CHEMICAL BIOLOGY

Solid-Phase Synthesis of Biomolecules

 

Rolf Breinbauer, University of Leipzig, Germany

doi: 10.1002/9780470048672.wecb552

 

The synthesis of biopolymers, tool compounds, and molecular probes lies at the heart of any effort in chemical biology. Solution-phase strategies are well established but usually require considerable efforts in time and labor during workup and purification steps. In contrast, solid-phase synthesis, in which the substrate is covalently bound to an insoluble support, allows the simple removal of excess or consumed reagents by simple filtration. In the cases of oligonucleotides and peptides, the synthetic operations can be performed by robotic systems allowing the high-throughput synthesis of these biopolymers at low cost within a short time. The solid-phase synthesis of oligosaccharides and small-molecule probes has made significant progress during the last several years.

 

This article gives an overview of the main technical aspects of solid-phase synthesis and reviews the application of this technique for the synthesis of oligopeptides and proteins, oligonucleotides, oligosaccharides, and small molecules.

Biologic Background

From the mid-nineteenth century onward, researchers have made tremendous strides in understanding the molecular nature of living organisms. The discovery that cellular catalysts (“enzymes”) are composed of oligomers of amino acids; that genetic information is encoded in oligonucleotides; the solving of the puzzle of metabolic pathways; and the investigation of hormonal and cellular signaling have shaped the modern view of what has become molecular and cellular biology. These discoveries have been accompanied by progress in both analytical techniques as well as in the synthesis of these molecules. Even today, the investigation of cellular organisms offers new surprises, such as the catalytic nature of RNA (“ribozymes”), the pathogenic nature of proteins (“prions”), or the silencing of genes via RNA interference. The synthesis of biopolymers and biologically active molecules remains an important task in chemical biology, as it 1) gives access to natural compounds, which otherwise could be isolated only in small and insufficient amounts; 2) allows the synthesis of modified or non-natural analogs or intermediates of natural compounds; and 3) provides tool compounds for the investigation of biologic mechanisms.

Technical Background

Although Emil Fischer had already pioneered the synthesis of sugars, nucleobases, and oligopeptides at the beginning of the twentieth century, the synthesis of these biomolecules in solution involved multistep sequences with many tedious workup and purification operations. Bruce Merrifield recognized that their syntheses can be simplified if a substrate is immobilized on an insoluble polymeric support (“resin”) via a linker unit (1). Once immobilized the substrate can be easily modified by adding an excess of reagents and building blocks to ensure complete conversion. Consumed and excess reagents are then easily removed by washing the support and by simple filtration. Finally, cleavage of the linker releases the desired product into the solution allowing its isolation in pure form (Fig. 1).

 

Figure 1. General flow scheme of solid-phase synthesis. The solid support is represented by the gray ball. In this cartoon, only one functional group is depicted instead of billions on a real bead.

 

Solid Support

A wide range of support materials exists for the heterogeneous immobilization of substrates in solid-phase synthesis, each offering certain advantages in different applications (2, 3). Parameters to be considered are 1) loading (= mmol of functional groups per gram support), 2) bead size (measured in “mesh”), 3) swellability in solvents (degree of cross-linking and hydrophilicity), and 4) mechanical stability (mechanical abrasion interferes with filtration workup). As more than 99.99% of the substrate molecules are buried within the gel matrix of a polymeric bead, the resin must be swollen by solvent molecules to allow access to reagents. Polystyrene (PS) resins (cross-linked with 1-2% divinylbenzene (DVB), loading up to 2 mmol functional group/g) are the resins most commonly used for oligopeptide and small-molecule solid-phase synthesis. These resins swell in most organic solvents but not in very polar solvents such as alcohols or water. If reactions need to be performed under polar conditions, the use of Tentagel (PS-resin with grafted polyethyleneglycol chains), PEGA, Pepsyn, or Argogel is recommended because these substances are distinguished by polar, flexible, polymeric chains. Highly cross-linked macroporous resins (e.g., Argopore) exhibit a solvent-independent permanent pore structure and can be used in any solvent. For oligonucleotide synthesis, controlled pore glass (CPG), which is an inorganic support with well-defined, large pores, is widely used. The low loading and high cost of this material are compensated by the operational advantage of good accessibility in any solvent. Recently, the synthesis of biopolymers on planar surfaces, such as glass (DNA-chips) (4) or cellulose membranes (SPOT-synthesis), (5) has gained increasing attention.

