Liquid-phase peptide synthesis

Initially, the term Hquid-phase synthesis was used to contrast the differences between soHd-phase peptide synthesis and a method of synthesis on soluble polyethylene glycol (PEG) [5, 6]. Although soluble polymer-supported synthesis is less ambiguous than Hquid-phase synthesis, the latter term is more prevalent in the Hterature. In-keeping with previous reviews [7-12], the phrases classical or solution synthesis will be used to describe homogeneous reaction schemes that do not employ polymer supports while liquid-phase synthesis will be reserved... 

As the intermediate products resulting from individual synthetic steps cannot be purified, a virtually 100% selectivity is essential for the synthesis of larger-peptide molecules. Even at a selectivity of 99% per reaction step, the purity will drop to less than 75% for a dekapeptide (30 steps) It is practically infeasible to go beyond 10-15 amino acid peptides by using the solid-phase method. In order to prepare larger peptides, individual fragments are first produced, purified, and then combined with the final molecule by liquid phase synthesis. This combination of methods is listed under chemical hybrid in Table 4.2. 

Thus, for the production of Roche s anti-AIDS drug Fuzeon (enfuvirtide), three fragments of 10-12 amino acids are first made by solid-phase synthesis and then linked together by liquid-phase synthesis. The preparation of the whole 35 amino acid peptide requires more than 130 individual steps ... 

The 9-fluorenylmethoxycarbonyl group, developed by Carpino and co-workers in 1972 [257], has become one of the most widely used protective groups for aliphatic or aromatic amines in solid-phase synthesis. For solid-phase peptide synthesis in particular, this protective group plays an important role [258] (Section 16.1). The Fmoc group is not well suited for liquid-phase synthesis because non-volatile side products are formed during deprotection. 

To study the nucleation step for the folding of collagen, a protocol has been developed for the liquid-phase synthesis by which three peptide strands are covalently linked via a C-terminal branch. 70-73 The C-terminal branch is expected to enhance triple-helical thermal stability, and to provide a model of the disulfide-linked C-terminus of type III collagen. 71 ... 

Bayer E, Mutter M, Liquid phase synthesis of peptides, Nature, 237 512-513, 1972. 

Aminomethyl-3-nitrobenzoylpolyethyleneglycols are another class of photosensitive soluble polymeric supports designed for the liquid phase synthesis of protected peptide amides 192,193) (scheme 8). These supports have been very recently used for efficient synthesis of three biologically active 14-peptideamides corresponding to the wasp venom peptides, mastoparan, mastoparan X and Polistes mastoparan 198). 

Monofunctional polyethyleneglycol supports containing benzyloxycarbonylhydra-zide, p-benzyloxybenzyloxycarbonylhydrazide and t-butyloxycarbonylhydrazide anchoring groups, 22, 23 and 24 respectively have been developed recently for the liquid phase synthesis of protected peptide hydrazides 199). 

The solubilizing effect of PEG on the attached peptide and the absence of any direct influence of the polyoxyethylene chain on the physicochemical properties of the peptides provide a wider range of possibilities for analytical control during the liquid phase peptide synthesis than those in the solid phase method. The reactions employed in the stepwise liquid phase synthesis can thus be quantitatively monitored by several analytical methods. 

Automation of the liquid-phase synthesis, at its present stage, is advantageous only when the optimum reaction conditions for the synthesis of a specific peptide have been elucidated in advance, and the synthesis of small- to medium-sized peptides appears to be within the scope of this automated method. 

The liquid phase synthesis on PEG has also been used for the conformational analysis of collagen-like sequences by CD studies 237). The attachment to PEG has also permitted the CD spectral delineation of the specific interactions between the polypeptide chains and sidechain groups 238,239). Thus, Anzinger et al. observed that onset of local ordered structures in the mesogenic side chains of polylysine blocks attached to PEG leads to significant, specific alteration in the backbone conformations of the peptide chain 239). 

Linear polystyrene can be functionalized by various methods . The functional group capacity in these polymers diould not be too high otherwise, steric complications may arise. Poly(ethylene ycol) has been found to be most suitable for liquid-phase synthesis. This linear polyether and the block copolymers with functional groups at defined distances are chemically stable and soluble in a large number of solvents including water and can be precipitated selectively. Partially hydrolyzed poly(vinylpyrrolidone) and its copolymers with vinyl acetate were successfully applied in peptide synthesis. Poly(acrylic acid), poly(vinyl alcdiol), and poly-(ethylenimine) are less suitable for the sequential type synthesis because of the... 

