Specifically, we will showcase an example of solid-phase peptide synthesis SPPS to demonstrate this concept. Organic Chemistry II. Solid Phase Synthesis. Principles Solid-phase synthesis is a method used to streamline the synthesis of molecules.
It is often used in combinatorial chemistry a technique used to prepare a large number of molecules in a short period of time , to generate libraries of compounds due to the ease of purification, and overall chemical synthesis.
Solid-phase synthesis typically involves the use of a resin; a non-soluble, polymer-based material, which is pre-functionalized so the starting building blockcan easily bind. The building blocks are generally protected once they are added onto the resin, and they can be easily deprotected and treated with the next desired building block in solution Figure 1.
Once the desired molecule has been synthesized, it can easily be cleaved from the resin. Because it is robust, solid-phase synthesis has been used to synthesize nucleic acids, oligosaccharides, and most commonly, peptides. Discovered and reported by Robert Bruce Merrifield in , SPPS has become the most widely used method to generate libraries of peptides. SPPS can easily take advantage of Fmoc base sensitive or Boc acid sensitive N-protecting groups on the amino acids to build up libraries of peptides in a short amount of time.
Fmoc protecting groups can be removed by 4-methylpiperidine, while Boc protecting groups can be removed by strong acids such as trifluoroacetic acid. In this experiment, we will demonstrate SPPS through the synthesis of a dipeptide. We will use the Kaiser test, a qualitative method to test for the presence of primary amines, to monitor the progress of the reaction.
Figure 1. Concept behind the solid phase peptide synthesis SPPS. Procedure 1. Drain the beads under vacuum and add 10 mL DMF. Add mg Fmoc-Ala-OH 1. Drain the solvent under vacuum and repeat the loading with Fmoc-Ala-OH for 15 min.
Drain the solvent under vacuum and repeat the deprotection. One test tube will be the control while the other will monitor the reaction. If the deprotection is incomplete or failed, the solution will remain yellow. Compare the reaction test tube with the control test tube. Coupling the Next Building Blocks Drain the solvent under vacuum. Wash the beads with 10 mL N-methylpyrrolidone under N2 and drain the solvent under vacuum.
Drain the solvent under vacuum. Perform the Kaiser test see steps 3. The beads and solution in the test tube should be yellow. Place a new receiving flask on the peptide synthesizer and drain theTFA solution containing the desired peptide under vacuum into the new flask. Decant the remaining TFA and ether solution from the conical vials and concentrate the peptide precipitate to afford the desired dipeptide as a white solid.
Solid phase synthesis is a method in which the product is synthesized while bound to an insoluble material. Solid phase synthesis is often used to produce biological oligomers and polymers such as peptides, nucleic acids, and oligosaccharides.
These molecules are composed of chains of smaller molecular subunits, called monomers. Synthesizing an oligomer or polymer takes many steps, as the monomers must be added in the correct order. An issue with multi-step syntheses is that purification and isolation of the stable products of each step, called intermediate products, decreases the overall yield. In solid phase synthesis, the intermediate product remains bound to the solid support throughout synthesis.
This allows solution-phase reagents, solvents, and byproducts to be washed away, eliminating the need to purify and isolate each intermediate product between steps. This video will illustrate the procedure for solid phase peptide synthesis and introduce a few applications of solid phase synthesis in chemistry. In solid-phase synthesis, a molecule is synthesized on a solid support in a sequence of reactions. For instance, an oligomer or polymer will be synthesized one monomer at a time to form the final product.
The growing oligomer or polymer remains strongly bound to the solid support until it is separated, or cleaved, from the support with reagents. Each monomer must have at least two binding sites to be part of the polymer chain, but only one binding site can be available at a time to ensure that the monomer binds to the correct atom. This is achieved with protecting groups, which are functional groups that are not reactive during one or more steps of the synthesis.
The binding site is restored, or deprotected, by treating the molecule with specific reagents to convert the protecting group to a reactive functional group. To begin solid-phase synthesis, the starting material is bound to a specially designed resin or insoluble polymer at its only available binding site. Then, the bound starting material is deprotected to allow binding of the second monomer in the chain. Next, a solution of the second monomer in the chain is added, along with a coupling agent to facilitate bonding between the monomers.
Once the second monomer binds to the starting material, the resulting dimeric intermediate product is deprotected. This process is repeated until the target oligomer or polymer has formed. This synthesis was mediated through the use of microfluidic pumping systems that drive the material through various packed columns containing immobilized reagents, catalysts, scavengers or catch and release agents.
