Synthesis of an Enkephalinase Inhibitor, Gly-Gly-PheLeu, by Continuous-Flow Peptide Synthesizer Magdy N. Iskanderl a n d Paul A. Jones School of Pharmaceutical Chemistry, Victorian College of Pharmacy, Ltd., 381 Royal Parade, Parkville, Victoria. Australia 3052 Solid-base peptide synthesis (SPPS) was conceived in t h e early 1960's b y Merrifield2 a n d Letzinger a n d Kornet.3 After rapid development of t h e SPPS technique i n t h e 1 9 7 0 ' ~ t,h~e Fmoc-polyamide method of SPPS became a simple a n d efficient method for peptide synthesis. It involves a polymeric support i n a reaction column, a n d t h e amino acid derivatives, reagents, a n d solvents a r e passed through i n continuous stream. During t h e coupling t h e amino acid derivative solution is recycled through t h e column, while during t h e washine a n d d e ~ r o t e c t i o noeriods. t h e column eluant is passedbirectly waste. ~ i assemhly e of t h e continuousflow DeDtide svnthesizer was carried o u t i n our lahoratorv with diffic;lty a n d has generated great interest among our students. T h i s experiment has many educational merits:
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(1) The chemical synthesis of peptides is an expanding and evolving aspect of hiotechnology (2) It is an introduction of SPPS for undergraduate students. (3) I t is an opportunity for students to experience current methods of SPPS. (4) The synthesis protocol for the tetrapeptide and identification by TLC can be done within 5 h; the HPLC purification and biochemistry (not included in this paper) can he performed in another 4. (5) Gly-Gly-Phe-Leu is one of the biologically active pep tide^.^ (6) The PMR spectrum (Fig. 1) of this peptide provides diversity in chemical shifts and couplings for different protons. W e therefore recommend this experiment a s a n undergradua t e medicinal chemistry exercise. Instrumentation This manually operated peptide synthesizer is constructed in our laboratory workshop and is an effective instrument of remarkable simplicity. Equipment, Specifications, a n d Prices6 1, PVC box (hekht 36 em,base 25 X 25 em, top 12 X 25 X 5 em, no back), $60. 2. Pressure displacement pump, FMIT lab pump PRP #A04040 or equivalent, e.g., Edlex Model E, $1,875. 3. UV monitor, ISCO type 6-1140001-16 (filter 280-310 nm) or equivalent, e.g., JASCO Model UVIDEC-100-V, $2,850. 4. UV chart recorder, ISCO VA-5 or equivalent, e.g., Hitachi, Perkin-Elmer 165, $1,800. 6. Six-pas. Teflon rotary valve (0.8 mm bore), four-pos. Teflon rotary valve (0.8 mm bore), one female leur, Teflon tube-connec-
' Author to whom correspondence should be addressed.
Marrifieid, R. 6. Fed. Prm. 1962, 21, 412. Letsinger, R. L.; Kornet, M. J. J. Am. Chem. Soc. 1963, 85,30453046. Sheppard, R. C.. Ed. Peptides 1971: Nesvndba: Amsterdam. 1973: -. a I l l . Malfroy. 8.; Swerts. J. P.; Guyon, A,; Roques. 6. P.; Schwartz. J. C. Nature (Lond.)1978. 276, 523-526. All prices listed are for new equipment and in Australian dollars. The prices of the pump. UV monitor; and UV chart recorder can be reduced dramatically by purchasing them a s secondhand or demonstration models, as we did.
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Journal of Chemical Education
Figure 1. PMR specnum of H-Gly-Gly-Phe-Leu-OH
in 020at 400 MHz.
