Note. Optimization of protocols for solid-phase synthesis on a flexible crosslinked support: Synthesis of [Leu 5 ]enkephalin

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1 Indian Journal of Chemistry Vol. 428, April 2003, pp Note Optimization of protocols for solid-phase synthesis on a flexible crosslinked support: Synthesis of [Leu 5 ]enkephalin I M Krishna Kumar & Beena Mathew* School of Chemical Sciences, Mahatma Gandhi University, Kottayam Kerala, India nl.net.in Received 28 August 200 I; accepted (revised) 8 November 2002 An optimized protocol for the solid-phase synthesis of peptides on high capacity I,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) resin is illustrated by synthesizing I Leu 5 ]enkephalin and hydrophobic tripeptides in both free and protected form using t-butyloxycarbonyl (Boc)/benzyl (Bzl) chemistry. The improvements and developments made in the field of solid-phase peptide synthesis (SPPS) and commercial availability of synthesizers and reagents have helped much in selecting this method for the synthesis of any medium to large sized peptides. But the success of this method is often complicated by the compatibility difference of the polymer support with the growing peptide chain 1 and formation of rigid secondary structures on the polymer suppore 3. Among the various support materials developed, polar and flexible supports reported by Pillai et a/. 45 were found to be efficient for the synthesis of hydrophobic peptides. Since these are styrene based supports, they possess all the characteristic features of conventional Merrifield resins (divinylbenzene-crosslinked polystyrene, DVB-PS). In the present note, we report the synthetic protocol for the use of a new amphiphilic support, BDDMA-PS, for the synthesis of several model hydrophobic peptides and [Leu 5 ]enkephalin. Results and Discussion Microporous spherical beads of copolymer BDDMA-PS was prepared in 90% yield by the suspension po lymerization technique (Scheme 1). The resin swells better in both polar and nonpolar solvents. At 2 mol % crosslinking, the dry resin swelled to an extent of 1.2 times its dry volume in water. The swelling capacity of the resin was measured by placing 1 g resin in a sintered disk (G3) immersed in solvents. After 24 hr, it was transferred to a centrifuge tube and excess solvent was removed. The stick and its contents were then weighed. A blank experiment was also conducted using an empty sintered stick. The solvent absorption (ml/g) of dry resin in various solvents are DCM 8.7; NMP 9.2; DMF 7.5; toluene 10.1 and methanol 2.8. This amphiphilic nature is highly favoured in peptide synthesis, since solvents of widely varying polarities are to be repeatedly used for the synthesis. The resin was characterized by IR and 13 C-CP-MAS-NMR techniques. IR (KBr): 1720 em ' (carbonyl group of BDDMA), 13 C-CP-MAS-NMR: an intense peak at ppm (aromatic carbons), a small peak at ppm (para carbon of styrene) and a singlet at 40.4 ppm (methylene carbons of the polymer backbone). The physical stability and morphology of the resin was followed by scanning electron microscopy. The fresh resin beads were with shiny smooth surface. The Scheme I - Preparation of BDDMA-PS

2 NOTES 917 continuous shaking of the DCM swelled resin for 48 hr did not make any change in the surface characteristics showing its extreme stability. The resins were scanned under microscope (18.0x magnification) to find out the clumping nature, since clumped resins may impart synthetic problems especially during the draining of solvents and reagents. The polymer was then exposed to rigorous SPPS condition to evaluate the chemjcal stability. The resin showed no change in its FfiR spectrum even after treatment with reagents such as 30% TFA/DCM, 20% piperidine/dmf and neat TFA for 24 hr. The resin was also treated with 6 N HCI/propionic acid (1: 1 v/v), 2M NaOH at 120 C for 24 hr. Thus, it can be seen that the diacrylate crosslinks in the polymer network were extremely stable. The BDDMA-PS resin was functionalised to -2 mmol of chlorine/g level by chloromethylation 6. Usually such resins gave poor synthetic results due to the site-site interactions and local cluster formations in the resin matrix 7. To synthesize the peptides, their C terminal amino acid was first anchored to the chloromethylated resin. Both cesium salt method 8 and triethyl amine (TEA) method 9 were employed for this purpose. The cesium salt method gave more reliable results than the TEA method as the later reaction was time consuming, not quantitati ve and causes a certain degree of quatemisation leading to the formation of anion exchange resins (Table 1). A time-dependent esterification of Cssalt of Boc-amjno acid (Boc-Ala) to chloromethyl BDDMA-PS and 