Amino Acid Tester libraries
Determination of amino acid composition
of bioactive components
of soluble peptide libraries
Sets of amino acid tester libraries, like sets of omission libraries, can be used for determination of the amino acid composition of bioactive components of peptide libraries (Campian et al. 1998). An amino acid tester library comprises all peptides of a full peptide library in which a certain amino acid (e.g. Ala) is present in any position. For this reason if Ala happens to be present in the bioactive peptide the Ala tester library is expected to show activity when tested. If Ala is not present in the bioactive peptide, no activity is expected. An amino acid tester library contains all those peptides which are missing from an omission library. This is demonstrated in the figure where the amino acids are symbolized by yellow, blue and red circles. Thus the number of components of an amino acid tester library (E, F and G) can be calculated by subtracting the number of peptides of an omission library (B, or C or D) from that of a full library (A). For example the number of components of E is 19 (27 - 8).
A B C D E F G
The table below shows that normally the number of components in amino acid tester libraries is considerably smaller than that in omission libraries.
Library Tri Tetra Penta Hexa _
6859 130321 2476099
AA Tester 1141 29679 723901 16954119
Synthesis of the amino acid tester libraries
Amino acid tester libraries belong to those partial libraries
that are not simple to synthesize. They can usually prepared by pooling
several simple libraries. This situation is demonstrated by preparation
of the alanine tester library of a special full tripeptide amide library.
The full library is prepared by using 19 amino acids in the first and second
coupling position (cysteine is omitted in all positions) and 20 amino acids,
including pyroglutamic acid in the N-terminal position (see omission libraries).
The full library contains 7220 peptides. In this case the amino acid tester
libraries are composed from 3 libraries. In the case of alanine tester
libraries, as the figure shows, the component libraries are:
Library 1. Coupling position 1 (CP1) is occupied in all peptides by alanine. In CP2 19 amino acids are varied including alanine. In CP3 20 amino acids are used including alanine.
Library 2. In CP1 alanine is omitted, so only 18 amino acids are varied. In CP2 only alanine is used. In CP3 again the 20 amino acids are applied.
Library 3. In both CP1 and CP2 the Ala lacking 18 amino acids were used. The last coupling position is unvaried, only alanine is used. The number of peptides in library 1, 2 and 3 are 380, 360 and 324, respectively. The total number is 1064.
Component libraries of alanine tester library
Blue circle: 18 amino acids excluding Ala, green circle: 19 amino
acids including alanine, red circle: 20 amino acids including pyroglutamic acid,
yellow circle: only alanine
It would have taken too much work to prepare the three component libraries for all 19 tester libraries of the set. For this reason we devised a special chemfile (synthetic program) for the Advanced ChemTech 357 automatic synthesizer that made possible to prepare the three component libraries in a single run. The figure below shows the flow diagram of the procedure. Libraries 1, 2 and 3 formed via routes 1, 2 and 3, respectively, and the resin was distributed among the three routes according to the number of peptides in the three component libraries.
Synthesis of alanine tester library in a single run
Red numbers show the number of amino acids used in split-mix coupling operations,
black numbers indicate quantities of resin in milligrams
Screening with amino acid tester libraries
Applicability of amino acid tester libraries is demonstrated by a model experiment similar to that used for omission libraries: competitive inhibition of binding of LH-RH to its polyclonal antibody. The 19 amino acid tester libraries were submitted to binding experiments using radioimmunoassay in determination of binding of radiolabeled LH-RH to the antibody. For better visualization in the figure below, 100 - LH-RH binding % was plotted instead of LH-RH binding %.
It can be clearly seen that glycine, proline and arginine
tester libraries are the most effective ones in inhibiting LH-RH binding.
This confirms previous findings with omission libraries that the composition
of the active peptide is: Gly, Pro, Arg. The sequence of these amino acids
is not known. It has to be determined in a separate experiment. It is known,
however, that is a tripeptide amide library is prepared using these three
amino acids in all the three coupling position, the library is expected
to contain the active peptide. This "occurrence
library" is composed from 27 peptides. The nine sub-libraries of this library
can be used to determine the sequence of the three amino acids of the active
Synthesis of the sub-libraries of the occurrence library
The nine sub-libraries of the ocurrence library are: 1G, 2G, 3G, 1P, 2P, 3P, 1R, 2R and 3R, where G, P and R are the symbols of the non varied amino acids and the numbers show their coupling position. For the synthesis of the nine sub-libraries a special procedure was developed which made possible to prepare all of them in a single run using Advanced ChemTech 357 automatic synthesizer. The flow diagram below shows the amino acids used in the three coupling steps as well as the products. The number of operations are optimized to the possible minimum.
2G 2P 2R 3G 3P 3R 1G 1P 1R
Synthesis of 9 sub-llibraries in a single run
Positional scanning with the nine sub-libraries
of the occurrence library
The results of the experiments is illustrated in the figure. The LH-RH binding is reduced most effectively by: 1G, 2P and 3R. Consequently, the sequence of the active peptide is: R-P-G-NH2.