As exemplified, among others,  by the peptide hormones discovered so far, the shorter-lenghtier peptides take part in a number of important functions in the living organism. It can be supposed, that only a small fraction is known of these biologically active peptides having potential therapeutic effect. This fact motivates the intensive international and domestic research activity in this field.
        Two, in principle different, approaches offer themselves for searching for peptides bearing new biological effects:

If the n-residue peptides are synthesized stepwisely and independently, the number of the require synthetic steps (S_n) can be calculated as follows:

 It is noted, that a synthetic step means a complete coupling cycle, that is, in addition to the coupling step itself incorporates the operations connected with the protecting groups, too.
        With good organization, that is, choosing a systematic synthesis route the number of synthetic steps can be reduced. The minimum number of synthetic steps is:

        The synthesized peptides are supposed to be submitted to screening tests. Since several tests have to be done on each peptide, the total number of the required screening tests is hopelessly large. If the number of kinds of screening tests is denoted by t, the total number of screening tests is expressed by the following equation:

       Table 1 shows the the possible number of peptides depending on the number of residues, the number of synthetic steps required for their synthesis, and number of the screening tests, calculating with 10 different tests (t=10). The figures - which are rounded - clearly show, that even the synthesis and testing of all tripeptides would be an almost hopeless venture.
 
 

    Because of the very large number of possible peptides, the stepwise synthesis of all peptides - even in the case of small ones - is an unrealizable task. The large number of the screening experiments constitutes a further problem. The proposal to be outlined on the next pages will try to somewhat improve this almost hopeless situation.

The proposal to be outlined here constitutes a research project which makes possible to search for biologically active peptides with much greater chance than before. When I write down this project I'm fully aware of its potential importance in industry. It is also clear, that it's realization is possible only through cooperation of different institutions. Primarily the participation of the pharmaceutical industry is desirable since the investments can be recovered through pharmaceutical industry.
  The essence of the proposal is that instead of one by one synthesis of peptides, peptide mixtures should be prepared containing several hundred or several thousand peptides in approximately 1 to 1 molar ratio, and these peptide mixtures should be submitted to screening tests. It will be shown that on this way much labor can be saved both in the synthetic work and in the screening experiments. In the first stage one has to determine whether or not the mixture shows any biological effect. If biological effect is observed, of course, it has to be determined which component (or which components) are responsible for the activity.

        Since not single peptides but rather mixtures of peptides are synthesized, post synthetic purification and removal of by-products are out of question. Because of this, the classical method of synthesis (in solution) can not be used either. In the synthesis of peptide mixtures the solid phase method have to be applied. It is noted here, that in the syntheses not necessarily the 20 amino acids are used. In some cases more than 20 amino acids may be used, for example if - in addition - non-common amino acids are intended to be used as building blocks. Less than 20 amino acids may be used, for example, in decapeptides, since the synthesis of all peptides seems to be unrealistic and have to compromise with the use of fewer kinds of amino acids. Let denote by k  the number of the amino acids intended to vary in the i   position. The numbers of amino acids varied in the C-terminal and N-terminal position are k₁ and k_n , respectively.

        Realization of the synthesis. The resin is divided into k₁  equal portions (that is to as many portions as many amino acids are intended to vary at the C-terminal of peptides). Then each portion of resin is coupled with one of the k₁  kinds of amino acids then the amino-protecting group is removed from every sample. A small quantity is removed from every sample and they are taken aside for later use, then the samples are thoroughly mixed. Then the mixture of aminoacyl resins is divided into k₂  equal portions and each of them is coupled with one of the k₂  kinds of protected amino acids then the amino-protecting groups are removed from each sample. Before mixing, again small samples are removed and taken aside. The mixture of dipeptides is cleaved from a small portion of the mixed resin to use it in biological tests. The rest of the mixed resin is divided into k₃  equal parts and the amino acids intended to occupy the third position are coupled to them. Then the synthesis is likewise continued until the mixture of n-residue peptides is reached.
        It is worthwhile to add some notes. As in an ordinary solid phase synthesis, one has to make an effort to achieve good conversion by applying the reagents in excess. Fortunately, however, conversions lower than 100%, or minor unwanted splitting reactions do not cause so serious problems like in ordinary syntheses. The labour requirement could be significantly reduced by using mixtures of properly protected amino acids in acylation reactions. This, however, does not seem to be an acceptable solution because of the differences in the reactivity of the activated amino acids which would lead to the formation of peptides in significantly different concentrations thus causing problems in the screening experiments. Formation of peptides in equal concentrations can only be assured by mechanical mixing of samples followed by dividing into equal portions. This makes possible a complete conversion for every amino acid component. Possibility of acylations with mixtures of several amino acids of identical reactivity might be a matter of further considerations. Smaller differences in reactivities could be compensated by properly selected molar ratios of the amino acid derivatives of the mixture. In the following calculations the possibility of acylations with the mixtures of amino acid derivatives will be left out of considerations.
        The number of peptides formed in the synthesis, that is, the number of components in the peptide mixtures - in a general case - can be calculated by the following formula:

If the same number (k) of amino acids are varied in every position

The number of synthetic steps in the synthesis of a peptide mixture containing N_n  peptides (considering the attachment of the first amino acid to the resin as separate step) is:

If the same number (k) of amino acids are varied in each position,

The formulae show the advantage of the synthesis of peptide mixtures: the number of the synthetic steps can be calculated by summing the numbers of the varied amino acids, while the number peptides is given by the product of the numbers of the varied amino acids.
        One example: synthesis of the mixture of tetrapeptides prepared by varying the 20 kinds of amino acids, needs only 80 synthetic steps! It is noted, that in the same run all shorter peptides - that is the 400 dipeptides and the 8000 tripeptides - are formed, too. The traditional synthesis of these peptides would need 168 400 synthetic steps. A different comparison: in the traditional method with 80 steps only about 30 tetrapeptides can be synthesized.

   Screening of peptide mixtures

        Peptides mixtures - in the first approximation - are synthesized to determine whether or not they contain biologically active component. It is supposed - although it needs experimental verification - that screening experiments can be made with mixtures, too. This offers great advantage over the traditional method since the number of screening tests is reduced by a factor equal to the number of components of the mixture. For example, the mixture of the 8000 tripeptides can be examined by a single series of tests. If there is active peptide among them, one of the executable t tests gives positive result. If the number of active peptides is more than one, then, of course, more tests may give positive result. In the synthesis of the mixture of n-residue peptides it is wortwhile to test the shorter peptides, too. The synthesis is so designed to allow for this. Taking this requirement into account, and the number of kinds of tests being t, the total number of the executable tests is:

Although this equation certainly holds, its realizability in practice deserves some notes. There is - without any doubt - an upper limit in the number of components of the peptide mixtures to be submitted to screening tests. It is difficult to estimate this number without experiments. The mixtures may probably contain many thousands of components, and as it can be judged today, the method outlined above is rather limited by possibilities of screening tests than by the number of the required synthetic steps. If there are too many components in the mixture, too large samples have to be applied in the screening experiments to achieve observable effect for a single component. The mixture supposedly contains a number of more or less active analogs and their effect is probably summarized. Nevertheless, an unsurpassable limit in the number of components certainly exists. Therefore in certain cases may prove useful to examine the effect of the n-residue mixtures without final mixing. In other cases the synthesis should be designed so not to surpass the optimal number of components.

        "Backsearching" for the active peptide. If the peptide mixture is detected to contain active component, that is, if the mixture shows a new type biological effect, then the further task is the isolation and structure determination of the active peptide followed by its synthesis. Once the mixture containing the active component or components is in our hand the isolation can be carried out using the effective separation methods, since these make possible to separate the active compound even from thousands of inactive components. It is possible, however, to follow a different method, too. This will be outlined here. This approach to the identification of the active peptides is supposed to be less tedious then the isolation method, moreover it supplies additional information concerning the structure-effect relationship. Applicability of the method requires a procedure for quantitative determination of activity. For the sake of simplicity let's suppose that the mixture contains a single effective component (besides analogs having the same kind of effect but smaller activity).

        Backsearching step No. 1. The experiments are started with the k_n samples taken aside in the synthesis of the n-residue peptides before final mixing. The mixtures of n-residue peptides are cleaved from each resin sample. The mixtures of peptides differ from each other in the n-th   (that is the N-terminal) residue of their component peptides. Each peptide mixture is submitted to a quantitative activity determination. This shows how the activity depends on the terminal amino acid residue, that is, this way we can determine the N-terminal residue of the active peptide, and in addition it will show the effect of its replacement by other amino acid residues. Let's suppose, for example, that the N-terminal residue in the sample showing the highest activity (as well as in the active peptide) is Phe (phenylalanine). It is noted here that if there are several samples showing equally high activity it is practical to choose as the N-terminal residue of the active peptide the cheapest or the synthetically less problematical amino acid. This note holds for the subsequent backsearching steps, too.

