4, 1992
Summary.
Gamma-turnsare regular secondarystructure elements,found with somefrequency in small peptides, that have been implicated in the biologically active conformations of several systems.This report describesthe design,synthesisand conformational analysisof a non-peptide y-turn mimetic. Low energy conformations of the mimetic systemexhibit good conformational agreementwith an experimentally observedpeptide y-turn. The mimetics were incorporatedinto the nonapeptide bradykinin, for which a y-turn, formed by residuesSer 6 to Phe 8, has been hypothesized to be a bioactive conformation. The resultsindicate that a bioactive conformation of bradykinin may include a reverse turn at this position. 0 1992Acadrmlc Press,Inc.
Bradykinin is a nonapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) with potent vasodilative and bronchoconstictive properties(1). Despiteextensive investigation, the bioactive conformation of bradykinin remainsto be determined (2-7). Proposedbioactive conformations for bradykinin include a y-turn incorporating residues Ser 6 - Phe 8 (2,3), or a p-turn incorporating residues 6-9 (5-7). Additionally, bradykinin is rapidly inactivated in vivo via proteolytic cleavage of the amide bond between Pro 7 and Phe 8 (8) thus making this segment an attractive candidate for the development of mimetics. It hasalsobeen suggestedthat y-turns are presentin the solution conformations of several other peptides, including substanceP (9) cyclic somatostatinanalogs(lo), and cyclolinopeptide §PresentAddresses: M.S., Lion Akzo Co. Ltd., No. 13-12,7-Chome, Hirai, Edogawa-Ku, Tokyo 132, Japan; J.Y.-H.L., Abbott Laboratories, Abbott Park, IL 60064; H.N., Molecumetics Ltd., 2023 120th Ave. N.E., Suite 400, Bellevue, WA 98005; R.A.C., Searle Research and Development, St. Louis, MO; M.K., Department of Pathobiology, SC38, University of Washington, Seattle, WA 98195. *To whom correspondenceshould be addressed. 0006-291X/92
999
$4.00
Vol.
187,
No.
2, 1992
BIOCHEMICAL
RESEARCH
COMMUNICATIONS
F
R2
w
N-C,/
Hr;J
cap0
BIOPHYSICAL
0
RI
R"\
AND
N-b-O--Me // 0 0
.-Ii
I Peptide y - turn
Ph
Figure 1. Schematicrepresentation of a peptidey-turn andlactam(1) mimeticstructures.
A (11). Additionally, it has been proposedthat enkephalin assumesa ‘y-turn conformation in binding to membranesand to the b-opioid receptor (12-15). Gamma-turns are elements of peptide secondary structure characterized by a 3 + 1 hydrogen bond between the CO group of amino acid residuei and the NH group of amino acid residuei+2, as shown schematically in Fig. 1. Two types of y-turns exist -- the conventional or classic (16) y-turn with ($,I# values generally in the range, (70 to 95, -75 to -45). and inverse y-turns with ($,v) values generally in the range, (-95 to -70, 45 to 75) (16,17). The main chain conformations of the two y-turns are mirror imagesof each other. Although rare in proteins, a recent analysis of 54 proteins with high resolution X-ray crystal structures has shown the existence of approximately ten conventional y-turns, with all but one producing chain reversal at the endsof P-hairpins; inverse y-turns are about six times more common than conventional “Iturns in proteins, but produce chain kinks, and are not usually involved in chain reversal (16). However, y-turns are found more commonly in small cyclic peptides(lo-12,17-23). The use of conformationally constrained peptides or conformationally defined peptide mimetics has proven to be a very useful approach for defining the biologically active conformation of peptides(24-28). Thus, as an initial approach to further investigate the role of y-turns in defining the biologically active conformations of peptides, we have designeda cyclic mimetic, 1, of a peptide y-turn (Fig. 1) that provides for the facile developmentof hybrid peptideturn mimetic systems. To investigate the proposedbioactive conformation of bradykinin, and generate a degradation resistant analogue, we have synthesized‘y-turn mimetic 1 (Fig. I), and have incorporated it into the bradykinin sequence.