Linker

A linker can be considered as a polymeric (or, more general, immobilized) protecting group. It is a functional group responsible for the covalent attachment of the substrate onto the support that will be cleaved from the product at the end of the synthesis sequence. The following features have to be considered when choosing an appropriate linker: 1) The attachment of substrate should proceed in high yield, 2) the linker should be stable under all reaction conditions considered for the synthetic sequence (“orthogonality”), and 3) the linker should be cleaved under mild conditions in quantitative yield without obscuring the integrity and purity of the product. More than 200 different linkers have been described exhibiting different degrees of orthogonality and releasing different functionalities at the product after cleavage (6, 7). Cleavage of the linker can be initiated by the addition of acid (commonly used in oligopeptide synthesis), base (used in DNA-synthesis), metals, oxidation, reduction, or light (photocleavable linker). Linkers that release their product without any visible functional group are named “traceless linkers.” “Safety-catch linkers” require two different chemical reactions in order to undergo cleavage, which offers a unique degree of stability. In Fig. 2 some frequently used linkers and the corresponding cleavage conditions are depicted.

 

Figure 2. Representative linkers frequently used in solid-phase synthesis. The molecular moiety depicted in blue represents the molecule that is released upon cleavage of the linker. DCM = dichloromethane, TFA = trifluoroacetic acid.

 

Automation

As solid-phase synthesis basically involves only three types of operations (addition of excess of reagent; shaking the beads in the reagent cocktail; filtration and washing) machines have been designed to perform solid-phase synthesis automatically (8). Automated oligonucleotide and oligopeptide synthesis has become routine.

Synthesis of Biopolymers

The solid-phase synthesis of biopolymers via iterative coupling of monomeric building blocks has found widespread application. It can be adapted easily for the incorporation of non-natural building blocks or labeled monomers, which will be used as probe molecules in chemical biology.

 

Synthesis of Oligopeptides and Proteins

Oligopeptides are synthesized on solid phase from the C- to the N-terminus. Depending on the N-terminal protecting group of the amino acid building blocks, two different strategies can be followed. In the “Boc-strategy,” the tert.-butyloxycarbonyl-protecting group is cleaved by addition under acidic conditions (trifluoroacetic acid), which requires a linker that is resistant to these conditions because it is either cleaved only by very strong acids (HF, trifluoromethanesulfonic acid) or by a different agent (Fig. 3) (1). The “Fmoc-strategy” is based on the fluorenylmethoxycarbonyl-protecting group, which is cleaved by addition of 20% piperidine/DMF. It has the advantage that the important class of acid labile linkers (e.g., Wang-linker or Rink-linker) is compatible with this approach.

 

 

Figure 3. Flow scheme of a solid-phase synthesis of an oligopeptide following the Boc-strategy. The first amino acid is attached to the Merrifield linker by alkylative esterification of its cesium-salt. After acidic deprotection of the Boc-group and neutralization, the N-terminus undergoes DCC-mediated coupling with another Boc-protected amino acid building block. This sequence can be iterated, leading to even longer oligopeptides. Finally, the protect is released upon cleavage with HF. DCC = dicyclohexylcarbodiimide.

