Bioconjugate Chem. 2000, 11, 679−681
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Caged Chemotactic Peptides Michael C. Pirrung* and Sandra J. Drabik Department of Chemistry, Levine Science Research Center, Box 90317, Duke University, Durham, North Carolina 27708-0317
Jasimuddin Ahamed and Hydar Ali Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Received January 11, 2000; Revised Manuscript Received May 31, 2000
This work has as its ultimate goal the creation of a concentration spike of a chemoattractant peptide in a time-resolved and spatially defined way using a light pulse. This strategy requires “caging” the peptide with a photochemically removable group. Model studies used alanine ethyl ester in reductive amination with nitrobenzaldehydes to form two different N-nitrobenzyl derivatives. An fMLF peptide bearing these two N-terminal nitrobenzyl groups was also prepared. The yield and kinetics of their deprotection to return the fMLF peptide were determined. It was established that the caged peptides have vastly reduced biological activity as chemoattractants, as designed.
INTRODUCTION
Chemotaxis is the directed movement of cells along a chemical gradient. It is displayed by a variety of prokaryotic and eukaryotic cells including bacteria, cellular slime molds, and leukocytes (neutrophils) (1), which respond to factors derived from complement, bacterially derived lipids, or N-formylated peptides. The cellular mechanism by which neutrophils rapidly move to sites of infection signaled by chemoattractants remains enigmatic. Even though the receptors involved have been sequenced and many of the molecules involved in neutrophil adherence and traction identified, the essential mechanisms that control and regulate the neutrophil motor remain obscure. The ability of short N-formylated peptides to induce chemotaxis in mammalian leukocytes was originally discovered by Schiffmann et al. (2). Extensive follow-on biological studies have ensued (3). Chemotactic peptides act at a highly specific receptor, the formyl peptide receptor (FPR), located in the plasma membrane of neutrophils (4). Interaction of the peptide with the G-protein-linked receptor can initiate a number of intracellular events including lysosomal enzyme release, superoxide formation, and redistribution of cations, especially Ca2+. Much of the early research focused on the rabbit neutrophil, while more recently the human receptor has been under investigation (5). It has been cloned and expressed (6), and receptor subtypes of the hFPR are known (7).
Numerous reports have discussed structure/activity relationships and conformational studies of N-formyl peptide analogues, including backbone-modified peptides (8). A variety of synthetic peptides have been prepared and tested for chemotactic activity, and novel agonists have been discovered using autocrine peptide library methods (9). The most active chemotactic peptide is N-formyl-L-methionine-L-leucine-L-phenylalanine (fMLF) which has an ED50 for chemotaxis of 8 × 10-11 M. Freer and co-workers provided detailed studies of peptides with substitutions at positions one (Met) and three (Phe) (10), as well as at position two (Leu) and C-terminal modifications (11). Biological activity appears to reside, in order of importance, in (a) the formyl group, (b) methionine at position one, and (c) phenylalanine at position three. The leucine residue can be replaced by other aliphatic residues. Strategies for conjugation of chemotactic peptides to reactive or fluorescent groups based on these structure/ activity relationships have been developed (12). Because of the role of the chemotaxis receptor in time/ space functions of the cell, techniques that address time/ space relationships are needed to study the FPR. As an example, the intracellular movement of the FPR during chemotaxis has been monitored through fluorescence microscopy (13). Once occupied by ligand, the receptor rapidly signals and is then desensitized and internalized, making it difficult to study. Novel chemotactic peptide analogues would allow a closer focus on the cellular functions of neutrophil leukocytes. For example, a caged (inactive) peptide that could be activated rapidly in situ would allow the regulation of these cellular events to be studied with greater precision. This concept draws on earlier studies of bacterial chemotaxis using caged amino acids (14). The caging of peptides and proteins has been a subject of significant recent interest (15). The peptides that have been caged include those that inhibit cAMP-dependent protein kinase, calmodulin, myosin light chain kinase, and neuropeptide Y. The proteins that have been caged include G-actin, myosin, thrombin, and ion channels. As
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680 Bioconjugate Chem., Vol. 11, No. 5, 2000
powerful as these methods have been in time-resolved studies of cell motility, inter alia, in most cases the caged derivative can only be prepared by direct reaction of the native biomolecule with a chemical derivatizing reagent. As this process is generally conducted in aqueous solution, the diversity of chemical reactions that can be used for derivatization is limited, limiting the types of linking chemistry and photoremovable groups that may be used. In the work described here, chemical synthesis enables the preparation of an optimal compound that would be unavailable from the native peptide. On the basis of earlier work showing that substitutions of photoremovable groups at the C-terminus of the chemotactic peptide do not affect its activity (W. A. Sherwin, unpublished material), direct functionalization of the N-formyl residue was instead performed. Early efforts to exploit the dimethoxybenzoin carbamate photoremovable group previously developed in our laboratory (16) failed at the final formylation stage, requiring an alternative protecting group. Some of the most effective photoremovable groups are nitrobenzyl derivatives, which upon irradiation undergo an internal redox process to produce a hemiaminal (17). This decomposes to provide the free amide and a nitrosoaldehyde. To maximize the long-wavelength absorption of the arene, electron-donating groups are used.