MATERIALS
AND
METHODS
IWR spectra were measuredin CDCl, or in D,O at either 24 or 70°C; conditions are noted where appropriate. Resonanceswere assignedusing double quantum filtered COSY and 1000
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
heteronuclear correlation pulse sequences on Nicolet NMC360 or Bruker AM400 spectrometers. IR spectra were recorded on a Pet&n-Elmer Model 137 infrared spectrophotometer. Low resolution mass spectra were recorded on an HP5985 system. All reactions were monitored by thin-layer chromatography carried out on 0.25 mm E. Merck silica gel plates (60F-254) with UV light and 7% ethanolic phosphomolybdic acid and heat as the developing agent. E. Merck silica gel 60 (particle size 0.040-0.063 mm) was used for flash chromatography. All reactions were carried out under an argon or nitrogen atmosphere with dry, freshly distilled solvents under anhydrous conditions unless otherwise noted. Yields refer to chromatographically and spectroscopically (lH NMR) homogeneous materials, unless otherwise noted. Bradylcinin binding assays were performed by Dr. Jan Rosenbaum at Procter & Gamble following a published protocol (29). Structures of the y-turn mimetics studied here were built using the MacroModel molecular modeling program (30), employing the MM2 force field. Conformational searches for low energy ring conformers utilized the MacroModel Batchmin program, retaining only methyl groups at positions of chain extension from the ring. Each conformation was minimized using the MacroModel-MM2 force field by the block diagonal Newton-Raphson method, and compared with idealized y-turns. Solvent effects were roughly taken into account by employing a distancedependent dielectric constant with a cutoff distance of 20 A.
RESULTS
AND DISCUSSION
Conformational
Analysis.
In comparing mimetic structures with that of a peptide y-turn,
we have concentrated on three parameters that particularly reflect the positioning of the peptide chain as it extends away from the turn (or the mimetic): carbons; the torsional angle between the C,‘C’(=O) bonds in the mimetics);
the distance between the C,’ and CoLi+2
and Ni+2Cai+2 bonds (and the equivalent
and the 4-atom root-mean-square
distance between the peptide C&,
C’(=O), Ni+2, and Cai+2 atoms and the equivalent y-turn mimetic atoms. For comparison, we have used the ‘y-turn observed in the crystal structure of a cyclic pentapeptide, cycle-(Gly-ProGly-D-Ala-Pro)
(31). In calculating energy minimized mimetic ring conformations,
chain extension was truncated with single methyl groups corresponding
the peptide
to the C,’ and C&+’
carbons. Each isomer conformations,
with
of the lactam mimetic, the ring exhibiting
1, can exist in three relatively
various puckering
low energy
patterns that position the chain
extension bonds in differing orientations. Table 1 lists the conformational energies, the CaiCi(=O)Ni+2Cai+2 torsional angles, the CaiC,i+2 distances, and the 4-atom C&Ci(=O)Ni+2Coi+2 rms differences between the y-turn peptide crystal structure and the four lactam conformers for each of the two isomers. From the values listed in Table 1, it can be seen that the C,‘C’(=O)Ni+2Ca’+2 torsional angle and C,iCai+2 distances for the first and third conformers of the S-isomer are closer to those observed in the peptide y-turn than are those for the other conformers.
The rms deviation from the peptide crystal structure ‘y-turn for these conformers is
also smaller than for the other conformers.
An overlay of the low energy lactam conformer with
the peptide y-turn region is shown in Fig. 2. The energy separation between conformers 1001
1,2 and
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Table 1. Conformr+tipns of lactam y-turn mimetics, as calculated with MM2 force fields. For comparison, the Ca’C’(=O)Ni+2(H)C,i’2 torsional angle and C,‘-C, i+2 distances are also listed for the y-turn in the crystal structure of cyclo-(Gly-Pro-Gly-D-Ala-Pro).29 System/ Conforme?
Energy (kcal/Mol)
C,iCi(=O)Ni+2C,i+2
c,i-cai+2
Torsional Angle
Distance (A)
CaiCi(,0)Ni+2Cai+2
RMS (A)
Lactam 1 S (R)
0.0
46” (-46’)
4.9
0.29 (0.3 1)
Lactam 2 S (R)
1.35
82” (-82’)
4.7
0.42 (0.44)
Lactam 3 S (R)
1.51
16” (-16”)
5.0
0.29 (0.29)
Lactam 4 S (R)
4.29
107” (-1070)
4.6
0.52 (0.54)
Pentapeptide Crystal29
--
+8”
5.2
-.
%alues for R-isomers are shown in parantheses. The S and R isomers form mirror images, thus the conformational energies and C&-C,i+2 distances are equal, and dihedral angles simply reverse sign.
3 is relatively
low, and the lactam ring is reasonably
flexible, thus it is probable that there will
be some conformational averaging between the three low energy conformers. Based on this analysis,the low energy conformers for the lactam mimetic 1 provide good agreementwith the observedy-turn conformation. The CaiCai+:!distance is only slightly shorter than that observed experimentally, the rms deviation is about 0.3 A, and the torsional angle is reasonably close to that observed experimentally. Synthesis. The synthesisof 1 is shown in Scheme1. The acid chloride of 5bromovaleric acid participates in a [2+2] cycloaddition via its derived ketene with the N-benzylimine of formaldehydeto provide the bromoazetidinone2 in 88%. Condensationwith glycine methyl ester proceededsmoothly to provide 1 in 45% yield, via the intermediacy of azetidinone 3.
