Solid-phase syntheses

The solid-phase synthesis of glycopeptides was first realized by applying the polymeric benzyl ester principle of Merrifield. According to this methodology, Lavielle and associates (50) used 7V-(tm-butyloxycarbonyl)- 9-glycosyl serine derivative 153 for condensation with resin-linked alanine 154. 

Removal of the Fmoc group was achieved by treatment with morpholine-dichloromethane, leaving the allylic anchor and the 0-glyco- 

The allylic HYCRAM derivative was subsequently modified by insertion of a standard amino acid between the aminomethyl resin and the hydroxy butenoic acid moiety. Using this allylic anchor, the resin-linked, glycosylated HIV peptide T-derivative 164 was synthesized by application of Fmoc amino protection and sidechain protection with lert-butyl groups. The lac-tosamine peptide T (165) could be released from the resin by application of the palladium(0)-catalyzed allyl-transfer reaction to V-methyl aniline as the allyl acceptor. 

Similarly, the glycosylated peptide T derivative 166, carrying one jV-gly-cosyldically and two O-glycosylically linked saccharide side-chains was successfully synthesized (40,81). 

Liming and coworkers (56) also used the Fmoc group for temporary amino protection and employed a commercial resin (SASRIN , trademark of Bachem, Switzerland) having a dialkoxybenzyl anchoring group. The synthesized glycopeptide could be cleaved from the resin with 1% trifluoroacetic acid in dichloromethane. 

Despite the relative infancy in the development of solid phase reactions, a wide range of functionalised resins are commercially available. The main uses of these functionalised resins can be roughly classified as follows  

PRACTICAL ASPECTS OF COMBINATORIAL SOLID-PHASE SYNTHESIS 

3 History of Combinatorial Chemistry Breakthrough Discoveries That Shaped the Future of the Combinatorial Chemistry Field 

Although no single invention triggered scientific and research activities in combinatorial chemistry, there were several milestone discoveries that helped reveal the concepts of combinatorial technologies and prepare the general chemical community for the combinatorial way of thinking.  

1 Solid-Phase Synthesis. The first, and possibly the most crucial, was solid-phase peptide synthesis by Bruce Merrilield published in 1963. The solid-phase synthesis of peptides by Bruce Merrilield is undoubtedly considered to be a landmark in chemistry as a whole and had a critical impact on combinatorial chemistry. Although the concept of solid-phase peptide synthesis is, similar to many other ingenious ideas, very simple and straightforward, Bruce Merrilield was the first to recognize its potential and develop a workable method. 

Solid-phase synthesis in general has several inherent characteristics. As it is not possible to putify the growing substrate during solid-phase synthesis, the reaction has to be driven as close to completion as possible. Therefore, a large excess of reagents is often used. Suppose each reaction proceeded with 95% conversion, a very acceptable yield for solution-phase synthesis. However, for a 10-step synthesis, the purity of the final product would be 63% (0.95 x 100). Therefore, an efficient synthesis must be developed before library synthesis. 

With a view to application in combinatorial chemistry and to reduce solubility problems, Muller et al. [26] developed a solid-phase approach to SPHs in 1995. Uronic acid 7 was coupled to benzhydrylamine polystyrene functionalized with 

With the piperidine carboxylic acid 42 and two related unhydroxylated amino acids (Boc-4-piperidine carboxylic acid and Boc-i -pipecolic acid) a small library was constructed on a 4-methyl benzhydrylamine resin by use of HATU activation in DMF-dichloromethane 1 1. 

For combinatorial chemistry to deliver on its promise, general methods need to be discovered to accelerate soHd-phase reactions to the point at which they are equivalent in rate to, or faster than, homogeneous reactions. Microwave-assisted organic synthesis has had considerable success in achieving this goal. Since 1990, many organic transformations have been accelerated by subjecting them to microwave (MW) irradiation. In most cases reaction times of hours to days have been reduced to seconds to minutes. 