1 mol % DVB-crosslinked polystyrene (DVB-PS) resins in N-methylpyrrolidone (NMP) at 50 C showed that the reactions on BDDMA-PS are fast. The results showed that I 00 % incorporation takes place within 6 hr whereas the DVB-PS resin required 24 hr for quantitative incorporation (Figure 1). The high reactivity of BDDMA-PS resins can be due to the flexible and polar crosslinker which may allow free interaction between the reactive centers on the support and reagents in NMP. Taking [Leu 5 ]enkephalin as the test peptide, the following coupling procedures were applied to optimjze the solvent and coupling method compatible to BDDMA-PS support. All the couplings used 2.5 meg. excess of Boc-amino acid and extended for I hr. The completion of each amino acylation was checked by Kaiser test 10 and picric acid titration 11 The coupling methods used in the present study are: (a) Direct DCC mediated coupling in DMF; (b) Direct DCC mediated coupling in NMP-DMSO (5 %, v/v); (c) Preformed DCC/HOBt active ester coupling in DMF; and (d) Preformed DCC/HOBt active ester coupling in NMP DMSO (5 %, v/v) A comparison of these methods showed that active ester coupling is more effective than DCC mediated direct coupling. Among DMF and NMP-DMSO, the former was the best solvent for the direct DCC coupling, but both the solvents were equally effective for the active ester coupling. This is clear from the stepwise and total acylation time required for the chain assembly. In method (a), attachment of Tyr to Gly was extended to 60 min. In (b), coupling of Gly to Gly and Tyr to Gly were repeated twice. But in the methods (c) and (d), all the couplings were equally effective and completed within 30 min. The total time required for these methods are 3, 3.5, 2.5 and 2.5 hr, respectively. The superiority of DMF may be due to its comparable polarity with the growing peptide chain and BDDMA-PS support. So, an effecti ve swelling and solvati on of the functional si tes and polymer chain was effected which lead to the quantitative reaction. It was found that in method (a), a signifi cant chain loss of about 14.6 % was observed mainly due to racemisation. Table I - Details of C-terminal amino acid attachment to chloromethylated BDDMA-PS Chlorine capacity (mmollg) Amino acid Reaction time(hr)" Amino acid substi tution level ( mmollg)b Cesium salt method TEA method Gly 20 (48) 1.92 Ala 20 (48) 2.23 Leu 20 (48) 1.75 Val 30 (48) 1.75 lie 30 (48) 1.92 Pro 24 (48) "the reaction time required for TEA method is given in brackets b% conversion is confirmed by elemental analysis 1.97 (98 %) 1.87 (92 %) 1.80 (97 %) 1.60 (83 %) 2.04 (97 %) 1.43 (67 %) 1.53(91 %) 1.20(71 %) 1.58 (94 %) 0.81 (47 %) 1.74 (94 %) 0.98 (5 1 %)

3 918 INDIAN J. CHEM., SEC B, APRIL I. DVB-PS BDDMA-PS 1 ~ ~ c 0 2 ~.0 ::l en "0 "[) co 0 40 c.e <( Time (h) Figure 1-T ime-dependent Soc Ala incorporation into chl orometh ylated BDDMA-PS and DVB-PS resins by Cs-salt method The finished peptide was cleaved from the support by both acidolysis (TFA) and transesterification (TEA/methanol) methods. The weight increment of the peptidyl resin after the chain assembly was in accordance with the quantitative coupling at all stages (99.89 %). Yield from 100 mg peptidyl resin on TFA cleavage was 105 mg (96.7 %) and on transesterification was 116 mg (86.1% ). The hi gh cleavage yield especially on transesterification can be attributed to the considerable swelling and solvation of the resin in methanol (3.8 ml/g). Usually transesterification reactions gave onl y 30 to 35 % cleavage yields when the stericall y hindered C-terminal amino acid was attached by benzyl ester linkages on DYB-PS supports1 2. On TLC analysis using (i) pyndine:water:acetic acid (8: I: I; v/v) and (ii) n butanol:water:acetic acid (4:2: 1; v/v), the peptide obtained from all the coupling methods gave a single spot at Rf(il 0.66 and Rf(iil 0.49 corresponding to the target peptide. This is further confirmed by HPLC antilysis (Figure 2), amino acid analysis and mass spectrometry. [Amino acid analysis: Leu 1. 0 ( 1.0), Phe 0.94 ( 1.0), Gly 1.98 (2.0), Tyr 0.21 ( 1.0)]. The low value of Tyr is due to its destruction during hydrolysis. This is clear from electrospray ion ization t'.- 11.) (.) c:: i l..0 <( I 1-b..,.-.., Time (min) Figure 2 - Analytical HPLC profile of : rude (a) free and (b) protected [Leu 5 ]enkephalin. Solvents used : (A) 0.1% TFA I Water (B) 0. 1% TFA in 90% acetonitrile /water. Bondapack CI S column (4.6 x 250 mm, 5 ~t) ; I ml!min now rate. Gradi ent used : 0 % 8 for 5 min, 80 % 8 in 40 min. mass spectrum [m/z (100%), C 2RH37Ns0 1 requires in the free form (m.p C, [a] , c l in DMF) and m/z (100%), c~, H 56N s0 9 l equires for protected peptide (transesterificati on) (m.p C, [a] , c I in DMF)].

