Since its inception,1) the solid-phase peptide synthesis (SPPS) methodology has been systematically improved as the result of a wide variety of experimental investigations. These efforts have ranged from optimizing the coupling reac- tion itself (through the use of efficient acylating reagents, mi- crowave irradiation and variations in temperature)2—5) to broadening our knowledge of the complex peptide-resin sol- vation process.6—9) Predictably, methods such as nuclear magnetic resonance10,11) and Fourier transform infrared spec- troscopy12,13) have also been tested in attempts to further im- prove SPPS. In our case,14—17) we pioneered the application of the electron paramagnetic resonance (EPR) technique, which is based on the use of a previously developed amino acid-type marker.18,19) All of these efforts have led to ongoing improvement of the SPPS method. Intriguingly, little attention has yet been given to the possibility that incomplete cleavage occurs or that there is premature removal of peptide chains from the solid support. Within this context, we previously proposed some rules for the selection of resins used in the Na-tert- butyloxycarbonyl (Boc) chemistry. Our proposal was based on the stability of model peptide-resin linkages toward exist- ing acid cleavage procedures,20,21) as well as on the degree to which premature peptide chains are removed during trifluo- roacetic acid (TFA)/Boc removal of peptide-resins. The re- sults of other studies22,23) have indicated the need for caution in selecting the type of resin to be used in the synthesis of peptide sequences containing C-terminal residue in either the a-carboxamide or the a-carboxyl function. In addition, ap- propriate final cleavage experiments, which are typically car- ried out either in anhydrous hydrogen fluoride24) or in a triflu- oromethanesulfonic acid/TFA/thioanisole cocktail,25) should be performed, since a significant decrease in the amount of cleaved peptide can occur in this step. The type of the C-ter- minal residue and the length of the peptide sequence seem to affect the overall cleavage yield (which, surprisingly, can be as high as 30%) as a consequence of incomplete final chain removal accompanied by premature loss from the resin dur- ing TFA removal of peptide-resins. The results of a comparison between benzhydrylamine-resin (BHAR)26) and methylbenzhydrylamine-resin (MBHAR),27) both used for the synthesis of a-carboxamide peptides in the Boc-chemistry, led us to conclude that the latter is the resin of choice mainly when the resin-bound amino acid is of the hydrophobic type. However, in the presence of a hydrophilic residue at the C-terminal position, the difference between the two aminated resins in terms of their efficiency depends on the length of the peptide. When the peptide sequence is longer, BHAR produces higher yields than does MBHAR. This is a consequence of the greater stability of the peptide- resin linkage of the former toward successive TFA treatments in each synthesis cycle.20,21) In the present study, we aimed to take a similar approach to establishing rules for the base-labile Na-9-fluorenylmeth- yloxycarbonyl (Fmoc)-protecting group synthesis strategy.28) Since it is impossible to occur premature peptide chain cleav- age from the resin during the Fmoc group removal in piperi- dine/dimethylformamide (DMF) solution, other parameters were varied. The first was the cleavage capacity of different acid cocktails used routinely for the final peptide cleavage in 468 Vol. 55, No. 3 Comparative Investigation of the Cleavage Step in the Synthesis of Model Peptide Resins: Implications for Naa-9-Fluorenylmethyloxycarbonyl-Solid Phase Peptide Synthesis Guita Nicolaewsky JUBILUT,a Eduardo Maffud CILLI,b Edson CRUSCA, Jr.,b Elias Horacio SILVA,a Yoshio OKADA,c and Clovis Ryuichi NAKAIE*,a a Department of Biophysics, Universidade Federal de São Paulo; Rua 3 de Maio 100, CEP 04044-020, SP, Brazil: b UNESP, Department of Biochemistry and Chemistry Technology; Rua Prof. Francisco Degni S/N, CEP 14800-900, Araraquara, SP, Brazil: and c Faculty of Pharmaceutical Sciences, Kobe Gakuin University; Nishi-ku, Kobe 651–2180, Japan. Received October 7, 2006; accepted December 21, 2006 Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Naa-tert-butyloxycarbonyl (Boc)-peptide syn- thesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Naa-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to es- tablish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, de- pending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve com- plete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50—60%) than that observed with the Gly-bearing sequences at the same position (70—90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin. Key words peptide synthesis; peptidyl resin; cleavage; linker group Chem. Pharm. Bull. 55(3) 468—470 (2007)Notes © 2007 