In the Laboratory
Solid Phase Organic Synthesis and Combinatorial Chemistry: W A Laboratory Preparation of Oligopeptides George A. Truran,*† Karelle S. Aiken, Thomas R. Fleming, Peter J. Webb, and J. Hodge Markgraf** Department of Chemistry, Williams College, Williamstown, MA 01267; *
[email protected], **
[email protected] Background The initial report of solid-phase peptide synthesis appeared in 1963 (1). Over the past four decades the general field of solid-phase organic synthesis (SPOS) has grown enormously. The fundamental technique is based on polymeric resin beads to which a reactive functional group is bonded covalently. This immobilized group serves as the starting unit for the linear assembly of additional covalently bonded units. When the desired sequence of molecular building blocks is completed, the original covalent bond to the resin is cleaved to generate the new molecule as a separate entity. The original solid support reported by Merrifield was a copolymer of styrene and divinylbenzene. Currently a wide range of functionalized polystyrenes and polystyrene copolymers is available. The immobilized functional group typically is an amino, carboxy, halo, hydroxy, or phosphino group. The polymer is joined to the immobilized functional group by a connecting section known as a linker. The linker provides the covalent bond that is cleaved in the final step. The selection of polymer support and linker is dictated by the choice of reagents needed to deblock protecting groups, effect the couplings, and cleave the linker bond—for example, stability and lability to acids or bases. In the present set of experiments a copolymer of polystyrene (PS) and polyethylene glycol (PEG) is joined to an amine linker (Rink Tentagel amide resin) capped with a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group (2, 3) (see structure). A glossary of all abbreviations is included in the supplemental information.W
the result of these advances in strategies and instrumentation. Vast arrays of systematically varied structures can now be made and evaluated for pharmaceutical and agricultural chemicals, catalysts, polymers, pigments, and other industrial materials. At Williams College and the University of Connecticut (G.A.T., 1999–2001) we have developed an introduction to these new concepts for our undergraduate students. At Williams we have offered a month-long winter term on combinatorial chemistry. At UConn we have included these new experiments in an advanced laboratory course. In both settings the laboratory component involves the preparation of oligopeptides. The general scheme for a tripeptide (Scheme P , linker I) is based on the following conventions: polymer L , amino acid AA with carboxy terminus on the right and amino terminus on the left; a is piperidine, DMF; b is HBTU, DIEA, DMF; and c is TFA, H2O. The final product is shown with the carboxy terminus as an amide. 1
a
Fmoc–AA –OH
L – P → H2N–NH– L – P → Fmoc–NH– b 2
a
Fmoc–AA –OH
L – P → H–AA1–NH– L – P → Fmoc–AA1–NH– b a
L – P → Fmoc–AA2–AA1–NH– Fmoc–AA3–OH
L – P → H–AA2–AA1–NH– b a
L – P → Fmoc–AA3–AA2–AA1–NH– c
Fmoc
NH
CH
O
CH2
CO
NH
CH2
PSPEG
L – P → H–AA3–AA2–AA1–NH2 H–AA3–AA2–AA1–NH– Scheme I
MeO
Experimental ProcedureW
OMe
The principal advantage of SPOS is that the product is always associated physically with the resin bead. Thus reactions can be driven to completion by using excess reagents, and products can be separated readily and repeatedly from reactants by simple filtration. Among the notable advances in SPOS are the concepts of parallel synthesis and divergent synthesis. These strategies permit the rapid production of large collections of compounds (known as libraries). New technologies have been developed based on miniaturization, automation, and robotics. Advances in analytical methods have kept pace with these developments, and high-throughput screening is now standard practice. Combinatorial chemistry is †
Current address: Department of Physical Science, Westfield State College, Westfield, MA 01086.
