COMBINATORIAL CHEMISTRY - MODERN SYNTHESIS APPROACH

 

* The t-butoxy carbonyl (Boc) group.
An alternative choice for amino group protection is the Boc group. Its advantage is that can be removed under milder conditions than the Z group.

Protection of amino group by using t-butoxy carbonyl (Boc) group

The Boc group is completely stable to catalytic hydrogenolysis and as such is orthogonal to the Z group. Basic and nucleophylic reagents are no effect on the Boc group and its removal can be carried out by TFA at room temperature. The most convenient reagent that can be used in the protection reaction is the Boc anhydride (Boc2O).

The 9-fluorenyl methoxy carbonyl (Fmoc) group.
The Fmoc group differs from both Z and Boc groups since it is very stable to acidic reagents.

Protection of amino group by using 9-fluorenyl methoxy carbonyl (Fmoc) group 

The Fmoc group can be removed under basic conditions. Usually 20% piperidine dissolved in DMF is used as reagent. One of the reagents for introducing the Fmoc group is the FmocCl.

Protection of carboxyl groups:
Carboxyl groups are most often protected by converting them to benzyl esters or t-butyl esters.

Protection of carboxyl groups by (A) benzyl esters and (B) t-butyl esters.

The benzyl esters are cleaved by saponification, HBr/AcOH, HF and catalytic hydrogenation but not by TFA. Their response to acids is similar to that of the Z groups but somewhat less sensitive.

The t-butyl esters, unlike benzyl esters, are stable to bases or nucleophilic attack. The properties of t-butyl esters are somewhat similar to those of the Boc groups although they are less sensitive to acidolysis. They can be cleaved by TFA.

Protection of other functional groups: The alcoholic and phenolic hydroxyl groups are protected by converting them to benzyl ether or t-butyl ether. The former protecting group can be cleaved by HF, HBr/AcOH or by catalytic hydrogenolysis and the latter one by TFA. Thiol groups can also be protected by benzyl ether formation or by tritylation.

The guanidino group (present in arginine) can be protected by nitration or by arylsulphonyl groups. The nitro group resists HBr/AcOH and can be cleaved by liquid HF. Among the arylsulphonyl groups the tosyl (Tos) group can be cleaved by liquid HF or sodium in liquid ammonia. Two other arylsulfonyl groups are more sensitive to acidic conditions. 

The 2, 2, 5, 7, 8-pentamethylchroman- 6-sulphonyl (Pmc) group can be cleaved by TFA under conditions similar to the removal of the Boc group. The 4-methoxy-2, 3, 6 tri-methyl benzenesulphonyl (Mtr) group is also cleaved by TFA but is less sensitive and requires a few hours for cleavage.

Protection by (A) Pmc group  (B) Mtr group (C) Nitro group 

The amide groups (in side chains of asparagine and glutamine) can be protected by tritylation. The trityl protecting group is stable to base, catalytic hydrogenolysis, very mild acid but is cleaved with TFA. It is used in conjunction with the Fmoc amino group protection strategy. 

The NH group of the imidazole ring (in the side chain of histidine) is protected in conjunction with the Fmoc strategy by tritylation. The trityl protecting group can be removed by TFA at room temperature.

The indole ring (in tryptophane) can be protected by Boc group that can be removed by TFA.

[Furka A. (2007)]5

(2) SOLUTION PHASE COMBINATORIAL CHEMISTRY
The solution phase synthesis involves conducting chemical reaction simultaneously, preferably in well-ordered sets (arrays) of reaction vessels in solution. [Pandeya S.N. and Thakkar D. (2004)]16  Most ordinary synthetic chemistry takes place in solution phase. The use of solution phase techniques has been explored as an alternative to solid-phase chemistry approaches for the preparation of arrays of compounds in the drug discovery process. Solution phase work is free from some of the constraints of solid-phase approaches but has disadvantages with respect to purification. 