The active ester methodology has been much applied mainly to form peptide bonds under mild conditions in both liquid- and solid-phase synthesis. In liquid-phase synthesis, most frequently the activation of a carboxyl group has employed HOSu, followed by esters of electron-withdrawing-containing phenols (nitrophe-nols of pentafluorophenols), the most reactive HOBt esters usually being unstable and used in solid-phase synthesis. The preparation of these active esters requires the presence of a coupling reagent such as DCC or DIC. 

K. Wiithrich, a leader in the field of determination of peptide-architecture by two-dimensional NMR spectroscopy, is also at the ETH in Zurich. At the University of Basel, professor Max Brenner (Plate 12) focussed his interest on several fundamental aspects of peptide synthesis, such as the problem of overactivation or peptide bond formation via rearrangements. One of his coworkers, Iphigenia Photaki (Plate 35) has already been mentioned in the section on Greece. Brenner s successor at the University of Basel, Manfred Mutter, excels with his work on liquid phase synthesis (cf. p. 111). Recently he accepted an invitation to the Free University of West Berlin and from there to Lausanne. 

Beyond these three main concepts, the inverse bioconjugation approach offers another strategy to connect peptides or proteins with synthetic polymers. Using a solid support, which is preloaded with a polymer block, the biological molecule can be assembled in a stepwise fashion through solid-phase synthesis. Mutter and coworkers first showed the attachment of PEO to a poly(styrene) resin via a benzyl ether linker. This concept was finally developed further by Bayer and Rapp leading to a commercially available PAP resin, which is widely applied in solid-phase peptide synthesis. In a similar approach. Lutz, Borner, and coworkers demonstrated the preparation of cleavable and non-cleavable soluble polystyrene supports by ATRP for the liquid-phase synthesis of peptide-polymer conjugates. ... 

In another study Tjoeng et al. [80] showed successfuUy that low yields of cleaved peptide obtained by the usual methods can be significantly improved in liquid-phase synthesis by employing this type of photocleavable polymer support. 

Comparable to peptide synthesis, the liquid-phase synthesis of oligonucleotides is based on the sequential repetition of reaction steps, thus adding one monomeric nucleotide to the other (Scheme 4). 

The preparation and availability of suitable soluble polymer supports are basic requirements for the successful application of liquid-phase synthesis of peptides and nucleotides. Many parameters have to be considered when designing appropriate supports and the route of synthesis and the target product of synthesis must also be considered. Liquid-phase synthesis requires optimum solubility properties of the polymer supports and therefore the adaption of the functional capacity to the solubilizing power of the polymer backbone and side-chains is the determining step before synthesis. The main factors of evaluation are the solubility characteristics of the polypeptide or oligonucleotide to be synthesized. 

Diphthalimidocarbonate is a new reagent for the synthesis of active esters. A new photolabile support and a new technique for the hold-in-solution method provide improvements in the liquid-phase synthesis of peptides. The carboxy-group of an A -acylated amino-acid is protected by conversion into an oxazole ring photo-oxygenation regenerates the carboxy-group as a triamide, which is activated for peptide synthesis (Scheme 148). ... 

This review presents a survey on functional soluble polymers in view of their use as supports for liquid-phase synthesis. The gen al aspects of syntheas in homogeneous media as well as analytical and separation problems are discussed, focussing on the role of the polymer in the synthetic cycle and the problems associated with polymer-supported reactions. A survey of polymeric carriers in respect of their functional groups and badcbones is provided with an emphasis on poly(oxyethylene), polystyrene, and poly(vinyl alcohol) suf rts. Combined methods using solid and sduble supports are al highli ted. Ihe polymeric carriers are discussed and evaluated for their use in peptide and nudeotide synthesis. Finally an outlook into future developments is attempted. 

Attachment of the first amino add to the polymer Incomplete polymer -analogous reaction - in most cases esterification - is not as important as it was considered at the beginning of the liquid-phase synthesis, but is still as essential as it is for matrix reactions [67]. The unreacted functional grou K can be blocked to prevent the formation of unwanted peptides. However, for particular amino adds (e.g. proline) the fixation to the polymer still represents a crucial step. 

Studies by Bayer et al. [22] and Mutter [69] have shown that liquid-phase synthesis of peptides exhibits kinetic behaviour analogous to classic peptide synthesis. The reaction rates of both methods are of the same order of magnitude, and using poly(ethylene glycol) esters, the polymer reaction proceeds at an even higher rate than the reaction involving the corresponding low-... 

Solution ( = Liquid-Phase) Methods for Peptide Synthesis... 

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