Pirrung et al.
Formylation by the formic anhydride procedure produces Nv-CHO-MLF and Pip-CHO-MLF as the methyl esters, which are readily hydrolyzed to the acids (∼70%). The photochemical deprotection reaction was shown to return the fMLF peptide ester by its isolation in good yield (reaction 5). Negligible amounts of methionine
RESULTS
To assess the utility of nitrobenzyl chemistry for protection of the formyl peptide N-terminus, alanine ethyl ester was used as a model. The most reliable method for preparation of its N-nitrobenzyl derivatives proved to be reductive amination with nitroveratraldehyde (reaction 1) or nitropiperonaldehyde (reaction 2).
The resulting secondary amines can be formylated with in situ-generated formic anhydride. Because of the superior photochemical deprotection properties of nitrobenzyl derivatives with benzylic methyl substitution (18) or electron-withdrawing groups (19), a number of efforts were made to prepare such derivatives of Ala-OEt. However, it was not possible to reductively aminate AlaOEt with nitroacetophenones to incorporate a benzylic methyl group, and while nucleophiles such as cyanide could be added to the nitrobenzaldehyde imines, the resulting secondary amines could not be formylated. Photochemical deprotection of the two N-nitrobenzyl formylalanines in acetonitrile in a Rayonet photochemical reactor with 350 nm lamps proceeded cleanly and at similar rates (0.42 s-1), as analyzed by HPLC. The preparation of the caged peptides was accomplished by applying a similar reductive amination protocol to synthetic MLF-OMe (reactions 3 and 4) to produce the N-nitroveratryl or N-nitropiperonyl peptides.
oxidation, a side reaction sometimes of concern in photochemical deprotection processes, were observed, as determined by HPLC and MS analysis in comparison to the known compound (20) (authentic sample synthesized from methionine sulfoxide). The rates of the deprotection of Nv-CHO-MLF-OH and Pip-CHO-MLF-OH were determined using HPLC analysis. A modest but interesting 4-5-fold acceleration of deprotection is observed (to 1.9-2.4 s-1) as compared to the simple alanine ester model. The quantum yields of deprotection of the two chemotactic peptides were determined using HPLC analysis of two-component photochemical reactions containing a nitrobenzyl amide whose deprotection has a known quantum yield. While Φ for the caged chemotactic peptides is relatively low (2 and 4%), these quantum yields are comparable to those of other nitrobenzyl-caged biological effectors and should not limit the utility of these caged peptides in studying leukocyte chemotaxis. The ability of the peptide methyl esters to trigger β-hexosaminidase secretion in rat basophilic leukemia (RBL-2H3) cells stably expressing fMLF receptors was evaluated using an assay previously reported (21). Figure 1 shows the data for NV-fMLF-OMe and fMLF-OMe. All data are summarized below. The N-substitution leads to a 3-4 orders of magnitude drop in activity, meaning that photochemical deprotection of these caged chemo-
Caged Chemotactic Peptides
Figure 1. Secretion of β-hexosaminidase by N-formyl peptides in RBL-2H3 cells expressing fMLF receptors. RBL-2H3 cells were stimulated with fMLF-OMe and NV-fMLF-OMe compounds at different concentrations for 10 min. The OD was taken at 400 nm wavelengths in a ELISA reader. Values are the mean of single triplicate determinations and representative of three separate experiments.
tactic peptides should permit activation of leukocytes to chemotaxis on microsecond time scales, the rate of photochemical removal of nitrobenzyl groups after a light pulse. ACKNOWLEDGMENT
Financial support provided by NIH AI-42151. The assistance of L. LaBean in administrative support of this work is greatly appreciated. Supporting Information Available: Experimental descriptions. This material is available free of charge via the Internet at http://www.pubs.acs.org. LITERATURE CITED (1) Harvath, L. (1989) Annu. Rep. Med. Chem. 24, 233-241. Didsbury, J. R., Uhing, R. J., and Snyderman, R. (1992) In Mononuclear Phagocytes (R. van Furth, Ed.) pp 413-424, Kluwer, Amsterdam, . (2) Schiffmann, E., Corcoran, B. A., and Wahl, S. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1059-1062. (3) Prossnitz, E. R., and Ye, R. D. (1997) Pharmacol. Ther. 73102. Hallett, M. B. (1997) Bioessays 19, 615-621. Snyderman, R., Smith, C. D., and Verghese, M. W. (1986) J. Leukoc. Biol. 40, 785-800. Ali, H., Richardson, R. M., Haribabu, B., and Snyderman, R. (1999) J. Biol. Chem. 274, 6027-6030. (4) Becker, E. L., Bleich, H. E., Day, A. R., Freer, R. J., Glasel, J. A., Latina, M., and Visintainer, J. (1979) Biochemistry 18, 4656-4668. (5) Bommakanti, R. K., Klotz, K.-N., Dratz, E. A., and Jesaitis, A. J. (1993) J. Leukoc. Biol. 54, 572-577. Lala, A., Gwinn, M., and De Nardin, E. (1999) Eur. J. Biochem. 264, 495499. (6) Boulay, F., Tardif, M., Brouchon, L., and Vignais, P. (1990) Biochemistry 29, 11123-11133.
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