Thermally demanding reactions, such as the Diels-Alder reaction, are often completed in hours in solution but when performed on a solid phase can take several days. If such soHd-phase reactions are performed with MW irradiation, the reaction times can be reduced from days to minutes. 

For example, Yu et al. showed that polystyrene-bound peptides could be hydrolyzed in 7 min in a domestic MW oven, a process normally taking 24 h. Furthermore, traditional soHd-phase peptide couplings were achieved in 4 min in 99-100% conversion with no detected racemization. A broad range of solid-phase reactions was found to undergo substantial rate acceleration, including Claisen and Knoeve-nagel condensations, nucleophilic substitutions, sucdnimide and hydantoin formation, and Suzuki coupHngs. 

Sequence-specific heteropolymers, as a class of synthetic molecules, are unique in that they must be made by chemical steps that add one monomer unit at a time. Moreover, to create truly protein-like structures, which typically have chain lengths of at least 100 monomers and a diverse set of 20 side chains (or more), extremely efficient and rapid coupHngs under general reaction conditions are necessary. For these reasons, soHd-phase synthesis is typically used, so that excess reagents can be used to drive reactions to completion, and subsequent reaction work-ups are quite rapid. 

Specifically, the carboxylates of N -Fmoc-protected (and side chain-protected) N-substituted glycines were activated and then coupled to the secondary amino 

In 2001 Sharpless proposed this method - in which smaller units are joined together by way of heteroatom bonds (C-X-C X=heteroatom) - as a fast track route to compounds [16]. The criteria for a click reactiorf were recently summarised broad applicability with high yields readily accessible starting compounds readily separable by-products or none at all straightforward reaction conditions easy product isolation stereospecific 

Unprotected glycol-dendrimers, peptido- and redox-dendrimers and dendro-nized polymer organogels can also be prepared by click chemistry [18 c]. 

Solid-phase methodology was established in 1963 in pioneering work conducted by Merrifield in the area of peptide synthesis [19]. Interest in this synthetic strategy continues unabated to this day, particularly in connection with the production of new active components for drugs, since the repetitive amide bond formation performed in automated synthesisers lends itself ideally to the construction of extensive substance libraries by combinatorial chemistry [20]. 

Recent solid-phase syntheses utilise light-sensitive linkers which can subsequently be cleaved photochemically. And oxidative and reductive cleavage concepts are also used. The support material is subsequently filtered off and reused after washing. 

In contrast to reactions in solution, solid-phase synthesis has the advantage that excess amounts of reactants can be used and the yields thus increased. Work-up and purification processes - which often prove difficult in homogeneous solution - are rationalised as straightforward washing or filtration. Recycling of the support material after cleavage of the product from the solid phase also has cost benefits. 

FIGURE 3.41 Examples of enzymatically derived types of polymers. 

Proteins, as a biopolymer class, are one of the main structural and regulatory units in living organisms. Proteins consist of linear chains of alpha amino acid 

FIGURE 3.42 Structure and the three-letter code of the main mammalian amino acids. The amino acids are aligned toward the increasing relative hydrophobicity under physiological conditions. 

SPPS was originally developed by Robert Brace Merrifield in 1963 and involves stepwise additions of protected amino acids to a growing peptide chain, which is covalently bound to a solid resin particle. The solid support for peptide synthesis must maintain a stable physical form that allows filtration in all of the 

FIGURE 3.43 Synthetic scheme for sohd phase peptide synthesis. 

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Then N-Boc-O-benzylserine is coupled to the free amino group with DCC. This concludes one cycle (N° -deprotection, neutralization, coupling) in solid-phase synthesis. All three steps can be driven to very high total yields (< 99.5%) since excesses of Boc-amino acids and DCC (about fourfold) in CHjClj can be used and since side-reactions which lead to soluble products do not lower the yield of condensation product. One side-reaction in DCC-promoted condensations leads to N-acylated ureas. These products will remain in solution and not reaa with the polymer-bound amine. At the end of the reaction time, the polymer is filtered off and washed. The times consumed for 99% completion of condensation vary from 5 min for small amino acids to several hours for a bulky amino acid, e.g. Boc-Ile, with other bulky amino acids on a resin. A new cycle can begin without any workup problems (R.B. Merrifield, 1969 B.W. Erickson, 1976 M. Bodanszky, 1976). 

