The split and pool (split-mix) synthesis is a method in combinatorial chemistry that can be used to prepare combinatorial compound libraries. It is a stepwise, highly efficient process realized in repeated cycles. The procedure makes it possible to prepare millions or even trillions of compounds as mixtures that can be used in drug research.

History

According to traditional methods, most organic compounds are synthesized one by one from building blocks coupling them together one after the other in a stepwise manner. Before 1982 nobody was even dreaming about making hundreds or thousands of compounds in a single process. Not speaking about millions or even trillions. So the productivity of the split and pool method invented by Prof. Á. Furka (Eötvös Loránd University Budapest Hungary), in 1982 seemed incredible at first sight. The method had been described it in a document notarized in the same year. The document is written in Hungarian and translated to EnglishFurka Á. Tanulmány, gyógyászatilag hasznosítható peptidek szisztematikus felkutatásának lehetőségéről (and Study on the possibility of systematic searching for pharmaceutically useful peptides) https://mersz.hu/mod/object.php?objazonosito=matud202006_f42772_i2 Motivations that led to the invention are found in a 2002 paperÁ. Furka, Combinatorial chemistry: 20 years on …Drug Discovery Today 2002, 7, 1-2. and the method was first published in international congresses in 1988Á. Furka, F. Sebestyén, M. Asgedom, G. Dibó, Cornucopia of peptides by synthesis In Highlights of Modern Biochemistry, Proceedings of the 14th International Congress of Biochemistry, VSP. Utrecht, The Netherlands, 1988, Vol. 5, p 47. then in print in 1991.Furka Á, Sebestyén F, Asgedom M, Dibó G ( 1991) General method for rapid synthesis of multicomponent peptide mixtures, Int J Peptide Protein Res 37; 487-93.

The split and pool synthesis and its features

The split and pool synthesis (S&P synthesis) differs from traditional synthetic methods. The important novelty is the use of compound mixtures in the process. This is the reason of its unprecedentedly high productivity. Using the method one single chemist can make more compounds in a week than all chemists produced in the whole history of chemistry. The S&P synthesis is applied in a stepwise manner by repeating three operations in each step of the process:

Dividing a compound mixture into equal portions Coupling one different building block (BB) to each portion Pooling and thoroughly mixing the portions

The original method is based on the solid-phase synthesis of MerrifieldMerrifield R.B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide (1963) J. Am. Chem. Soc. 85, 2149-2154. The procedure is illustrated in the figure by the flowing diagram showing of a two-cycle synthesis using the same three BBs in both cycles. Choosing the solid phase method in the S&P synthesis is reasonable since otherwise removal of the by-products from the mixture of compounds would be very difficult.

Efficiency

The high efficiency is the most important feature of the method. In a multi step (n) synthesis using equal number of BBs (k) in every step the number of components in a forming combinatorial library (N) is:

N=kn

This means that the number of components increases exponentially with the number steps (cycles) while the number of the required couplings increases only linearly. If a different number of building BBs are used in the cycles (k1, k2, k3....kn) the number of the formed components is:

N=k1.k2.k3...kn.

This feature of the procedure offers the possibility to synthesize a practically unlimited number of compounds. For example, if 1000 BBs are used in four cycles 1 trillion compounds are expected to form. The number of needed couplings is only 4000!

The reason of the high efficiency

The explanation of the extraordinary efficiency is the use of mixtures in the synthetic steps. If in a traditional reaction one compound is coupled with one reactant and one new compound is formed. If a mixture of compounds containing n components is coupled with a single reactant the number of new compounds formed in the single coupling is n. The difference between the traditional and the split and pool synthesis is convincingly shown by the number of coupling steps in the traditional and the split and pool synthesis of 3,2 million pentapeptides.

Conventional synthesis: 3,200,000x5=16,000,000 coupling steps cca 40,000 years S&P synthesis: 20x5=100 coupling steps cca 5 days

It is possible to conduct the conventional synthesis rational way as is shown in the figure. In this case, the number of coupling cycles is:

20+400+8,000+160,000+3,200,000=3,368,420 cca 9,200 years

The theoretical upper limit of the number of components As often mentioned the split and pool method makes it possible to synthesize an unlimited number of compounds. In fact, the theoretical maximum number of components depends on the quantity of the library expressed in moles. If for example, 1 mol library is synthesized the maximum number of components is equal to the Avogadro number:

6,02214076·1023

In such a library each component would be represented by a single molecule.

Components of the library form in equal molar quantities

As far as the chemistry of the couplings makes it possible the components of the libraries form in nearly equal molar quantity. This is made possible by dividing of the mixtures into equal samples and by homogenization of the pooled samples by thoroughly mixing them. The equal molar quantity of components of the library is very important considering their applicability. The presence of compounds in unequal quantities may lead to difficulties in evaluation of the results in screening. The solid phase method makes it possible to use the reagents in excess to drive the reactions close to completion since the surplus can easily be removed by filtration.