 

In addition, measurement of the UV-absorption of the cleaved protecting group allows the determination of the yield of the previous coupling operation. In practice, oligopeptides with up to 50 amino acids can be synthesized on solid phase. Instrumental for achieving reasonable yields for long oligomers is an almost perfect conversion for each deprotection and coupling step, for which a plethora of modern peptide coupling reagents are available; the coupling reagents also minimize epimerization (9). The synthesis of larger proteins (>60 aa) may be accomplished by preparation of fragments on solid phase that are then assembled using solution-phase ligation reactions (10), such as the native chemical ligation using an N-terminal Cys-residue (11). Table 1 lists representative examples of de novo synthesized proteins using solid-phase peptide synthesis. Recently, the combination of expressed protein ligation and solid-phase peptide synthesis has emerged as a powerful tool for the preparation of proteins with unnatural modifications or labels (12). Solid-phase peptide synthesis has reached a level where the ton-scale synthesis of peptide drugs, such as the HIV drug Enfuvirtide (a 36mer peptide), has been established.

 

Table 1. A selection of notable examples for the total solid-phase synthesis of proteins

 

Author

Year

Protein

Size

Merrifield et al.

1969

Ribonuclease A

124 aa

Sigler et al.

1976

Apolipoprotein CI

57 aa

Fairwell et al.

1983

Human parathyroid hormone

84 aa

Blake

1986

S -carbamoylmethyl bovine apocytochrome c

104 aa

Wlodawer et al.

1989

HIV-Protease

202 aa

Briand et al.

1989

Ubiquitin

76 aa

Di Bello et al.

1992

Horse heart cytochrome C

104 aa

Lu et al.

1998

Bovine pancreatic trypsin inhibitor

58 aa

Kaiser et al.

1999

Macrophage migration inhibitory factor

115 aa

Canne et al.

1999

Human group V secretory phospholipase A2

118 aa

Hackeng et al.

2001

Human matrix Gla protein

84 aa

Miranda et al.

2001

Human S100A12

91 aa

Low et al.

2001

cytochrome b562

106 aa

Becker et al.

2003

Ras + Raf

166 + 81 aa

Kochendoerfer et al.

2003

Erythropoietin

166 aa

Kochendoerfer et al.

2004

Vpu

81 aa

Cabrele et al.

2006

Id2

119 aa

aa = amino acid.

 

Synthesis of Oligonucleotides

The solid-phase synthesis of DNA oligomers has reached a high level of maturity and is extensively used for the synthesis of primers for polymerase chain reaction or the design of new genes (13). In contrast to the cellular process, the synthetic route forms the polymer in 3’-to-5’ direction. The oligomerization is carried out on CPG using a base labile linker. Typically, the nucleobases are introduced as acyl-protected phosphoramidites, which will be oxidized to phosphates after each elongation step (14). Treatment with base leads to global deprotection (nucleobase protecting groups and the cyanoethyl protecting group at phosphate) and cleavage of the oligonucleotide from the solid support. The conversion of each coupling step can be monitored by UV measurement of the dimethoxytrityl-carbocation, which is generated by the deprotection of the 5'-OH before each new coupling cycle (Fig. 4). The introduction of non-natural or modified nucleobases can be easily accomplished by using a corresponding phosphoramidite building block during the synthetic sequence.

 

 

Figure 4. Flow scheme of the synthesis of DNA-oligonculeotides using the phosphoramidite method. DMT = dimethoxytrityl, TCA = trichloroacetic acid.

 

The synthesis of RNA-oligomers (e.g., needed for applications in RNAi, aptamers, or as ribozymes) follows the steps described above for DNA but requires the protection of the 2'-OH group, for which the 2'O-TOM- and the 2'O-ACE protecting groups are most often used (Fig. 5) (15).

PNA-oligomers are synthesized using methodology developed for peptide synthesis. The large-scale synthesis of oligonucleotides has been established for the preparation of sufficient quantities for clinical trials of antisense drugs.

 

 

Figure 5. Most frequently used 2'-OH protecting groups in RNA-oligomer synthesis. DMF = N,N-dimethylformamide, PG = protecting group.

Synthesis of Oligosaccharides

The automated solid-phase synthesis of oligosaccharides is expected to have a tremendous impact in glycobiology as many natural proteins are decorated by complex oligosaccharide conjugates that strongly influence the biologic activity of the conjugate. Recently, the first examples of automatically synthesized oligosaccharides have been reported (16). The availability of appropriately protected building blocks and the creation of certain linkage patterns remain as challenges in this field.