A standard cycle of coupling, washing, deblocking, and washing requires approximately one hour. We find it expedient for students to work in pairs, an arrangement that facilitates weighing and mixing of reagents. The yields are good. The purity of the peptides is excellent. The apparatus is fashioned from a plastic syringe barrel and a detachable PTFE stopcock from a buret. An adapter is made from a short section of PTFE rod. The inside of the syringe barrel is fitted at the bottom with a polypropylene frit. The apparatus is illustrated in figures accompanying the supplemental material. Hazards Protective gloves and safety glasses should be worn at all times. The usual precautions should be observed for working with chlorinated, volatile, flammable, or acidic
JChemEd.chem.wisc.edu • Vol. 79 No. 1 January 2002 • Journal of Chemical Education
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In the Laboratory
liquids and for working at reduced pressure with suction filtration and rotary evaporation. None of the reaction procedures involves inherently dangerous operations. Results and Discussion These preparations of oligopeptides by SPOS illustrate the dual strategies of parallel and divergent syntheses in combinatorial chemistry. The former technique is utilized to generate a small library of tripeptides. The latter process is used to create a pair of pentapeptides, one of which is an enkephalin neurotransmitter known to be an opioid peptide in the brain (4). The resin of choice is Rink Tentagel amide resin (see above), which is suitable for chain building by Fmoc-protected amino acids. These two components constitute an orthogonal protection scheme in which the linker bond is base stable and acid labile, while the Fmoc group is acid stable and base labile. Another example of this protection scheme is the protection of the phenolic group of tyrosine as a tert-butyl ether, which is stable until the final step when treatment with TFA cleaves both it and the linker. Fmoc-protected amino acids are commercially available and are stable, crystalline solids. Amino acids without functionalized side chains are the most straightforward to use: Ala, Gly, Ile, Leu, Phe, Val. Most amino acids containing amino, hydroxyl, carboxy, or amide side chains can be used, if proper side-chain protection is utilized: Lys (Boc), Tyr (tBu), Ser (tBu), Asp (tBu), Gln (Trt). These derivatives, which are orthogonally protected, are stable to Fmoc/HBTU coupling cycles; the side-chain protection is removed at the final step during TFA cleavage. Certain amino acids are unsatisfactory because their side chains introduce complications that require specialized reagents and conditions: Cys, Met, Arg, His, Pro. In this project the three tripeptides prepared by parallel syntheses are H–Phe-Leu-Ala–NH2 (1), H-Gly-Phe-Val-NH2 (2), and H-Val-Ala-Tyr-NH2 (3). In all cases, the carboxy terminus is generated as an amide. In each case, one of the amino acids incorporates an aromatic ring, which permits TLC visualization by ultraviolet light. The two pentapeptides prepared by divergent syntheses start with the same pair of amino acids. The resin bed is then divided in half, and the sequences are completed in the standard manner to generate H-Tyr-Gly-Gly-Phe-Leu-NH2 (4) and H-Tyr-Ala-Ala-PheLeu-NH2 (5). The naturally occurring analgesic agent is 4.
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The tripeptides can be prepared and analyzed over the course of two laboratory periods; the pentapeptides require an additional period for isolation and complete analysis. We have found it convenient to have students conduct the tripeptide TLC and NMR analyses in a subsequent experiment during time afforded by recrystallization processes, melting-point determinations, reflux periods, etc. Conclusions This project provides an introduction to SPOS and combinatorial chemistry. Oligopeptides are prepared using modern techniques of polymeric resins as solid supports. Synthetic strategies, use of protecting groups, and the creation of libraries are incorporated into the experiments. Among the compounds synthesized is an endogenous painkiller. Acknowledgments We are indebted to Louis A. Carpino (University of Massachusetts-Amherst), William L. Scott (Eli Lilly & Co.), and Chia-Yu Hwu (University of California-San Diego) for encouragement, supplies, and helpful correspondence. We thank Lawrence J. Kaplan for assistance with the figures and Deborah Morandi for preparation of the manuscript. We are grateful for generous financial support from Pfizer Inc. W
Supplemental Material
Supplemental material for this article is available in this issue of JCE Online. It includes information on chemicals and equipment, purification of reagents, experimental procedures, yields, TLC data, NMR spectra, reaction mechanisms, notes for the instructor, a glossary of terms, and a list of general references to SPOS and combinatorial chemistry. Literature Cited 1. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154. 2. Carpino, L. A. Acc. Chem. Res. 1987, 20, 401–407. 3. Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161–214. 4. Bilsky, E. J.; Egleton, R. D.; Mitchell, S. A.; Palian, M. M.; Davis, P.; Huber, J. D.; Jones, H.; Yamamura, H. I.; Janders, J.; Davis, T. P.; Porreca, F.; Hruby, V. J.; Polt, R. J. Med. Chem. 2000, 43, 2586–2590.
Journal of Chemical Education • Vol. 79 No. 1 January 2002 • JChemEd.chem.wisc.edu