,’
.’
Figure 2. Overlay of low energy lactam conformers with crystal structure y-turn.
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0
NH*.HCI (5 eq.) M&&C KHC03 (5 cq.) CH3CN + Hz0 (9: 1) * retlux, overnight
1) (COCI), CHzCllL reflux. 2 hr
OH
2) Phpy?IAph c
Br
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N ‘Ph BF3-OEt7 Et,N CH,Cl, -78 c r.t.
2
cc
NH
N
\
0
Ph
COzMe
CO?Me
1
3
Scheme 1. Synthesis of y-turn mimetic ring system.
We prepared
hybrid mimetic 4, shown in Fig. 3, by solid phasepeptide synthesison an
Advanced Chemtech200 synthesizerusingstandardprotocols(32) from Boc-Arg-(Tos)-Pam resin (Advanced Chemtech). The y-turn mimetic intermediate 5, preparedin analogousfashion to 2,
Arg -Pro --Pro -Gly
-Phe
4b
0
N
Boc-N I H
OH
$
0
‘-’ I Ph
H
5 Figure 3. Structures of y-turn mimetic intermediate, incorporating the y-turn mimetic.
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Table 2. Displacement of [3H] BDK in NC10815 cell cultureassaya
Concentration(M)
% ControlSpecificBinding(* SEM)b
10-s
91 *3
10-6
54 f 0
10-s
28 * 1
lOA
8*1
10-5MBDK
0 fO.l
aReceptorbindingassaywasperformedin membranes preparedfrom NG108-15cells. The competitionbindingassaywasconductedat 25 “C for 20 min in the presenceof a protease inhibitorcocktail,followingpublishedprocedures (33). bControlSpecificBinding= 2998f 192 DPM, NSB: 10e5BDK = 82 f 4 DPM. All samplesweredonein triplicate. SEM represents standarderror of the mean.
was incorporated by preactivation (5 minutes, O-5” in DMF) with BOP reagent (33). Protected amino acids were incorporated by double couplings of their respective symmetrical anhydrides or, for Boc-Arg(Tos), as its HOBt ester. Couplingsjudged incomplete by qualitative ninhydrin assay were capped with acetic anhydride. The peptide was removed from the resin using anhydrousHF/anisole (9: 1) at O-S’, and purified by reverse-phaseflash chromatography (bonded phaseethyl (C,), Merck) followed by HPLC on a C,, column using a water/acetonitrile gradient in 0.1% trifluoroacetic acid to separatethe two diastereomersof 4. Retention time for the first diastereomer was 16 m, corresponding to an elution at 42% CH,CN, that for the second diastereomerwas 19.9 m, correspondingto elution at 48% CH&N.
The ‘H and PDMS data for
the two diastereomerswere consistentwith their structures. Mimetic Activity.
The two diastereomersof 4 were assayedfor their ability to bind to
NG108-15 cells (29). The 19.9 min retention time diastereomerproduced a concentrationdependentdisplacementof 3H-labeledbradykinin (BDK) in the micromolar range (TabIe 2). The other diastereomerdid not bind to this preparation.
CONCLUSIONS In summary, we have described the synthesisof a y-turn mimetic. Incorporation of mimetic 5 into the bradykinin sequence,thereby mimicking a proposedbioactive conformation, leads to an analog that possesses micromolar binding affinity for the bradykinin receptor in NG108-15 nerve cells. Although the affinity of 4 for the BDK receptor is significantly lessthan bradykinin itself [Kd = 3 x 10“’ M (29)], this result lends further support to the presenceof a reverse turn (either y or, more probably, p (5-7)) at this site. Further investigations with second generation hybrid mimetics (26,28) are in progressto clarify the bioactive conformations of bradykinin, and will be reported in due course. 1004
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ACKNOWLEDGMENTS This research was supported in part by NIH grant GM38260. Additionally, MK wishes to thank the Camille and Henry Dreyfus Foundation, the Searle Scholars Program/The Chicago Community Trust, the NSF (Presidential Young Investigators Award), Monsanto, Procter and Gamble, Schering, Searle and Syntex for matching funds, and the American Cancer Society (Junior Faculty Fellowship) for generous financial support. The molecular modeling facilities were provided, in part, by a BRSG shared instrumentation grant. We thank Dr. Clark Still, Columbia University, for providing the MacroModel and Batchmin programs for our use, Dr. David O’Conner (Procter and Gamble) for obtaining the PDMS spectra for the two diasteromers of 7, Dr. Jan Rosenbaum (Procter and Gamble) for the results of the bradykinin assay, and Richard Shen for HPLC purification.
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