        Backsearching step No. 2. The experiment is continued with the k_n-1 samples taken aside in the synthetic stage of the (n-1)-residue peptides. The amino acid determined before, that is Phe in our example, is coupled to each sample. Cleavage of the peptides from the support gives k_n-1 different peptide mixtures. Their common feature is that every peptide has Phe in the N-terminal position. By submitting the peptide mixtures to quantitative screening experiments one can determine the amino acid residue occupying position n-1 (that is, the pre-N-terminal position) in the active peptide. This experiment also shows the effect on activity of substitution of this amino acids with other ones. Let's suppose that the pre-aminoterminal amino acid is Arg (arginine). It should be noted that in this backsearching step the Phe is coupled to k_n-1 samples and the same number (k_n-1) of screening experiments have to be done. Not all of the t kinds of tests are required, only the one proved before to be positive. Consequently the number of the synthetic steps and the number of screening experiments are the same: k_n-1. It is also noted that in the previous backsearching step only screening test are done (their number is k_n) synthetic steps are not needed.

        Backsearching step No. 3. This, and the subsequent backsearching steps may be realized using two different approaches. The peptides in samples taken aside during the synthesis have to be elongated to contain n residues, in such way, to carry on their N-terminal section the amino acid residues assuring activity. This can be realized on two ways. Either by stepwise coupling with amino acids (in our example with protected Arg then Phe) or by coupling in a single step with a previously synthesized oligopeptide having the required sequence (in our example Phe.Arg). The required synthetic steps in the two approaches significantly differ. The number of the screening experiments, however, are the same in both cases. Let's turn now to the No. 3. backsearching step.

        Stepwise elongation. Let's take the k_n-2 samples taken aside in the synthesis of (n-2)-residue peptides. Each sample is coupled first with protected Arg then with protected Phe. After cleaving the peptides from the support each of the k_n-2 peptide mixtures are submitted to activity tests to determine the amino acid residue occupying the third position counting from the N-terminal end. The number of screening tests to be executed is k_n-2. The number of the required synthetic steps is: 2k_n-2. The multiplying factor preceding k  is the bigger the shorter are the peptides to be elongated. The numerical value of the factor is equal to the number of amino acids to be coupled with in the elongation process.

        Elongation with oligopeptide. A previously synthesized dipeptide (in our example Phe.Arg) is coupled to each of the k_n-2 samples taken aside, then the process is continued as described above. The number of screening test is also k_n-2. The number of synthetic steps (leaving out of consideration the synthesis of the oligopeptide) is also k_n-2. This procedure seems to be more economical. In practice it means that the active peptide is synthesized in parallel with the screening tests using the classical method started from the N-terminus. Small fractions of the growing peptide are sacrificed in the backsearching steps. This backsearching method has the great advantage (in addition to the fact that it needs less synthetic steps) that when the backsearching procedure is finished the active peptide is synthesized, too.

        Backsearching of more than one active peptide. In the synthetic peptide mixtures several active peptides may be present, showing different effects. In these cases the number of backsearching steps will be bigger by a factor equal to the number of the differing active peptides. That is, if the number of the active peptides is "a" the values deduced above are multiplied by a. It is noted that the presence in the mixture of peptides having different effects may complicate the backsearching process especially in the case of peptides with opposing effects. This, however, is not treated in details.
        The backsearching process ends when the sequences of all active peptides are determined by applying either the oligopeptide or the stepwise elongation method.
 
 

Number of synthetic steps using oligopeptide elongation

         In synthesis:

Total in synthesis and backsearching:

        If k amino acids are varied in each step:

Number of synthetic steps using stepwise elongation.

         In synthesis:

         If k amino acids are varied in each step:

Number of screening tests equally valid using the oligopeptide and stepwise elongation

         In synthesis:                                                                              T_n = t(n-1)

         In backsearching:

         If k amino acids are varied in each step:

An example: preparation and screening of all pentapeptides

                                               N₅ = 320000,     n=5  k=20 t=10     a=1

         Total number of synthetic steps

                                               Ogopeptide elongation          180
                                               Stepwise elongation              300
                                               Number of tests                    140

        Applicability of the method outlined before is not restricted for only the systematic searching for active peptides. The same principle applies to all other sequential types of compounds, that is, when the compounds belonging to this type of compounds differ from each other only in their building blocks or the sequences of these building blocks. Among them may occur natural compounds like oligosaccharides or oligonucleotides but synthetic products may be taken into account, too. Among these later ones one may think about sequential copolymers or sequential polycondensates.

                                                                                                               Dr. Arpad Furka
                                                                                                               university professor
 
 

File number 36237/1982
I certify this stitched document comprising 14, that is, fourteen pages was subscribed in my presence by Dr. Arpad Furka, university professor, with his own hands.
Budapest, 1982. Nineteen hundred and eighty two, June 15, (fifteen).
                                                                                                                 Dr. Judit Bokai
                                                                                                                state notary public

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