tor kit in. polypropylene (each kit contains tube-end fittings with lock nuts, 10 gripper fittings, and five coupling bodies), complete column: Omnifit 10 X 100mm, and Teflon tubing L/3zin. X %s in. X 10 f t (all from Activon Scientific Products, Victoria, Australia), $661. (Omnifit's address in the United States is P.O. Box 56,2W5 Park Street, Atlantic Beach, NY 11509.) Construction of the Peptlde Synthesizer The peptide synthesizer parts (valve 1, valve 2, pump, column, UV detector, two solvent reservoirs, and injector port) are all connected with Teflon tubing (% in. and Ks in.), and the end of each joint fitted with tube-end fitting. If necessary, any two end fittings can be connected through a coupling body. As shown in Figure 2, valve 2 (six ways) is connected to thedimethylformamide bottle, the 20% piperidine in dimethylformamide bottle, and the amino acid injection port. This injection port is composed of a female leur and a syringe filter. Only three of the six ways in valve 2 are used. The centerway in valve 2 is connected to valve 1through way 6. Valve 1is also connected to the waste bottle outlet of the UV detector, and the pump inlet through ways 2, 3, and 4. Ways 1and 5 in valve 1 are connected. The pump outlet is connected to the top of the reaction column, which in turn is connected to the inlet of the UV detector. Valve 1in Figure 2 is in the flow position. Description of the System The solid-phase peptide synthesis resin (derivatized pepsyn K)is placed in the reaction column, where the assembly of the peptide takes place. This column is located on the front panel of the instrument (Fig. 2). The 20% pipDMF, DMF, and amino acid derivative to be passed through the resin are selected by the six-way solventselect valve (V2), and delivered by the pump (placed inside the PVC box). The flow and recirculate modes are selected by the flow1 recirculate valve (Vl). In the flow mode (3.5 mLlmin), the washing of the resin (with DMF), deprotection (treatment with 20% piperidine in DMF), and Loading of amino acid derivative selected are pumped through the reaction column and UV flow cell and out to waste. In the recirculate mode, the amino acid derivative and 1hydroxybenzotriazole (HOBT) are continuously recycled through the reaction column, flow cell, and the second valve by the pump (durine cou~lineof the amino acid derivative). The amino acid derivative t o be coupled to the resin is introduced through the 5-mL glass syringe located on the front of the instrument. Both the DMF ~
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Figure 2. Diagram of and picture of the peptide synthesizer.
and the 2Wc piplnMF reservoirs are 1-L hott1e.r (Duranl. Horh are placed in a tray on the top of the instrument. The wastr b o t t l ~rs located on the left-hand side of the mrtrument.
Pre-class Preparations Stortingmoteriols and reagents. The Fmoc and Boc amino acids were purchased from Auspep Pty LM; pepsyn K, Milligen, ethanediamine, BDH, 4-hydroxymethyl-phenoxyacetieacid, Milligen (fully derivatized pepsyn K-type resin is available from Milligen). Dimethylformamide was distilled at 15 mmihp 50-60 'C through a fractionating column, and piperidine was distilled over sodium hydroxide in a nitrogen atmosphere. Other reagents, such as dicyclohexylearhodiimide (DCCI), 4-dimethylaminopyridine (DMAP), and HOBT were purchased from Fluka. Resin deriuntizotion. If the derivatized resin is not available, the followingsteps need to he followed. Pepsyn K (10 g) was mixed with ethylenediamine (100 mL) with occasional swirling. (The resin is rather fragile and may break up during prolonged shaking.) After 20 h, the excess reagent war decanted off, and the resrn gently waahed wrthDMFfJ X:iOmL).TothrruspendedresininDMFr3 X30ml.l r055y. 3 m h 0 and wereadded I-hvdraxvmrthsl~h~nox~~reticacid HOBT (0.41 g,-3 m ~ )with , bceasi&a~ swirling, and ieft for 18 h. The suspension was decanted and washed with DMF (5 X 30 mL). Coupling the C terminal of the first amino ocid. A solution of Fmae leucine anhydride (3.40 g, 5 mM) in DMF (10 mL) was added to the resin suspension (10 g, 1.0 mM) in DMF (80 mL). After a 2min premix, the DMAP solution (0.12 g, 1.0 mM) in DMF (5 mL) was added, followed by the N-methyl marpholine solution (0.51 g, 5 mM) in DMF (5 mL). The reaction mixture was left for 2 h with occasional swirling, and the reagents were then decanted. The derivstized resin was washed with methanol and dried under vacuum. t'rrparorwn 01 p~nrn/llrorophenylrstrr o/ Fmoc ominu ocid ~ e n e r a prorrdure,. l T o n stirred, ice-cooled solution of the Fmuc nmrnoacid (2 mMl and penrafluoroph~nolt2m.MI in dry erhylar~. ~
~
Figure 3. Derlvatization of the resin and synlhesis protocol. Abbreviations: Fmoc: fluorenylme~oxycarbonyl;Bac: t-butoxycsrbonyl; Leu: leucine; Phe: phenylalanine: Gly: glycine.