The major disadvantage of solid-phase peptide synthesis is the fact that ail the by-products attached to the resin can only be removed at the final stages of synthesis. Another problem is the relatively low local concentration of peptide which can be obtained on the polymer, and this limits the turnover of all other educts. Preparation of large quantities (> 1 g) is therefore difficult. Thirdly, the racemization-safe methods for acid activation, e.g. with azides, are too mild (= slow) for solid-phase synthesis. For these reasons the convenient Menifield procedures are quite generally used for syntheses of small peptides, whereas for larger polypeptides many research groups adhere to classic solution methods and purification after each condensation step (F.M. Finn, 1976). 

We shall describe here one step in the total synthesis of a protected heptatetracontapep-tide ( = 47 amino acids) by K. Hofmann (H.T. Storey, 1972), and compare the techniques and the results with those of solid-phase synthesis. 

Sections 27 15 through 27 17 describe the chemistry associated with the protection and deprotection of ammo and carboxyl functions along with methods for peptide bond formation The focus m those sections is on solution phase peptide synthesis Section 27 18 shows how these methods are adapted to solid phase synthesis... 

Memfield s concept of a solid phase method for peptide synthesis and his devel opment of methods for carrying it out set the stage for an entirely new way to do chem ical reactions Solid phase synthesis has been extended to include numerous other classes of compounds and has helped spawn a whole new field called combinatorial chemistry Combinatorial synthesis allows a chemist using solid phase techniques to prepare hun dreds of related compounds (called libraries) at a time It is one of the most active areas of organic synthesis especially m the pharmaceutical industry... 

E. C. Blossey and D. C. Neckers, Eds., Solid Phase Synthesis, Halsted, New York, 1975 P. Hodge and D. C. Sherrington, Eds., Polymer-Supported Reactions in Organic Synthesis, Wiley-Interscience, New York, 1980. A comprehensive review of polymeric protective groups by J. M. J. Frechet is included in this book. 

The major impetus for the development of solid phase synthesis centers around applications in combinatorial chemistry. The notion that new drug leads and catalysts can be discovered in a high tiuoughput fashion has been demonstrated many times over as is evidenced from the number of publications that have arisen (see references at the end of this chapter). A number of )proaches to combinatorial chemistry exist. These include the split-mix method, serial techniques and parallel methods to generate libraries of compounds. The advances in combinatorial chemistry are also accompani by sophisticated methods in deconvolution and identification of compounds from libraries. In a number of cases, innovative hardware and software has been developed tor these purposes. 

A SAMPLE OF REVIEWS ON SOLID PHASE SYNTHESIS General... 

K. Gordon and S. Balasubramanian, Solid phase synthesis - designer linkers for combinatorial chemistry A review J Chem Technol Biotechnol 74 835-851 7999. 

J.W. Labadie, Polymeric Supports for Solid Phase Synthesis, Curr Opin Chem Biol 2 346-352 1998. 

Rapid purification Stir over CaH2 (5% w/v) overnight, filter, then distil at 20mmHg. Store the distd DMF over 3A or 4A molecular sieves. For solid phase synthesis, the DMF used must be of high quality and free from amines. 

Oxytocin [50-56-6] M 1007.2, m dec on heating, [a] -26.2"(c 0.53, N AcOH). A cyclic nonapeptide which was purified by countercurrent distribution between solvent and buffer. It is soluble in H2O, rt-BuOH and isoBuOH. [Bodanszky and du Vigneaud J Am Chem Soc 81 2504 1959 Cash et al. J Med Pharm Chem 5 413 1962 Sakakibara et al. Bull Chem Soc Jpn 38 120 1965 solid phase synthesis Bayer and... 