Solid-Phase Synthesis of Small Molecules

Protein function can be modulated by small-molecule ligands. Offering complementary advantages to mutation genetics, the search for small molecules as molecular probes for the investigation of biologic systems has gained new interest (“chemical genetics”). In addition, high-throughput screening efforts in hit finding and lead optimization in drug discovery require large collections of small molecules (“molecular libraries”). Solid-phase synthesis has strongly contributed to these efforts as it gives fast access to large compound libraries.

Combinatorial Synthesis

In the early 1990s combinatorial chemistry was developed as a tool for the production of large oligopeptide libraries (17-19). Following a split-mix strategy depicted in Fig. 6, libraries with millions of different compounds can be created. This approach was extended by Ellman and colleagues for the synthesis of small-molecule libraries (20). Diversity-oriented synthesis (DOS) has been invented as a powerful tool for the synthesis of very large structurally diverse compound libraries (21). For the identification of the active compound, several strategies, such as on-bead-screening, deconvolution, or encoding, have been established. The use of “IRORI-kans” (penetrable little containers filled with beads and labeled either with a radio-frequency tag or an optical bar code on the container lid) allows the synthesis of mg-quantities of compounds in a split-mix -format, which offers the additional advantage of having the synthetic history of each kan monitored by a computer-based readout system of the radio-frequency tag or the optical bar code) (22).

 

 

Figure 6. Combinatorial synthesis using a split-mix format. Building blocks are added separately in different vials, but the beads with reaction products are pooled before being distributed again before the next reaction step. Each vial contains all combined products of the previous reaction steps, but each bead contains only a single compound.

Natural Product Libraries

Several of the most useful small-molecule probes are complex natural products isolated from marine, fungal, or plant sources. As natural products have built-in biologic properties efforts in the synthesis of compound libraries increasingly use natural product scaffolds as a starting material because they promise higher hit rates in biologic screens (23).

In Fig. 7 a few representative examples of molecular probes identified by screening of combinatorial libraries are depicted.

 

Figure 7. Selected examples of tool compounds identified by screening of combinatorial small-molecule libraries.

 

References

1. Merrifield RB. Solid-phase peptide synthesis. 3. An improved synthesis of bradykinin. Biochemistry 1964; 3:1385-1390.

2. Hudson D. Matrix assisted synthetic transformations: a mosaic of diverse contributions. I. The pattern emerges. J. Comb. Chem. 1999; 1:333-360.

3. Hudson D. Matrix assisted synthetic transformations: a mosaic of diverse contributions. II. The pattern is completed. J. Comb. Chem. 1999; 1:403-457.

4. Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed, spatially addressable parallel chemical synthesis. Science 1991; 251:767-773.

5. Frank R. The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports—principles and applications. J. Immunol. Methods. 2002; 267:13-26.

6. Guillier F, Orain D, Bradley M. Linkers and cleavage strategies in solid-phase organic synthesis and combinatorial chemistry. Chem. Rev. 2000; 100:2091-2157.

7. James IW. Linkers for solid phase organic synthesis. Tetrahedron. 1999; 55:4855-4946.

8. Merrifield RB, Stewart JM. Automated peptide synthesis. Nature 1965; 31;207:522-523.

9. Chan WC, White PD, eds. Fmoc Solid Phase Peptide Synthesis - A Practical Approach. 2000. Oxford University Press, Oxford.

10. Gutte B, Merrifield RB. The total synthesis of an enzyme with ribonuclease A activity. J. Am. Chem. Soc. 1969; 91:501-502.

11. Dawson PE, Muir TW, Clark-Lewis I, Kent SB. Synthesis of proteins by native chemical ligation. Science. 1994; 266:776-779.

12. Pellois JP, Muir TW. Semisynthetic proteins in mechanistic studies: using chemistry to go where nature can’t. Curr. Opin. Chem. Biol. 2006; 10:487-491.

