Solid-phase synthesis continuous-flow

The requirements for solid-phase synthesis are diverse. The support must be insoluble, in the form of beads of sufficient size to allow quick removal of solvent by filtration, and stable to agitation and inert to all the chemistry and solvents employed. For continuous-flow systems, the beads also must be noncompressible. Reactions with functional groups on beads imply reaction on the inside of the beads as well as on the surface. Thus, it is imperative that there be easy diffusion of reagents inside the swollen beads and that the reaction sites be accessible. Accessibility is facilitated by a polymer matrix that is not dense and not highly functionalized. A matrix of defined constitution allows for better control of the chemistry. Easier reaction is favored by a spacer that separates the matrix from the reaction sites. Coupling requires an environment of intermediate polarity such as that provided by dichloromethane or dimethylformamide benzene is unsuitable as solvent. 

To illustrate the diversity of strategies available for solid-phase synthesis, several fairly recent protein syntheses can be cited. SRY, an 80-residue DNA binding protein, was syn-thesized[62] on a Pepsyn support (polyacrylamide gel beads) in a continuous flow machine using Fmoc/tBu protection and TBTU/HOBt activation. HPLC gave a homogeneous product with molecular weight 10051 Da (calculated 10033 Da). It bound to DNA, as expected. 

The fact that macroporous, highly cross-linked polystyrene does not swell makes this support particularly interesting for continuous-flow synthesis in columns. This support has also been successfully used as an alternative to CPG for the solid-phase synthesis of oligonucleotides [90,91]. Furthermore, because reagents do not need to penetrate into the polystyrene network, enzyme-mediated reactions should also proceed smoothly on macroporous polystyrene [85]. 

Dryland, A. and Sheppard, R. C. (1986) Peptide synthesis. Part 8. A system for solid-phase synthesis under low pressure continuous flow conditions. J. Chem. Soc. Perkin Trans. 1, 125—137. 

In the absence of base, active esters of both N-alkoxycarbonyl amino acids and N -protected peptides undergo anninolysis with preservation of chiral integrity. There is ample evidence to substantiate this statementP d and no evidence to the contrary. But there is a wealth of accumulated evidence that active esters undergo isomerization when left in the presence of tertiary annine.t In this regard, 4-nitrophenyl esters are much more sensitive than trichlorophenyl esters and piperidino esters are exceptions in that they are unaffected by tertiary anoine.W The isomerization recently observed in the TBTU-mediated couplings of Fmoc-Cys(R )-OH in the presence of 1,2,3-benzotriazol-l-ol in continuous flow solid-phase synthesis is a further example of this phenomenon. The result can be attributed to the effect of tertiary amine on the benzotriazolyl ester that is formed by capture of the acyluronium intermediate before it has time to be aminolyzed. 

The advantage of these reagents relies on their simplicity of use. An equivalent of the reagent is added to a 1 1 mixture of the carboxylic component and tertiary base in an inert solvent at room temperature. The mixing can be carried out in the presence of the amino component. These coupling reagents are compatible with Z, Boc, and Fmoc chemistry, solution and solid-phase synthesis, polystyrene, polyamide, and continuous flow resins. 

SPPS. In contrast to OPcp esters, these latter OPfp esters derived from 27 and those derived from HODhbt are the most commonly used active esters in SPPS and have been applied to automated continuous flow Fmoc/tBu synthesis. Their use notably simplifies the course of the solid-phase synthesis by avoiding individual preactivation procedures. 

Similarly to the tea bag approach, pieces of any solid-phase support suitable for solid-phase synthesis can be used for parallel peptide preparation. One of the first materials used in this manner was paper. Frank used paper disks l (Whatman 3MM, 1.5-cm diameter) packed into the columns of a multicolumn continuous-flow synthesizer. Prior to the synthesis, the paper was derivatized with 4-alkoxybenzyl (Pab) linkage to allow for the cleavage with trifluoroacetic acid. The disks can be easily labeled with a pencil, sorted, and combined depending on the common amino acid to be coupled at the next step. 

However, the polyacrylamide resin beads are compressible and thus more suitable for batch-wise rather than continuous-flow solid phase synthesis. To make the support withstand pressure and thus perform well under continuous-flow... 

Beaded cellulose has been tested as a more hydrophilic support [168,169]. Although Merrifield [1] found beaded cellulose unsuitable for solid-phase synthesis, the Perloza beaded cellulose has shown promising results as a solid support. Perloza [168,169] is a beaded, non-cross-linked cellulose support that has adequate mechanical properties for the synthesis of small peptides by either batch or continuous-flow methods. It has good solvation properties in a wide variety of solvents including water, dioxane, DMF, dimethyl sulfoxide (DMSO), DCM, and THE Perloza must be maintained in a solvent swollen state at all times, as upon drying it will not reswell to its original volume. 

It was not until the introduction of more rigid solid supports that continuous-flow solid-phase peptide synthesis became practical. Initially, a support with polyacrylamide gel held within the pores of an inert macroporous matrix (diatomaceous earth) was used [15]. Although these fragile supports worked well for continuous-flow synthesis, careful handling was required to avoid generating fines that could clog the column frits and damage instrument components. 

While a large number of improvements in procedures for solid phase synthesis of oligonucleotides have been described, the techniques have not altered fundamentally from those described in previous reports. Solid-phase synthesis using a continuous-flow phosphotriester method on a kieselguhr-polyamide support has been described. Other solid phases used as supports for synthesis include derivatized h.p.l.c.-grade silica gel, . derivatized cross-linked poly-... 

Batchwise solid-phase synthesis, the original procedure of SPPS developed by Mer-rifield in 1966. Alternatively, SPPS can also be performed in a continuous-flow mode solid-phase peptide synthesis using resin-flUed columns. 

Because of the inherent ability of solid-phase synthesis to be integrated and automated, numerous instruments were built from the onset of solid-phase chemistry, and this development culminated after the introduction of combinatorial chemistry methods. Operational simplicity of solid-phase synthesis contributed to the development of multiple solid-phase synthesis, where numerous reaction vessels are handled at the same time. In 1989, Schnorrenberg and Gerhardt" introduced the automated multiple synthesis of peptides in parallel fashion. Multiple synthesis in a continuous flow manner was also later reported. 

Researchers have found that upon exposing polyethylene particles to the oxidating conditions of oxygen with transition metals or chromic acid alone, a network of large pores can be created [22]. These pores range in size from 900-5000 A. These supports which have a reported loading capacity in excess of 100 /xmole of functional groups/g were shown to be suitable for both batchwise and continuous flow solid phase synthesis. 

Gait, M. J, Matthes, H W. D., Singh, M., Sproat, B S., and Titmas, R. C. (1982) Rapid synthesis of oligodeoxyribonucleotides VII. Solid phase synthesis of oligodeoxyribonucleotides by a continuous flow phosphotriester method on a kieselguhr-polyamide support. Nucl Acids Res. 10, 6243-6254. 

E Atherton, E Brown, RC Sheppard. A physically supported gel polymer for low pressure, continuous flow solid phase reactions. Applications to solid phase peptide synthesis. J Chem Soc Chem Commun 1151, 1981. 

A Di Fenza, M Tacredi, C Galoppini, P Rovero. Racemization studies of Fmoc-Ser(tBu)-OH during stepwise continuous-flow solid-phase peptide synthesis. Tetrahedron Lett 39, 8529, 1998. 

V Caciagli, F Cardinali, F Bonelli, P Lombardi. Large-scale production of peptides using the solid phase continuous flow method. Part 2 Preparative synthesis of a 26-mer peptide thrombin inhibitor. J Pept Sci 4, 327, 1998. 

---