One widely used method involving protected compounds is solid-phase synthesis (polymer-supponed reagents). This method has the advantage of requiring only a simple workup by filtration such as in automated syntheses, especially of polypeptides, oligonucleotides, and oligosaccharides. 

Other PK variations include microwave conditions, solid-phase synthesis, and the fixation of atmospheric nitrogen as the nitrogen source (27—>28). Hexamethyldisilazane (HMDS) is also an excellent ammonia equivalent in the PK synthesis. For example, 2,5-hexanedione and HMDS on alumina gives 2,5-dimethylpyrrole in 81% yield at room temperature. Ammonium formate can be used as a nitrogen source in the PK synthesis of pyrroles from l,4-diaryl-2-butene-l,4-diones under Pd-catalyzed transfer hydrogenation conditions. 

The adaptation of the Bischler-Napieralski reaction to solid-phase synthesis has been described independently by two different groups. Meutermans reported the transformation of Merrifield resin-bound phenylalanine derivatives 32 to dihydroisoquinolines 33 in the presence of POCI3. The products 34 were liberated from the support using mixtures of HF/p-cresol. In contrast, Kunzer conducted solid-phase Bischler-Napieralski reactions on a 2-hydroxyethyl polystyrene support using the aromatic ring of the substrate 35 as a point of attachment to the resin. The cyclized products 36 were cleaved from the support by reaction with i-butylamine or n-pentylamine to afford 37. 

The von Richter cinnoline process was further extended to solid-phase synthesis. The route began from benzylaminomethyl polystyrene and the required diverse o-haloaryl resins represented by 21 were prepared from substituted o-haloanilines. A Pd-mediated cross-coupling reaction with 21 and the alkynes provided the alkynylaryl derivatives represented by alkyne 22. The von Richter cyclization reaction with hydrobromic or hydrochloric acid in acetone/HaO and cleavage from the resin occurred in the same step to furnish the cinnoline derivatives 23 in 47-95% yield and 60-90% purity (no yield reported for each entry). 

Solid-phase synthesis and transformations of heterocycles 97T5643, 98JCS(P1)3293, 98T15385. 

Solid-phase synthesis of heterocycles 98MI37, 980PP489, 98YGK2. 

More recently, Tietze and Steinmetz (96SL667) used the patented polystyrene-resin methodology for for the solid-phase synthesis of a large number of diverse )3-keto esters 40a-h. These were reacted with phenylhydrazine in THF at room temperature to give hydrazones 41a-h that were then cychzed into 2-phenyl 5-substituted pyrazol-3-ones 42a-h by heating in toluene at 1(X)°C (Scheme 12). 

Substituted 4-aryl-1 -oxo-1,2-dihydropyrazino[l, 2-i]isoquinolinium salts 402 were obtained when 3-substituted isoquinolines 401 were cleaved from a polymer by treatment 25% TFA (00MIP5). c/i-3,lla-H-3-Phenyl-1,2,3,4,11,11 fl-hexahydropyrazino[l, 2-i]isoquinoline-1,4-dione (404) formed when isoquinoline derivative 403 was cleaved from a resin with 25% TFA during an automated solid-phase synthesis (98BMCL2369). 

Preparation of Memfield resin-bound nitro acetates, which is a suitable bndding block for the development of combinatorial solid phase synthesis, is repotted. The anion of ethyl nitro acetate is generated in DMF by an electrochemical method using Pt cathode, magnesium rod anode, and tetrabutylairunonium bromide as an electrolyte. Alkylaton of this anion with alkyl hahdes gives mono-alkylated products in 80% yield." ... 

The classic Reissert indole synthesis, involving the reducdve cyclization of o-ni-trophenylpymvic acid, has been used for synthesis of 2-ethoxycarbonyl-4-alkoxymethylindo-les The modified Reissert reacdon, involving the reducdve cyclizadon of an o-rdtrophenyl acetoaldehyde, has been adapted to solid-phase synthesis... 

On-Bead Solid-Phase Synthesis of Chiral Dipeptides... 

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