13. Letsinger RL, Mahadevan V. Oligonucleotide synthesis on a polymer support. J. Am. Chem. Soc. 1965; 87:3526-3527.

14. Caruthers MH, Barone AD, Beaucage SL, Dodds DR, Fisher EF, McBride LJ, et al. Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. Methods Enzymol. 1987; 154:287-313.

15. Micura R. Small interfering RNAs and their chemical synthesis. Angew. Chem. Int. Ed. Engl. 2002; 41:2265-2269.

16. Plante OJ, Palmacci ER, Seeberger PH. Automated solid-phase synthesis of oligosaccharides. Science 2001; 291:1523-1527.

17. Furka A, Sebestyen F, Asgedom M, Dibo G. General method for rapid synthesis of multicomponent peptide mixtures. Int. J. Pept. Protein. Res. 1991; 37:487-493.

18. Lam KS, Salmon SE, Hersh EM, Hruby VJ, Kazmierski WM, Knapp RJ. A new type of synthetic peptide library for identifying ligand-binding activity. Nature. 1991; 354:82-84.

19. Houghten RA, Pinilla C, Blondelle SE, Appel JR, Dooley CT, Cuervo JH. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature. 1991; 354:84-86.

20. Bunin BA, Plunkett MJ, Ellman JA. The combinatorial synthesis and chemical and biological evaluation of a 1,4-benzodiazepine library. Proc. Natl. Acad. Sci. U.S.A. 1994; 91:4708-4712.

21. Schreiber SL. Target-oriented and diversity-oriented organic synthesis in drug discovery. Science 2000; 287:1964-1969.

22. Nicolaou KC, Pfefferkorn JA, Mitchell HJ, Roecker AJ, Barluenga S, Cao, GQ, et al. Natural product-like combinatorial libraries based on privileged structures. 2. Construction of a 10 000-membered benzopyran library by directed split-and-pool chemistry using NanoKans and optical encoding. J Am Chem Soc. 2000; 122:9954-9967.

23. Breinbauer R, Vetter IR, Waldmann H. From protein domains to drug candidates-natural products as guiding principles in the design and synthesis of compound libraries. Angew Chem Int Ed Engl. 2002; 41:2879-2890.

Further Reading

Balkenhohl F, Von dem Bussche-Hunnefeld C, Lansky A, Zechel C. Combinatorial synthesis of small organic molecules. Angew. Chem. Int. Ed. Eng. 1996; 35:2288-2337.

Caruthers MH, Beaton G, Wu JV, Wiesler W. Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs. Methods Enzymol. 1992; 211:3-20.

Dawson PE, Kent SB. Synthesis of native proteins by chemical ligation. Annu Rev Biochem. 2000; 69:923-960.

Dorwald FZ. Organic Synthesis on Solid Phase. 2nd ed.. 2002. Wiley-VCH, Weinheim.

Kent S. Total chemical synthesis of enzymes. J. Pept. Sci. 2003; 9:574- 593.

Knepper K, Gil C, Brase S. Natural product-like and other biologically active heterocyclic libraries using solid-phase techniques in the post-genomic era. Comb. Chem. High Throughput Screen. 2003; 6:673-691.

Merrifield RB. Solid-phase syntheses (Nobel lecture). Angew. Chem. 1985; 97:801-812.

Nicolaou KC, Hanko R, Hartwig W, eds. Handbook of Combinatorial Chemistry. 2 vols. 2002. Wiley-VCH, Weinheim.

Pirrung MC. Spatially Addressable combinatorial libraries. Chem Rev. 1997; 97:473-488.

Seeberger PH, Werz DB. Automated synthesis of oligosaccharides as a basis for drug discovery. Nat. Rev. Drug. Discov. 2005; 4:751-763.

See Also

Diversity-Oriented Synthesis of Small Molecules

Natural Product Inhibitors to Study Biological Function

Nucleic Acid Synthesis, Key Reactions of

Proteins: Structure, Function and Stability

Solution-Phase Synthesis of Biomolecules