Key steps include trapping previously prepared 4- 2-azidoethyl phenol with a polymer-supported phosphine on a column and subsequently coupling it with 3,4-dimethoxybenzaldehyde , which is prepared in a separate channel. The resulting imine intermediate is sequentially converted to an amide in steps that include changing solvents and a palladium-mediated hydrogenation that takes place in a flow-through unit. In the final stages, the third ring of oxomaritidine is formed and the amide is cleaved, promoting a bridging 1,4-addition to give the end product Scheme Krchnak and co-workers27 described the synthesis of new pyrrole and quinoxalinone analogues.
The strategy involved the cycloaddition of piperine, a natural conjugated diene, with polymer supported acyl- and arylnitroso dienophiles Scheme The arylnitroso dienophiles were prepared immobilized by using ethanolamine to a silyloxy linker.
Subjected HDA adducts to reducing conditions. Calver et al. Suzuki cross-coupling with aryl iodides followed by cleavage with acid completed the solid-phase synthesis of 2,5-disubstituted indoles. Recently, Larock et al. The chemistry has been successfully performed in a solid support using chlorinated Wang resin and diversity has been achieved at the 5-position by different cleavage reactions.
Hyacinthacine is a new set of alkaloids isolated from Hyacinthoides non-scripta and Scilla campanulata immature fruits and bluebell stalks which inhibit several glycosidase enzymes. A new solid-phase synthesis of pyrrolizidine nucleus related to hyacinthacine core using L-Boc-proline as the building block was reported Scheme The route includes an elegant cyclization cleavage strategy step, which releases product from the resin in good yields under Baylis-Hillman conditions.
Terpenes and Steroids Terpenoids, also referred to as terpenes, are the largest group of natural compounds over 55, members isolated so far. From simple structures as menthol until complex ones as taxol, terpenes are a ubiquitous group of molecules that have long provided humans with flavours, fragrances, hormones, medicines and even commercial products such as rubber.
Chemically, they are built from five-carbon isoprene units and so can also be called "isoprenoids". Molecules with multiples of 5 carbons and particularly with single carbon side chains belong to this group, even if they are cyclic. Based on the number of the building blocks, terpenoids are commonly classified as monoterpenes C10 , sesquiterpenes C15 , diterpenes C20 , and sesterterpenes C More complex terpenoids include the sterols.
Many terpenes have biological activities and are used for the treatment of human diseases, been active against cancer, malaria, inflammation, and a variety of infectious diseases. The current literature revealed a few examples. To input molecular diversity in hydroxysteroids primary alcohol, secondary alcohols, and phenol , Maltais and co-workers33 utilized solid-phase reactions where hydroxysteroids were linked to solid support using the butyldiethylsilane polystyrene PS-DES resin.
The synthesized compounds were made by running steroidal alcohols and through a model sequence of reactions: solid-phase coupling, aminolysis of oxirane or reduction of azide, amidation, and final cleavage, as seen in Schemes 23 and In ,35 the same group enlarged the library of estradiol 16p- azidopropyl compounds 34 with the synthesis of phenolic sterols derivatives.
The authors used the same approach in which they generated two levels of diversity from two functional groups, where they introduced commercially available building blocks, such as amino acids, acyl chlorides, and anhydrides Scheme Taxol paclitaxel is an antimicrotubule agent clinically available to treat ovarian, breast, lung cancer and also Kaposi sarcoma.
Secosteroids, as vitamin D, are steroids in which one of the bonds in the rings is broken. In , Hojikuro and Takahashi38 proposed a route where synthetic analogs of vitamin D were prepared by using a solid-supported CD-ring Scheme The vitamin D3 system was synthesized by Horner-Wadsworth-Emmons HWE reaction of the A-ring phosphine oxide to resin , followed by introduction of the side chain and cleavage from resin.
Squalamine Figure 1 is a water-soluble antibiotic, which exhibits potent bactericidal activity against both Gram-negative and Gram-positive bacteria, is fungicidal and induces osmotic lysis of protozoa. This approach was based on the 1,6-elimination process that uses an in-built amine 'activator' N from NHBoc in to cleave a phenoxy ester a and b and hence to activate the linker without giving rise to free quinone methide by-products , as seen in Scheme The utilization of this linker could be useful to accelerate the screening processes of libraries prepared by SPOS.