4 NOTES 919 Following the DCC/HOBt coupling protocols in NMP-DMSO (5% v/v), the tripeptides, Ala-Ala-Ala, Gly-Ala-Val, Ala-Val-/le and Gly-Ala-Pro were synthesized in high yield and in excellent purity. All the couplings were complete (99.9% as determined by quantitative ninhydrin test) within 30 min except Val to Ala in Val-Ala-Gly and Val to lie in Ala-Val-lle which were extended to 1 hr. On TFA cleavage all the peptides were obtained in > 90 % yield and transesterification yielded % of methylesters (yield based on the first amino acid substitution level). The synthetic products were characterized by TLC and elemental analysis (Tables II and III). The present investigation clearly points towards the development of a new class of styrene based flexible supports for peptide synthesis. The amino acids can be quantitatively attached to the chloromethylated resin by cesium salt method. The peptides can be obtained in high yield and purity in both protected and free form. The resin possesses all the physicochemical characteristics such as swelling ability, mechanical and chemical stability, flexibility, polarity and morphology essential for the synthesis of hydrophobic peptides even at high capacity. It showed no significant effect upon the degree of racemisation and gives better results on active ester coupling in NMP and DMF. Table II - Synthetic details of protected peptides obtained by TFA cleavage Peptide Weight of Cleavage Rf value< Found(%) calcdd resin (mg)" yieldb c H N Val-Ala-Giy (98%) ( ) Ala-Ala-Ala (98%) ( ) Gly-Aia-Val (93%) ( ) Ala-Val-lie (90%) ( ) Gly-Ala-Pro (91 %) ( ) Tyr-Giy-Giy-Phe-Leu (97%) ( ) "Increase in weight of I g of resin after the chain assembly is presented byield from 100 mg of peptidyl resi n is presented in mg.% was calculated on the basis of first amino acid substitution level <solvent system, pyridine: water: acetic acid (8 : I : I v/v) dtheoretical values are given in brackets Table III - Synthetic details of protected peptides obtained by transesterification cleavage Peptide Weight of Cleavage yicldb Rr Found(%) Calcdd peptidyl resin value< (mg)" c H N Boc-Vai-Aia-Giy-OMe (82%) ( ) Boc-Ala-Aia-Aia-Ome 6IO (86%) ( ) Boc-Giy-Aia-Val -Orne (76%) ( ) Boc-Ala-Val-lle-Ome (74%) ( ) Boc-Gly-Aia-Pro-Ome (79%) ( ) Boc-Tyr(OBzi)-GI y-gi y-phe-leu-ome I1.63 (78%) ( ) "Increase in weight of I g of resin after the chain assembly is presented byield from 100 mg of peptidyl resin is presented in mg.% was calculated on the basis of first amino acid substitution level <solvent system, pyridine: water: acetic acid (8 : I : I v/v) dtheoretical values arc given in brackets

5 920 INDIAN J. CHEM., SEC B, APRIL 2003 Experimental Section All chemicals and HPLC solvents were purchased from Merck (India) and amino acids, TFA, thioanisole and DCC from Sigma Chemical Company USA. HPLC analysis was carried out on a Shimadzu make LC-IOA model instrument fitted with a SPD-IOA UV/vis detector and Shimpack C18 column (4.6 x 250 mm) in the reverse phase. A binary gradient of 10 to 60 % B over 40 min using 0.1 % TFA containing water (A) and acetonitrile (B) was used for the elution. Amino acid analysis was conducted on Alpha-plus 4151 analyser using post column derivatisation with o phthalaldehyde. The peptide was hydrolysed with 6N HCl at ll0 C for 16 hr in a pyrex glass tube under nitrogen