Boc-group deprotection (15). After the entire chain was assembled, the tri- fluoroacetate form of PR was washed thoroughly, unloaded from the reactor.
Peptide Research A .Swellographic Approach to Monitoring Continuous-Flow Solid-Phase Peptide Synthesis
Igor L. Rodionov, Michael B. Baru and Vadim T. Ivanov Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences
ABSTRACT
A new type of practical low pressure continuous-j7ow reaction system has been developed. aI/owing continuous "dead I'olume free" handling of polystyrenehased peptide-resins (PR) under conventional BoclBzI solid-phase peptide synthesis (SPPS). The reaction system offers a unique opporlllnity for direct recording of hed volume changes accompanying chemical and physical manipulations with PRs. A new monitoring principle, called "swellography," based on a straightforward interpretation of PR hed volume dynamics, is descrihed. It seems to provide a hasis for a novel. automated . non-invasive fee.{ihack control system. This novel technique does not complicate SPPS practice; and. moreover. it provides useful information ahout the swelling behavior of PRs in the real time of SPPS. Although the approach is not directly applicable for quantitation of deprotection and coupling reactions. it does gil'e a good opportunity for real-time optimization of solvent and reagent usage during the washing and neutralization steps of SPPS. A numher of 8to 16-memhered peptides were synthesized manually with complete swel/ographic monitoring. The practical merits of the new approach are discussed in some detail.
Vol. S. No.2 (1992)
INTRODUCTION The lack of compatibility of the highly efficient continuous-flow (CF) technique (8) with Boc/Bzl SPPS employing conventional gel-type resins (24) is usually attributed to unpredictable swelling variations of the intermediate peptide-resins during synthetic manipulations (8). In fact, none of the attempts to use a fixed-volume CF reaction system with the abovementioned resins resulted in a fully reliable technique for SPPS (5,8,22). Moreover, unexpected bed volume increase has been encountered recentIy, even with Keiselguhr-supported "rigid" gel polymer under CF Fmoc SPPS (6). The usefulness of monitoring the resin-swelling behavior to gain better insight into solid-phase reactions and SPPS has been pointed out (10,28). This paper describes a novel, practical CF reaction system that seems to offer a solution to both of the abovementioned problems.
MATERIALS AND METHODS Chemicals N N-dimethylformamide (DMF), trifluoroacetic acid (TFA), acetic acid (AcOH), triethylamine (TEA), chloroform (CHCI3), dichloromethane (DCM), meta-cresole, tetrahydrofuran (THF), diethyl ether, isopropanol (i-PrOH), terr-butanol (t-BuOH) and propionic acid were obtained from Reachim (USSR) and redistilled before use. ex-
cept for the two latter solvents. t-BuOH was used after double freezing at AOC in a refrigerator. All the above solvents were stored over 4-A molecular sieves or calcium hydride in the refrigerator; ethers were additionally kept in a nitrogen atmosphere. Boc-His(Dnp). tetramethylsilane (TMS). trifluoromethanesulfonic acid (TFMSA). thioanisole (PhS Me), fuming hydrochloric acid (HC!), ortho-phosphoric acid (H3P04), I-hydroxybenzotriazole (HOBT) and HPLC-grade acetonitrile (MeCN) were purchased from Fluka AG (Buchs. Switzerland) and used without further purification. except for HOBT (vacuum dried in a Fischer apparatus at 78°C over KOH). Dicyclohexylcarbodiimide (DCC), chloromethylated polystyrene divinylbenzene 1% (I ~200 mesh, CI: 0.68 meq/g), Boc-Arg(Tos), Boc-Lys(2-CI-Z), BocTyr(2 -Br-Z), Boc-Asp(c-Hex) and Boc-Cys(Acm) were obtained from Peptide Institute (Osaka, Japan). Boc derivatives of Asp(BzI), Glu(Bz!), Ser(Bz!), Thr(Bzl) and the bifunctional amino acids were from Reanal (Budapest, Hungary). HPLC-grade water was prepared on a Milli-Q apparatus (Millipore, Bedford. MA). Sephadex G-IO and G-15 (Pharmacia Fine Chemicals. Uppsala. Sweden), Bio-Beads S-XI and Bio-Beads S-X I [chloromethylated. 1.34 meq/g], both 200-400 mesh (Bio-Rad Laboratories. Richmond. CA). were used_ Trimethylsilyl trifluoromethanesulfonate (TMSOTf) was synthesized from reagent-grade TFMSA and TMS, followed by vacuum distillation (bp II 39°-40°C).
SPPS Apparatus The general layout of the device is illustrated in Figure I; the practical reaction system used in this work is presented in Figure 2 and was originally developed in our laboratory (I). The CF reactor consists of a lO-ml glass syringe barrel (15.6 mm i.d.), Chirana 168 614 (Czechoslovakia), with a Teflon-made bottom fitting and a moving piston. both fitted with glass sinter frits (40-50 11m pore size; Shott). The syringe barrel was carefully silanized (29). The bottom fitting was also equipped with a Teflon sampler. facilitating resin sampling at any desired point during the synthesis. This reaction system (the swel/ograph) was
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filled with 0.3-1.5 g resin, which had a swollen volume of 0.5-10 ml. The minimum initial quantity of resin allowed during swellographic monitoring is determined by the reactor's i.d. and may be lowered to 100 mg. All tubing in the apparatus was made of 0.5- and I.O-mm i.d. Teflon. All experimental data was obtained at 62 kPa of external pressure exerted on the polymer via application of appropriate weight (1.2 kg) to the moving piston. This value also limited, the overpressure of liquid inside the reactor. Displacements of the moving piston (proportional to resin bed volume variations) were measured with potentiometric position pickup, with its output signal of 10-1000 mV being registered directly on a Kipp & Zonen (Holland) chart recorder. The position pickup feeding voltage was 1-3 V of stabilized direct current. A detailed technical description of the device will be published elsewhere. The liquid delivery system used in the experiments was assembled using a piston pump (MMC; Microtechna, Czechoslovakia) and an SRV -4 valve (Pharmacia, Uppsala, Sweden), allowing flow-through and recycling modes.
Solid-Phase Peptide Synthesis At the beginning of SPPS, the above-described CF reaction system
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was charged with 0.3-1.0 g of starting resin already esterified, unless otherwise stated. with the corresponding Boc-amino acid cesium salt by the Gisin method (14). Esterifications were repeated until only trace amounts of covalent chlorine could be detected on the resin (30). All amino groups were protected at the 'alpha-amino position with the Boc-group, and the following side-chain protecting groups were used: cyclohexyl ester (Asp followed by Asn and Ala), benzyl ester (Glu, Asp), acetamidomethyl (Cys), 2-chlorobenzyloxycarbonyl (Lys), benzyl ether (Ser, Thr), 2-bromobenzyloxycarbonyl (Tyr), tosyl (Arg). A typical SPPS cycle is denoted in the legend to Figure 6. Duration of individual operations in the cycle was determined in real time by visual judgment of the synthesis swellogram (2), except for deprotection (20 min) and coupling. The latter was carried out repeatedly, according to the sequence illustrated in Figure 6b;until a negative Kaiser's test (18) was obtained. Unless otherwise stated, pentafluorophenyl esters of Boc-amino acids (26) in a minimum volume of DMF were used, except for Asn and Gin, which were coupled as pnitrophenyl esters (obtained from Reanal) with an equivalent amount of HOBT (19). All couplings to the second amino acid (from the C-ter-
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