Jagtap42 and co-workers used as alternative solid-support a polystyrene-divinyl benzene resin functionalized with a PE-DES-Cl to obtain a series of acyl and 7,diacyl paclitaxel analogues Scheme The starting material was deacetylpaclitaxel that was bonded into resin to give and then converted to the correspondent acyl analogues by Holton's method.
Subsequent acylation at the C-7 position was performed using different carboxylic acid and the acyl and diacyl libraries were delivered from the resin under acidic condition. The sesquiterpenoid dysidiolide Figure 2 is an inhibitor of the dual-specificity Cdc25 protein phosphatase family that acts in the regulation of the cell cycle, isolated from the Caribbean sponge Dysidia etheria, Laubenfels.
Lupeol is a triterpene found in fruits and vegetable and recently it was discovered that it is capable to inhibit tumors growth,45 to act as anti-inflammatory,46 urolithic and anticalciuric and also express antimalarial activity against chloroquine-resistant Plasmodium falciparum.
These sites also served as point to introduce chemical diversity. The intermediates were used to prepared several derivatives The triterpenoids betulinic acid and ursolic acid were investigated as scaffolds for the generation of combinatorial libraries in parallel format using solid-phase organic synthesis. The synthesis consisted in inmobilization of or to pre-derivatized amino acid 2-chlorotrityl or Sieber amide resins, and then treated with a variety of aliphatic, aromatic and amino acids Scheme After cleavage of the trifluoroacetyl protecting group, sequential acylation reactions with five Fmoc-protected amino acids and five carboxylic acids were performed to introduce two levels of molecular diversity.
Analogues of geraniol, a monoterpene found in essential oils of fruits and herbs, have been studied by Subramanian and co-workers. The SPOS started with the attachment of synthetic intermediates to the tetrahydropyranyl THP resin prepared from the Merrifield resin , combining a five-fold excess of 8-oxo-geraniaol with the polymeric support in the presence of 0.
Anilinogeraniols attached to the resin were obtained by reductive amination of with 10 equiv. To obtain the diphosphates , compound reacted with 3 equiv. Another steroid synthesized by SPOS was a new type of cyclic peptidosteroid , in which a bile-acid-based scaffold was used for the conformational restriction of a loop-like peptide.
Peptide backbone cyclization was then carried out, giving rise to a ring size equivalent to approximately 12 amino acids Scheme Solanesol is a terpene isolated from tobacco and it is an important intermediate of nutrients such as vitamin K2 and coenzyme Q Besides the efforts to obtain it by solution-phase synthesis, which require expensive chromatographic purification, Yu et al. In the proposed route, a dihydropyran-modified resin is coupled to Merrifield resin and after some steps a polymer-supported bromide is prepared Scheme This bromide reacted to give polymer-support nonamer Finally, treatment with PPTS was performed and completes the total solid-phase synthesis of solanesol The triterpene maslinic acid is an oleanane isolated from olive oil which has been identified as antiproliferative agent of colon cancer cells54 and anti-HIV.
Due to the high hydrophobicity of maslinic acis, it is very suitable to be utilized in solid-phase. The strategy consisted on the coupling of one, two, or three amino acid fragments with the carboxyl group at C of maslinic acid.
The pathway employed for the synthesis of one dipeptide derivative is described in Scheme Recently, Nicolletti et al. The CD-side chain fragment was anchored to the solid support through an ester group at C25 and coupled to the A ring of building block to assemble the vitamin D triene system by Wittig-Horner reaction. Prior to functionalization at C25 of , the hydroxy group was deprotected and the cleavage was done by nucleophilic attack on the ester carbonyl group.
Coumarins Coumarins are structurally related to lactones from o-cinamic acid, where the 1,2-benzopyrone is the simplest representative. The most investigated in medicinal chemistry is the use of furanecoumarins, which possesses some biological properties like vasodilator and bronchodilator.
Because of this, the synthesis of coumarins by combinatorial chemistry has gained some interest including the synthesis of coumarins by SPOS or its use as scaffold to prepare combinatorial libraries of coumarins derivatives. The design of a reliable synthetic route for the solid-phase method for parallel synthesis of 2,3-disubstituted 6H-pyrano[2,3-f]benzimidazoleones 2,3-disubstituted imidazocoumarins has been achieved.