The possibility of using two mixtures in the synthesis

In principle, the use of two mixtures in the S&P synthesis can lead to the same combinatorial library that forms in the usual S&P method. The differences in the reactivity of BBs however, bring about large differences in the concentrations of components, and the differences are expected to increase after each step. Although a considerable amount of labor could be saved by using the two mixtures approach when a high number of BBs are coupled in each position, it is advisable to stick to the normally used S&P procedure.

The presence of all structural varieties in the library

Formation of all structural variants that can be deduced from the BBs is an important feature of the S&P synthesis. Only the S&P method can achieve this in a single process. On the other hand, the presence of all possible structural varieties in a library assures that the library is a combinatorial one and is prepared by combinatorial synthesis.

Forming of one compound in the beads

The consequence of using a single BB in couplings is the formation of a single compound in each bead. The formation of OBOC libraries is an inherent property of the S&P synthesis. The reason is explained in the figure. The structure of the compound formed in a bead depends on the reaction vessels in which the bead happens to occur in the synthetic route. It depends on the decision of the chemist to use the library in the tethered (OBOC) form or cleave down the compounds from the beads and use it as a solution.

Realization of the split and pool synthesis

The split and pool synthesis was first applied to prepare peptide libraries on solid support. The synthesis was realized in a home-made manual device shown in the figure. The device has a tube with 20 holes to which reaction vessels could be attached. One end of the tube is linked to a waste container and a water pump. Left shows loading and filtering, right coupling-shaking position. In the early years of combinatorial chemistry, an automatic machine was constructed and commercialized at AdvancedChemTech (Louisville KY USA). All operations of the S&P synthesis are carried automatically under computer control. At present, the Titan 357 automatic synthesizer is available at aapptec (Louisville KY, USA).

Encoded split and pool synthesis

Although in the S&P synthesis a single compound forms on each bead its structure is not known. For this reason, encoding methods had been introduced to help to determine the identity of the compound contained in a selected bead. Encoding molecules are coupled to the beads in parallel with the coupling of the BBs. The structure of the encoding molecule has to be easier determined than that of the library member on the bead. Ohlmeyer et al. published a binary encoding method.Ohlmeyer MHJ, Swanson RN, Dillard LW, Reader JC, Asouline G, Kobayashi R, Wigler M, Still WC (1993) Complex synthetic chemical libraries indexed with molecular tags, Proc Natl Acad Sci USA 90; 10922–10926. They used mixtures of 18 tagging molecules that after cleaving them from the beads could be identified by Electron Capture Gas Chromatography. Nikolajev et al. applied peptide sequences for encodingV. Nikolaiev, A. Stierandova, V. Krchnak, B. Seligman, K. S. Lam, S. E. Salmon, M. Lebl Pept. Res. 1993, 6, 161. Sarkar et al. described chiral oligomers of pentenoic amides (COPAs) that can be used to construct mass encoded OBOC libraries.Sarkar M, Pascal BD, Steckler C, Aquino C., Micalizio GC, Kodadek T, Chalmers MJ (1993) Decoding Split and Pool Combinatorial Libraries with Electron Transfer Dissociation Tandem Mass Spectrometry, J Am Soc Mass Spectrom 24(7): 1026-36. Kerr et al. introduced an innovative kind of encoding.J. M. Kerr, S. C. Banville, R. N. Zuckermann J. Am. Chem. Soc. 1993, 115, 2529. An orthogonally protected removable bifunctional linker was attached to the beads. One end of the linker was used to attach the non-natural BBs of the library while to the other end the encoding amino acid triplets were linked. One of the earliest and very successful encoding methods was introduced by Brenner and LernerS. Brenner and R. A. Lerner Proc. Natl. Acad. Sci. USA 1992, 89, 5381. in 1992. They proposed to attach DNA oligomers to the beads for encoding their content. The method was implemented by Nielsen, Brenner, and JandaJ. Nielsen, S. Brenner, K. D. Janda J. Am. Chem. Soc. 1993, 115, 9812. using the bifunctional linker of Kerr et al. to attach the encoding DNA oligomers. This made it possible to cleave down the compound with the DNA encoding oligomer attached to it.

Split and pool synthesis in solution

Han et al. described a method that made it possible to keep the advantages of both the high efficiency of S&P synthesis and that of a homogeneous media in the chemical reactions.H. Han, M. M. Wolfe, S. Brenner, K. D. Janda Liquid-phase combinatorial synthesis Proc Natl Acad Sci USA 1995, 92:6419. https://doi.org/10.1073/pnas.92.14.6419 In their method polyethyleneglycol (PEG) was used as soluble support in S&P synthesis of peptide libraries.

MeO-CH2-CH2-O-(CH2-CH2-O)n-CH2-CH2-OH

PEG proved suitable for this purpose since it is soluble in a wide variety of aqueous and organic solvents and its solubility provides homogeneous reaction conditions even when the attached molecule itself is insoluble in the reaction medium. Separation from the solution of the polymer and the synthesized compounds bound to it can be achieved by precipitation and filtration. The precipitation requires concentrating the reaction solutions then diluting with diethyl ether or tert-butyl methyl ether. Under carefully controlled precipitation conditions the polymer with the bound products precipitates in crystalline form and the unwanted reagents remain in solution.