tate with DMF (2-10 mL), DCCI (2 mM) was added, and stirring continued for 1h a t O'C and 1h a t room temperature. Dicyclohexylurea was filtered off, and the solvent was evaporated in vacuo at 40 "C using a rotary evaporator. The Boc-Gly was treated in the same way. Procedure Students will he provided with the derivatized resin (250mg, 0.07 mM/g) that has had an aeid-labile handle coupled with the first residue, leucine (Fig. 3). This resin is placed in an Omnifit reaction column ready for deprotection of the Fmoc group from the Leu. Stepwise elongation of the target chain is performed using the pentafluorophenyl ester of Fmoc-Phe, Fmoc-Gly, and Bac-Gly (Fig. 3). Synthesis Protocol 1. Turn on the UV monitor, chart-recorder, and pump. 2. Set valve 2 to DMF-WASH and valve 1 to FLOW for 10 min only. 3. Set valve 2 to 20% piperidinelDMF for 10 min only. 4. Set valve 2 to DMF-wash for 10 min only, and then turn off the pump. 5. Dissolve the second amino acid, N-Fmoe-L-phenylalaninepentafluorophenyl ester (29.3 mg, 0.053 mM), in diemthylformamide (1mL containing HOBT, 0.053 mM). (The molar excess of theaminoacid is threefold of the resin, i.e.,0.053 mM.) Without heating, transfer the clear solution into the syringe provided. The final residue, Gly, is also coupled as the Boc-Gly-Opfp. 6. Place the filled syringe in the injection port. 7. Set valve 2 to AMINO ACID and valve 1to FLOW, turn the pump on, and inject the amino acid. (Normally the amino acid solution gets into the system without putting pressure on the syringe.) Wash the syringe with a further 1mL of DMF. During Volume 67
Number 2
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171
DMF
DMF Wash
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-.+
Recirculate
/I
Amino Acid
-
DMF
* wash
I\
he done in a fume hood). Afrer 30 minuwr'stirring. the rerin wa* filtered on a sintered funnel in the fumehood, washed again with W.4, 12 ml.1, and the combined liltrace and washing evaporated using a stream of nitrogen gas in the fume hood
Identification by Thin-layer Chromatography The crude peptide from the cleavage and isolation procedure was dissolved in diethyl ether (5 mL). The Rf = 0.68 was determined using TLC plates (plastic sheets, silica gel 60, thickness 0.2 mm, Merck), solvent system CHCL:CH30H:ACOH:H20 (45:30:6:9). The developed TLC plate was visualized with ninhydrin spray.
Figure 4. Typical trace for one cycle of amino acid addition and deprotectian.
this operation the volume added t o the column r i a the syringe is disolaced into the waste container. 8. ~mkediatelyfollowing the last DMF wash, set valve 1to RECIRCULATE. Fmoc amino acid coupling should lie for about 25 min (Fig. 4). 9. Set valve 1 t o FLOW and valve 2 to DMF-WASH for 10 min only. 10. Repeat steps 3-9 far each amino acid addition.
Cleavage and Isolation of the Tetrapeptide from the BocAmino Acid-Resin Assembly The -~~~resin was transferred from the reaction column to a sintered funnel to filter off the remaining DMF and then washed with ethyl alcuhol t3 X 4 mL). The dried resin was mixed with 95% aqueous trifluoroacetie acid (TFA, 5 mLl at room temperature tthis should ~~
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Journal of Chemical Education
Purification by HPLC The residue was dissolved in water (10 mL) and freeze-dried. The crude product was applied in water t o a Brownlee semi-prep column and eluted with agradient of A 0.1% TFAA/H20and B 0.1% TFA/60% ACN/H20.The main peak was collected and freeze-dried, yielding 5 mg 73% (found: Gly, 2.03: Phe, 1.02: Leu, 0.94: and theor: Gly, 2.0: 1):60.78,d(3H,J8.4 Phe, 1.0:Leu,1.0).PMR(4M1MHzinD20,Fig. and J 14, CH3-),0.81, d (3H, J 6.6 and J 14, CH& 1.50-1.53, m (3H, CH-CH), 2.92, dd ( l H , J 14 and J 8.4, benzylic proton), 3.07, dd ( l H , J 14 and J 6.6, henzylic proton), 3.76, s (2H, - C H r o f terminal Gly), 3.85, s (2H,-CHrof middle Gly), 4.23, dd (lH, J 8.4and J 6.0, CH-CO2HL4.57, dd ( l H , J8.0and J 6.0, CCHz-CH), 7.17-7.30, m (5H, aromatic protons). Safety Precautions Trifluormretir orid. emrorive and toxic. N,N.Dimethylfmnamrde: irritant. Studentdshould beadvised to handle thesechemicals in fume hoods.
Acknowledgment We t h a n k Auspep Pty. Ltd. for carrying o u t the amino acid analysis.