The synthesis started with coupling of Rink amide resin MBHA with 7-fluoromethylnitro-coumarincarbocylic acid , after which furnished 26 tricyclic compounds in six steps with high purity and good yields. The synthesis employs available building blocks and the reactions were carried out under ambient conditions. In order to develop a synthetic approach to gain both efficiency and diversity around the isocoumarin scaffold, it was recently established a parallel solid-phase synthesis method for the production of 3-substituted 4-haloisocoumarins based on an electrophile-promoted halocyclization of supported 2- alkynyl benzoates Scheme This synthesis allowed large variations at positions 3 and 4 of the isocoumarin scaffold and on the aromatic moiety.
In addition, the authors could recycle the resin and apply one of well-known advantage of the solid support, which is the temporary protection of one functional group i. In fact, the traceless cleavage would ensure that only the desired cyclized product is released from the polymer, with no cleavage of side products arising from an incomplete Sonogashira reaction.
Flavonoids The benzopyranone-ring system found in flavonoids is a molecular scaffold of considerable interest. Medicinal chemistry efforts have focused on diversifying benzopyranones and utilizing combinatorial chemistry approaches to construct small libraries in order to be evaluated, for example as potential enzyme inhibitors.
Previous reviews have discussed the importance of combinatorial libraries of flavonoids prepared by solid-phase synthesis.
The key steps were the addition of Grignard reagents to salicyl aldehyde derivatives and the cyclization employing amide acetals which introduced the R2 substituents on the isoflavones Scheme Bhat et al. The synthetic strategy involved the one pot conversion of bis-silylated salicylic acids into alkynyl ketones via an acid chlorination and subsequent Sonogashira coupling with terminal alkynes.
Nicolaou et al. In one natural product based library, a chalcone family of compounds was synthesized in parallel approach by condensing a variety of 11 aldehydes to a set of three resin-bound benzopyrans containing a methyl ketone substituent Scheme On the other hand, treatment of supported chalcones with iodine, followed by oxidative elimination, furnished flavones Cheng et al.
The immobilized hydroxyacetophenones were submitted to a Claisen-Schmidt condensation with a series of arylaldehydes resulting in the chalcones which were then cleaved from the resin yielding chalcones Scheme Huang et al.
After the oxidative cleavage of selenium resin, the flavones were obtained in good overall yields and high purity Scheme A different strategy was employed by Yao et al. The functionalized flavone was prepared in solution phase by classical Claisen condensation, mixed acid nitration and oxidative cyclization reaction with iodine-DMSO.
Then, this flavone was employed as scaffold for the synthesis of a combinatorial library in solid phase Scheme The linker was constructed by coupling of Fmoc-glycine onto Rink resin, followed by Fmoc deprotection. After that, the flavone was readily attached to the resin using 1,3-diisopropylcarbodiimide DIC.
The aryl nitro group was then reduced with tin II chloride to aryl amine , which was employed for the construction of different heterocyclic rings.
For example, treatment with 1,1'-carbonyldiimidazole CDI , followed by cleavage from the resin with triflouracetic acid -TFA furnished a benzimidazolone flavonoid Arai et al. They have also examined the one-pot reactions in the solid phase by attaching an acetophenone derivative to 4-methoxyphenyl diisopropylsilylpropyl polystyrene beads leading to resin The use of substituted benzaldehydes and subsequent addition of heterocyclic aldehydes gave 3-pyridyl-substituted flavones To introduce an additional point of diversity into the molecules, Suzuki-Miyaura coupling was performed, followed by cleavage from the resin, furnishing flavone in good overall yields and high purity Scheme Chalcones are known to possess some pharmacological activities, among them antitumor.
The synthesis of new pirrolobenzodiazepine-chalcones conjugates by SPOS has been achieved. The starting material is prepared in solution and coupled with the resin intermediate in the presence of K2CO3.
Polyamines Polyamines are organic polycations present in all cells and biologic liquids of live organisms that play an important role in control the general mechanism of the cellular proliferation. Putrescine is involved in many important biological processes such as embryogenesis and proliferation of normal or cancer cells while spermine, a putrescine metabolite, is known to inhibit cellular proliferation.
In function of its considerable relation with the mechanisms involved in the proliferative process, the enzymes of the polyamines metabolism are potential targets for the design of new anticancer drugs and the knowledge of the mechanism implicated in the transport of polyamines can allow the development of polyamines analogues.
All of these considerations have led medicinal chemists to employ the polyamine core as building block to design several small libraries of compounds combining heterocycle natural products scaffolds and polyamines. An interesting divergent approach to built polyamines and polyamines derivatives was published by Manov and Bienz70 employing the Merrifield resin and protected amines.
