Solid Phase Organic Synthesis Of Informational Macromolecules Of Interest To Medicinal Chemists

That the solid phase synthesis of collections of launched this field is not intrinsically surprising. The basic methodology existed because of Merrifield and many others. The pep-tide linkage has notable advantages for this work because it is relatively chemically stable; non-chiral, constructible by iterative processes amenable to automation, the products are rarely branched, possess a variety of interesting biological properties, and can be constructed in great variety. The counterbalancing defects of these compounds are that they are not easily delivered orally unless they are end capped and rather small in molecular weight, are readily destroyed by enzymatic action, and fail to penetrate into cells. The physiological reason for this is readily understood. Peptides, and other informational macromolecules, function in the body to provide specific structure or to generate signals for cells to respond to according to their sequence and architecture. It would be dangerous if they were absorbed intact from ingestion of other life forms. To prevent cellular disruption they are first digested in the gastrointestinal tract, absorbed as monomers, and then reassembled after our own genetic pattern so that they join or supplement those already present without causing disruption in cellular architecture or function.

Nucleic acids have many of the same advantages and disadvantages, and their compound libraries came into being soon after the peptides. Oligosaccharides,on the other hand, have lagged considerably behind. They possess chiral linkages whose construction must be carefully controlled, often are branched, are fragile in the presence of acid conditions, are often highly polar, and are readily digested. Controlling these processes is much more difficult and it has taken longer to conquer these problems.

3.1 Peptide Arrays are prominent among the compounds of interest to biochemists but less so to most medicinal chemists for reasons explicated above. Construction of peptides of specified sequence is an iterative process whose complications largely consist of protection-deprotection steps to ensure that side-chain functionality does not interfere with orderly amide bond formation. When made on resin beads, there are limits to the quantities that can be made in part because of the geometric restrictions caused by bead size and the need to avoid adjacent molecules from interacting with each other, and the comparative lability of the beads to aggressive reagents places limitations on the chemical conditions that can be employed. Porous beads obviously can be loaded more heavily than those whose surface only is accessible after wetting by the reagents. Many different kinds of resins and other solid supports are now available, including some that are solvent soluble depending on the solvent and the temperature.

With their balance of advantages and disadvantages and the present gold standard being oral activity, peptide libraries are currently of primary value in lead seeking, in basic studies on cellular processes, or for the preparation of parenteral medications. Despite intensive study spanning several decades by some of the best minds of this generation, means of delivering thereputically significant blood levels of peptides through the oral route remain elusive. Translating the therapeutic message in peptide leads into oral non-peptide drugs through generally applicable systematic techniques has also been elusive. The several successes that have been achieved have been primarily the result of screening campaigns or serendipitous observations, and the results have so far not revealed an underlying tactical commonality that can be exploited in new cases. Perhaps the best known of these studies has been the translation of snake venom pep-tides, whose injection by serpents leads to a precipitous fall in blood pressure, into truncated pseudodipeptides like captopril, and then on to enalaprilat and lysinopril, which are pseudotripeptides. The basic lesson learned from all of these studies has been that the resulting agent should be as little peptide-like as possible and not exceed the equivalent of at most four amino acid-like residues. Examination of peptide structures in light of the well-known Lipinski rules provides a rationale for what experience has shown. Beyond about four residues, the molecular weight is becoming too high, the polarity is weighted too much toward water solubility, and the hydrogen-bonding inventory is excessive. Further, the compounds are excessively water soluble so that they do not pass through cellular membranes efficiently by passive diffusion.

An added feature to bear in mind is that the preparation of certain medically important polypeptide drugs, such as human insulin and growth hormone, through genetic engineering methodologies, is well developed and convenient so these substances can be used in parenteral replacement therapy. Their preparation through synthetic peptide chemistry represents important achievements in peptide intellectual technology but does not satisfy a commercial need.

Nonetheless, peptide compound libraries are very convenient for uncovering leads quickly for receptors where natural ligands or serendipitous drugs have not previously been found and large libraries of peptides continue to be made (50-65).

The number of peptides that could in principle be made is stupefymg. For example, given that there are approximately 20 common amino acids, and allowingfive post-trans-lational modifications (and ignoring the fact that there are more of these and that there are many wholly synthetic amino acids), the avail

3 Solid Phase Organic Synthesis of Informational Macromolecules of Interest to Medicinal Chemists Table 1.1. Number of Possible Peptide Products as a Function of the Amino Acids Used


(20 x 20)

= 400


(20 X 20 x 20)

= 8000


(20 x 20 x 20 x 20)

= 160,000


(20 x 20 x 20 x 20 x 20)

= 3,200,000


(20 X 20 X 20 X 20 x 20 X 20)

= 64,000,000


(20 x 20 x 20 x 20 x 20 x 20 x 20)

= 1,380,000,000

able synthons are at least approximately equal to the number of letters in the Western alphabet. Ey analogy with the number of languages that have been generated using this system, the potential number of peptides that could be made is clearly astronomical. It would require an incredible effort to make a library containing even only one molecule of each, and Furka has estimated that the mass of such a library would exceed that of the universe by more than 200 orders of magnitude (3)!

Were one to use just the common amino acids, the progression of peptides possible is enormous, as is shown in Table 1.1.

The simplest and least ambiguous method for constructing peptide libraries is the spatially separate or spatially addressed method. Here a single peptide is constructed on a single type of resin, and the resin/products are kept separate. No decoding sequence needs to be attached to the beads in this kind of library. This method was introduced by Geysen in 1984. To make 96 peptides at a time and to keep track cf the products and facilitate their screening, the reactions were run on resins attached to individual pins so constructed that they fit into individual wells of 96-well plates. (Fig. 1.3) (66). A convenient variation was developed for parallel synthesis in which beads were contained in porous bags and dipped into reagent solutions. These are called " tea bags." The identity of the peptide or peptides contained is recorded on the attached label. Sub-sequentiy Fodor et al. developed a very diverse library on silicon wafers using photolitho-


Geysen pins and wells Figure 1.3. Geysen pins and wells.

graphic chemistry for forming the peptides and controlled the specific place along an x-y axis, where each peptide would be located through use of variously configured masks (Fig. 1.4.) Photolytic protecting groups were employed followed by coupling the newly revealed "hot spots" with a suitable reactant. After this, the masks are moved as often as desired and the process repeated. In principle this method could produce thousands of individual peptides on a credit card-like surface. Although somewhat laborious, the synthesis can readily be automated. The method requires photosensitive protecting groups and testing methodologies compatible with support-bound assay methods and the libraries are geographically coded by the position of products on the x-y axis (67). These techniques are now widely employed for gene array amplification and identification experiments.

Synthesis of mixtures of peptides further enhanced the speed and convenience of library construction but required development of devolution methods so that active components in the mixtures could be identified. Direct methods of sequence analysis are available. Mass spectrometry is popular as are NMR methods (involving magic angle methods on single beads). Edman degradation of peptides can also be performed. These methods are popular when iterative methods result in linear polymers.

A further complication of simultaneous preparation of peptide mixtures is that individual amino acids differ greatly in their reactivity, so if one simply placed all of the potential reactants in a flask under bond forming conditions, they would not react at the same rate. With each iteration, the disparity between readily formed and poorly formed bonds would widen. One way to deal with this problem is to use less than fully equivalent

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