In solution phase synthesis we use soluble polymer as support for the product. PEG is a common vehicle which is used in solution phase synthesis it can be liquid or solid at room temperature and show varying degrees of solubility in aqueous and organic solvent. By converting one OH group of PEG to methyl ether (MeO-PEG-OH) it is possible to attached a carboxylic acid to the free OH and use in solution phase combinatorial synthesis. Another common support which is used in solution phase synthesis is liquid Teflon consisting mainly of long chain of (-CF2 -) groups attached to a silicon atom. When these phases are used as a soluble support for synthesis the resulting product can be easily separated from any organic solvent.  [Mishra A.K. et al. (2010)]15

The main disadvantage of this method is when number of reagents are taken together in a solution, it can result in several side reactions and may lead to polymerization giving a tarry mass. Therefore, to avoid this, the new approach is developed in which all chemical structure combinations are prepared separately, in parallel on a giving building block using an automated robotic apparatus.

For example: Hundreds and thousands of vials are used to perform the reactions and laboratory robots are programmed to deliver specific reagents to each vial. [Pandeya S.N. and Thakkar D. (2004)]16

Table- 2. Characteristics of solid phase and solution phase combinatorial chemistry

SOLID PHASE

SOLution PHASE

Make a mixture of products

Makes only one product

Small amounts of products formed

Large amounts of products formed

Simple isolation of product by filtration

Work-up and purification more difficult

Requires two extra reaction steps: linkage & cleavage

No extra steps for attachment & cleavage needed

Limits to chemistry which can be performed

Wide range of reactions can be utilized

Automation possible

Automation difficult

Large excesses of reagent can be used to drive the reaction to completion

Large excesses of reagent cannot be used as it causes subsequent separation problem.

Longer reaction time than in solution phase

Less reaction time

Monitoring of reaction very difficult

Monitoring of reaction easy

Split and mix technique as well as parallel synthesis can be applied

Split and mix strategy not possible

Parallel synthesis can be applied

[Leard L. & Hendry A. (2007)]13

SYNTHETIC METHODS USED IN COMBINTORIAL CHEMISTRY

There are two methods, which used for synthesis of compounds in combinatorial chemistry. They include:
(1) Split and mix synthesis or Split and pool synthesis or Portioning – Mixing (PM) synthesis (one bead-one compound library)
(2) Parallel synthesis (one vessel-one compound library)

(1) SPLIT AND MIX SYNTHESIS ‘OR’ PORTIONING–MIXING SYNTHESIS
This technique was pioneered by Furka and co-workers in 1988 for the synthesis of large peptide libraries. This approach is termed divide couple and recombine synthesis by other workers.  [Jung G. (1999)]8

In this technique, the starting material is split in ‘n’ portions, reacted with ‘n’ building blocks, and recombined in one flask. For the second step, this procedure is repeated. This method is particularly employed for solid phase synthesis.  [Pandeya S.N. and Thakkar D. (2004)]16

In following figure spheres represents resin beads, A, B & C represent the sets of building block and borders represents the reaction vessels. In the case, when three building blocks are used, in each coupling step after three stages (ie. divide, couple & recombine), a total number of 27 different compounds, one on each resin bead, are formed using 9 individual reactions (ignoring deprotection).

Figure- 6. Schematic diagram of split and mix synthesis  [Jung G. (1999)]8

On the resulting products from split and pool synthesis, bioassays is performed and active mixture is discovered. Once an active mixture has been discovered, the next task is discovering which individual compound(s) in that mixture are active. The process of determining these active compounds is known as deconvolution. 
Advantages:
a) Only few reaction vessels required
(b) Large libraries can be quickly generated (up to 105 compounds) 
Disadvantages:
(a) Threefold amount of resin beads necessary
(b) The amount of synthesized product is very small.
(c) Complex mixtures are formed. 
(d) Deconvolution or tagging is required.
(e) Synergistic effects may be observed during screening, leading to false positives. [Leard L. & Hendry A. (2007)]13

(2) PARALLEL SYNTHESIS
In this method, each starting material is reacted with each building block separately (ie. In separate vessel). After each reaction step the product is split into ‘n’ portions before it is reacted with n new building blocks.  [Pandeya S.N. and Thakkar D. (2004)]16

In following figure spheres represents resin beads, A, B & C represent the sets of building block and borders represents the reaction vessels. In the case, when three building blocks are used, in each coupling step after three stages, a total number of 27 different compounds, one on each resin bead, are formed using 9 individual reactions (ignoring deprotection).

Figure-7. Schematic diagram of parallel synthesis  [Jung G. (1999)]8

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