They synthesized a library of 28 polyamines in which Scheme 50 depicted part of the synthesis. The Merrifield resin was modified with primary amines and rapidly dialkylated. After selective deprotection of either of the two terminal amines, the specific prolongation or derivatization of the resin-bound triamines was performed. The resulting polyamines were easily released from the polymer by treatment with 1-chloroethyl chloroformate ACE-Cl and subsequent methanolysis.
The authors took advantage of the antibacterial activity of usnic acid amino derivatives and they functionalized this dibenzofuran with polyamines in order to improve the antibacterial activity and to overcome their poor hydrosolubility.
Two sets of usnic acid derivatives polyaminated were synthesized by a split-and-mix method using SynPhase supports. Scheme 51 depicts one set of usnic acid triamines derivatives. The supports were pooled for the mesylation then divided into four pools for the nucleophilic displacement by various diamines, and led to supported triamines The supports were mixed in two batches for the condensation with usnic acid and then the products af were delivery from the supports in acid conditions.
A method for the synthesis of cyclic polyamines based on solid-phase chemistry has been reported. The linear polyamines and have been synthesized stepwise on solid support in a convergent manner using Merrifield resin as solid support. Cyclisations on the resins were effected conventionally by direct intramolecular SN2 reactions between sulphonyl-protected terminal amino groups and primary alkyl bromides under basic conditions.
The release of products from the resin was carried out by treatment with ACE-Cl followed by methanolysis. Others Lactones are important classes of organic compounds due to their high occurrence in nature, and also because they are versatile building blocks in the synthesis of a wide range of natural and synthetic products. Macrolides are a class of antibiotic compounds that contain a large lactone macrolide ring of 12 to 16 atoms and several sugars linked to the ring.
Sowin and co-worker74 published the first example of a library that employs the macrolide core as a template for combinatorial synthesis Scheme The synthesis was carried out using the aldehyde as starting material, this core is accessible in large quantities from 6-O-allyl-erythromycin A. For the library synthesis they used aminonomethylpolystyrene resin containing a fluorine tag and an extended modified Wang type linker. The library introduces three sites of diversity to macrolide scaffold via reductive aminations.
In this work, the authors prepared a set of 5,6-dihydropyrones grafting the Brassard diene analogue on a modified Merrifield resin and reacted it with aldehydes or ketones. Benzoannelated butyrolactones phthalides are present in various natural products. Knepper and coworkes76 employed the Sakurai reaction for the solid-phase synthesis of phthalides Scheme For example, 2-formyl benzoic acids were immobilized on Merrifield resin using standard conditions.
Treatment of resin with allyltrimethyl silanes in the presence of five equivalents of titanium tetrachloride furnished the phthalides in good yields and excellent purity.Baxendale, I. Place a few loaded beads in a test tube and heat both tubes to degrees in an oil bath. This video will illustrate the procedure for solid phase peptide synthesis and introduce a few applications of solid phase synthesis in chemistry. Distamycin Scheme 58 is a natural compound present in Streptomyces species which have an ability of recognize AT-rich sequences through noncovalently binding in the minor groove of B-form DNA.
Then place 0.
They synthesized a library of 28 polyamines in which Scheme 50 depicted part of the synthesis. The amine is initially protected. The arylnitroso dienophiles were prepared immobilized by using ethanolamine to a silyloxy linker. Nowadays SPOS grows up with the development of new resins, linkers, synthesizers, methods to monitoring reactions and resins for scaling up. A method for the synthesis of cyclic polyamines based on solid-phase chemistry has been reported.
The amine is initially protected.
To generate the final product, remove the solvent with a rotary evaporator. Marfil, M.
Principles Solid-phase synthesis is a method used to streamline the synthesis of molecules. Krchnak, V. A member combinatorial library based on the structure of aeruginosin A was synthesized by Doi et al. Adriaenssens, L. Solid phase synthesis is a method in which the product is synthesized while bound to an insoluble material. Commercially available Wang resin loaded with Fmoc-L-Trp 1 was deprotected and coupled with anthranilic acid in the presence of 1- 3-dimethylaminopropyl ethylcarbodiimide EDC as activating agent, followed by coupling with Fmoc-Gly in pyridine.
Alkaloids Alkaloids are one of the most important classes of natural products due to their ability to modulate many pharmacological events. Decant the remaining TFA and ether solution from the conical vials and concentrate the peptide precipitate to afford the desired dipeptide as a white solid. The amine is initially protected.