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: clovis@biofis.epm.br the Fmoc-synthesis methodology. The second was the type of linker group used for separating the peptide chain from the resin matrix. These variations were therefore tested using two types of peptide sequences that differed in the hydrophobic- ity of their C-terminal amino acids. The vasoactive an- giotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were deliberately synthesized using different linker groups attached to the solid support and designed for the synthesis of peptides containing carboxamide or carboxyl groups at their C-terminal position. Besides the Wang resin29) that at- taches a p-benzyloxybenzyl alcohol spacer to a polystyrene- type solid support and is used routinely for the synthesis of peptide acid, the other three tested resins were all character- ized by containing different linker groups coupled to MBHAR support. Amongst these, the HMPA resin30) uses the 4-hydroxymethylphenoxyacetic acid linker (also for the synthesis of peptide acids) whereas the Knorr31) and Rink32) resins attach 4-[(R,S)-a-[1-(9H-fluoren-9-yl)-methoxy-form- amido]-2,4-dimethoxybenzyl-phenoxyacetic acid and 4- [(2�,4�-dimethoxyphenyl) Fmoc-aminomethyl] phenoxyac- etamido groups, respectively. Among the known acid cock- tails used for final cleavage, the following solutions were se- lected28,33): (1): TFA/water (9.5 : 0.5); (2): TFA/p-cresol/water (9 : 0.5 : 0.5); (3): TFA/ethanedithiol (EDT)/p-cresol/water (7.5 : 1.5 : 0.5 : 0.5); and (4): TFA/EDT/phenol/thioanisole/ water (8.25 : 0.25 : 0.5 : 0.5 : 0.5, reagent K). Table 1 compares the cleavage yields of AII and [Gly8]- AII, each submitted to four types of linker resins and to the TFA cleavage solutions mentioned above. Based on the re- sults of these experiments, several conclusions were drawn. First, according to previous results applied to Boc proto- col,20,21) or even to those obtained in the acid hydrolysis-re- lated investigation of the peptide resins necessary for further amino acid analysis,34) greater resistance to acid cleavage was observed with peptides containing Phe at their C-termi- nal extremities than with those containing Gly at the same lo- cation. The use of the former resulted in 50—60% peptide removal, compared with 80—90% for the latter. In addition, a higher yield was observed when solution 4 (reagent K) was used. Furthermore, faster removal of peptide chains occurred when those chains were bound to Wang-resin. Finally, even after 2 h of acid treatment, none of peptide resins presented complete cleavage of peptide chains from their respective solid supports. The mean purity of the cleaved peptides ranged from 70 to 80%, with molecular weights and amino acid compositions that were consistent with the theoretical values. To determine the time course of the process of peptide chain cleavage in the most stable AII-resins, the acid treat- ment (with reagent K) was extended for up to 6 h at 25 °C (Fig. 1). Even after this long cleavage time, only the peptide- Wang support presented near total peptide removal from the resin. The cleavage values for the peptides attached to other supports (Rink or HMPA resins) did not surpass 70%. These findings suggest that considerable caution should be taken in the planning of cleavage procedures. A significant (20— 30%) loss in the overall yield can occur during this step, de- pending on the type of peptide-resin pair and cleavage pro- cedure. Finally, the AII-HMPA-resin was tested in order to evaluate the known high stability of the Arg 2,2,5,7,8- pentamethylchroman-6-sulfonyl (Pmc) side-chain group in reagent K. Even after 1 h of treatment, approximately 12% of this protecting group remained attached to the peptide back- bone, and was only completely removed after 2 h. Concerning the low cleavage yield of peptide sequences attaching hydrophobic residues at the C-terminal position, some recent experiments (not shown) has indicated hat the use of anhydrous HF or TFMSA/TFA/thioanisole treatments allow, regardless of the type of resin and linker group, a cleavage yield of about 95% for this type of peptide se- quences. These findings thus suggest that these cleavage treatments, routinely applied only in Boc chemistry would be also applicable to overcome the mentioned chain removal shortcoming in the Fmoc synthesis strategy. In conclusion, the present study revealed significant varia- tion in the degree to which peptide chains were cleaved from the solid support in the Fmoc-peptide chemistry. Similarly to what had been observed for the Boc-synthesis strategy, the amount of peptide removed from the resin is strongly de- pendent upon the type of linker group, the cleavage solution and the type of C-terminal residue in the peptide sequence. Previous studies have demonstrated that crude peptide purity is dependent on the type of cleavage cocktail used.28,35) As