atmosphere. A triple quadrupole mass spectrometer was used for recording the mass spectrum. Preparation of BDDMA-PS support. Styrene and BDDMA were washed with 1% NaOH solution (2 x 50 ml) followed by distilled water (3 x 30 ml) and dried over anhydrous Na 2 S0 4 to remove the inhibitors ml of styrene, 0.44 ml of BDDMA and 500 mg dibenzoyl peroxide were dissolved in 20 ml toluene. The mixture was added to 175 ml of 1% polyvinyl alcohoh 5000 solution taken in the reaction vessel equipped with a stirrer, water condenser and nitrogen inlet and kept at 85 C on a water-bath. The mixture was stirred at 1500 rpm for 15hr and the precipitated copolymer was filtered, washed with hot water to remove the stabiliser, acetone (3 x 50 ml), toluene ( 3x50 ml) and methanol ( 3 x 50 ml). Finally the resin was Soxhlett extracted using toluene, acetone, dichloromethane and methanol. The polymer beads were dried under vacuum at 45 C, yield 9.2 g. The beads were sieved and mesh sizes were used for peptide synthesis. IR (KBr): 1720, 1490 (ester); 700,3010 (aromatic); 2910,2850 cm 1 (CH 2 str.). Ch1oromethylation of BDDMA-PS resin. The dry resi ns (1 g) was swelled in DCM (15 ml) for 1h and then 6 ml chloro methyl methyl ether (CMME), JM anhydrous zinc chloride (0.2 ml), in THF were added and kept the mi xture at 50 C. The resin was filtered and washed with THF (10 ml x 3, 3 min), THF/4N HCl (10 ml x 3 x 3 min), THF/water (1:1 v/v, lo ml x 3 x 3 min), water (20 ml x 5 x 3 min) and finally with methaltlol (3 x 30 ml). The resin was then Soxhlett extracted with THF and dried under vacuum. IR (KBr): 1254, 690 cm 1 (CHrCl). Solid-phase peptide synthesis - General procedure. C-terminal amino acids were attached to the support by cesium salt 8 and TEA methods 9. The amino capacity was then estimated using picric acid titration 11 The peptides were assembled manually on a filter-frit reaction vessel. 100 mg of amino acyl resin was used for synthesis. 30 % TFA/DCM (5 ml x 30 min) was used for Boc removal. 5 % DIEA/NMP (5 ml x 2 times) was employed for neutralization. Each coupling was conducted using 2.5 meq. excess of Soc-amino acids and a minimum time of 30 min was given for each coupling. The acylation time was extended to 1 hr in case of incomplete couplings. The completed peptide was cleaved from he support using TFA (0.1 %, v/v of thioanisole). The free peptide was precipitated by cold diethyl ether, lyophilized and characterized. Transesterification was conducted in dry methanolffea (5 %, v/v) at 60 C for 8 hr. Methanol was rotary evaporated, washed with ether, lyophilized and characterized. Acknowledgement One of the authors (KK) is grateful to the UGC, New Delhi, for financial assistance. References I Atherton E, Clive D L & Sheppard R C, 1 Am Chem Soc, 97, 1975, KentS 8 H, Annu Rev Biochem, 57, 1988, Sheppard R C, In Peptides, edited by Nesvabda (North Holland. Amsterdam), 1973, Ill. 4 Renil M, Nagaraj R & Pillai V N R, Tetrahedron, 50, 1994, Pillai V N R & Mathew 8, Indian 1 Tech, 3 1, 1993, Fe inberg R S & Merrifield R B, Tetrahedron, 30, 1974, Tam 1 P & Yi -An Lu, 1 Am Chem Soc, 117, 1995, Gisin 8 F, Helv Chim Acta, 56, 1973, Merrifield R 8, 1 Am Chem Soc, 85, 1963, Kaiser E, Colescott R, Bassinger C D & Cook P I, Anal Biochem, 34, 1970, 595. II Gisin B F, Anal Chim Acta. 58, 1972, Kai ser E T, Science, 243, 1989, 187.