a complement, the present study demonstrated the critical in- fluence of various factors affecting the overall synthesis yield, especially in terms of incomplete removal of peptide from the solid support, which has been ignored by some au- thors. These losses can be much greater than predicted but March 2007 469 Table 1. Percentage of Peptide Cleavage from the Resin in Different Acid Solution (at 25 °C, for 2 h) Yield of cleavage (%) Peptidyl-resin 1a) 2b) 3c) 4d) [Gly8]AII-Wang-R 86 83 74 93 [Gly8]AII-Rink-R 79 79 77 80 [Gly8]AII-HMPA-R 79 70 68 84 [Gly8]AII-Knorr-R 81 77 75 88 [Phe8]AII-Wang-R 64 65 64 76 [Phe8]AII-Rink-R 58 45 48 67 [Phe8]AII-HMPA-R 49 46 47 60 [Phe8]AII-Knorr-R 59 50 49 69 a) TFA/water (9.5 : 0.5); b) TFA/p-cresol/water (9 : 0.5 : 0.5); c) TFA/EDT/p- cresol/water (7.5 : 1.5 : 0.5 : 0.5) and d) TFA/EDT/phenol/thioanisole/water (8.25 : 0.25 : 0.5 : 0.5 : 0.5, reagent K). Fig. 1. Time-Course Study of AII Cleavage from Wang, Rink, Knorr and HMPA-Resins Using Reagent K, at 25 °C can be avoided by establishing the appropriate combination of resin type, linker group and cleavage protocol used. Experimental All Fmoc amino acids were purchased from Advanced Chemtech (Louisville, KY, U.S.A.) or Bachem Inc. (Torrance, CA, U.S.A.). Solvents and reagents were acquired from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.) and Fluka (Buchs, Switzerland). Peptide Synthesis The peptides were synthesized manually according to Fmoc chemistry. The following side-chain protecting groups were used: t-butyl for Asp and Tyr residue; Pmc for Arg residue; and trityl for His residue. In each synthetic cycle, the Na-Fmoc deprotection step was carried out in 20% piperidine/DMF for 20 min, followed by washings with dichloromethane (DCM) and DMF. The coupling reactions were performed with a three-fold excess of the acylating component diisopropylcarbodi- imide/N-hydroxybenzotriazole in DMF/DCM (1 : 1). After approximately 2 h of coupling, the ninhydrin test was performed to estimate the complete- ness of the reaction. Cleavage from the resin and removal of the side-chain protecting groups were simultaneously with different acid cocktails as de- tailed below. After the cleavage procedure had been completed, the crude peptides were precipitated with anhydrous ethyl ether, separated from the soluble nonpeptide materials by centrifugation, extracted into 5% acetic acid in water and lyophilized. Time-Course Cleavage Study In several small syringes, each equipped with a polypropylene filter, the cleavage solution was added to isolated por- tions (approximately 50 mg each) of protected peptide resins, stirred for 2 h at 25 °C and cleaved using solvents 1 through 4 (see text). After the cleavage reaction was complete, the resin was submitted to exhaustive washings with, consecutively, ethyl ether, DCM, methanol (MeOH), 10% acetic acid (AcOH)/water, water and MeOH to guarantee the removal of all cleaved peptides and other by-products of the reaction. After this treatment, small aliquots of each dried resin were hydrolyzed as previously reported34) for further amino acid analysis. The calculated peptide content of the cleaved resin was compared to the value obtained for the initial peptide-resin pair, taken as 100%, and checked against the amount of cleaved peptide. To eval- uate the purity of removed peptide, the cleaved peptide was isolated by pre- cipitation with cold ethyl ether in the resin, further extracted with 10% AcOH/water and lyophilized. Amino Acid Analysis As recently proposed,34) prior to cleavage, all peptide-resin pairs were hydrolyzed with a mixture of 12 N HCl/propionic acid for 100 h at 130 °C to guarantee quantitative removal of peptide chains from the resin. Pyrex tubes with plastic Teflon-coated screw caps (13�1 cm) were used for the hydrolyses, and the amino acid analyses were performed in a Biochrom 20 plus amino acid analyzer (Pharmacia LKB Biochrom Ltd., Cambridge, England) to determine the amount of peptide attached to the resin. Analytical RP-HPLC The RP-HPLC analyses were carried out in TFA/acetonitrile gradient using a Waters Associates HPLC system consist- ing of two 510 HPLC pumps, automated gradient controller, Rheodyne man- ual injector, 486 UV detector and 746 data module (Waters, Eschborn, Ger- many). We used Solvent A (0.1% TFA/H2O) and Solvent B (60% acetoni- trile/0.1% TFA/H2O with a gradient of 5—95% in 30 min) at a flow rate of 1.5 ml/min. A C18 column (0.46�25 cm, 5 mm particle size and 300 Å pore size; Vydac, Hesperia, CA, U.S.A.) was employed. Detection was at l� 210 nm. Liquid Chromatography/Mass Spectrometry The crude lyophilized peptides were analyzed on a system composed of a Micromass Platform LCZ Mass Spectrometer (Micromass, Manchester, U.K.), a Waters Alliance HPLC, a Waters 996 Photodiode Array detector, and a Compaq Workstation. 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