Oligonucleotide and peptide synthesis

Peter Nagler, Ian Grayson, Michael Schwarm, Guenter Knaup and Thomas Mueller from Degussa discuss the development and manufacture of new therapeutic products based on oligopeptide and nucleotide chemistry

The 21 natural amino acids and the 4 deoxynucleosides that make up DNA can rightly be described as the building blocks of life. From these simple molecules are derived the vast range of oligonucleotides and proteins that encode, transmit, and express biological information at the molecular level.One of the great achievements of the modern pharmaceutical industry has been to develop synthetic oligopeptide and oligonucleotide products, containing both natural and unnatural building blocks, which interfere with, alter and control the transmission of this information in the cell.

long experience

Degussa Exclusive Synthesis & Catalysts, with its many years of experience in the areas of amino acid and nucleoside chemistry, has developed a range of products to support the pharmaceutical industry in the development and manufacture of new therapeutic products based on oligopeptide and oligonucleotide structural units.

Degussa's peptide chemistry is based on long experience in the synthesis and production of enantiomerically pure amino acids.¹ At the site of Degussa's wholly owned subsidiary Rexim at Ham (France), protein hydrolysates - complex mixtures of proteinogenic L-amino acids - are separated and purified using large-scale ion-exchange chromatography, ultrafiltration and crystallisation to yield high purity amino acids for pharmaceutical, cosmetic and nutritional applications.

novel resolution

To extend its technological capabilities, Rexim has recently founded a joint venture with the Chinese company Nanning Only Time for the large-scale fermentative manufacture of amino acids such as L-valine and L-isoleucine.

Non-proteinogenic D-amino acids and other speciality amino acids are also of particular interest to the pharmaceutical industry. Some examples from Degussa's portfolio of speciality amino acids produced at scale are L- and D-penicillamine and D-proline, obtained by chemical resolution; L-norvaline or D-3-(3'-pyridyl)-alanine from enzymatic resolution; enantiomerically pure cyclohexylglycines and -alanines via catalytic hydrogenations of the corresponding phenylglycines and -alanines; or L-tert-leucine² and L-neopentylglycine, which are produced by enzymatic reductive amination, figure 1. Recently, D-tert-leucine has also become available via a novel chemical resolution procedure.

As well as the manufacture of the amino acids themselves, a wide range of N- and O-protected amino acids, derivatives such as esters and amides, and downstream products such as the chiral aminoalcohols, diamines and oxazolidones are produced at Degussa, many at a commercial scale. These are used for the construction of peptide building blocks for pharmaceuticals.

Dipeptides are by far the most important product group in the field of pharmaceutical peptides, based on manufactured volume. Some of them are active pharmaceutical compounds themselves, such as Ala-Gln, Gly-Tyr, or Gly-Gln, figure 2, which are ingredients of the infusion solutions Dipeptamin, Glamin and Nephrotect used in clinical parenteral nutrition.

Isolation of such dipeptides on an industrial scale presents a challenge because of their high water solubility, and difficulties in crystallisation from aqueous salt solutions.

Degussa has solved these problems by taking advantage of Rexim's many years of experience in large-scale ion exchange, which is a highly cost-efficient purification technology providing excellent product quality.

Other dipeptides are used, either in the free form or as various protected derivatives, as key building blocks in the synthesis of pharmaceuticals. Examples of these are Ala-Pro and e-Tfa-Lys-Pro, the core building blocks for the ACE-inhibitors enalapril and lisinopril, respectively, and protected derivatives of dipeptides such as Val-Pro, Asp-D-Ala, Tle-Pro and D-Phe-Pro, figure 3.

Another dipeptide unit, which is found in numerous natural products and is beginning to be incorporated into pharmaceutical syntheses, is the 2,5-diketopiperazine (DKP) group.

Although formation of cyclic peptides often occurs to a certain extent as an undesired side-reaction during peptide synthesis, the directed synthesis of homogeneous and pure diketo-piperazines is sometimes difficult to achieve and usually requires activation of the carboxylic group. Degussa has developed a process to cyclize free dipeptides to DKPs in high yield and purity, without the need for additional activation steps.³ Some examples are the proline-derived DKPs [cyclo-Val-Pro] and [cyclo-Phe-Pro], which are intermediates for dihydroergotoxin, and the glycine-derived [cyclo-Gly-Val] and [cyclo-Gly-Tle], which are starting materials in the Schoellkopf bislactim ether synthesis of enantiomerically pure amino acids,⁴ figure 4.

longer and shorter

Larger peptide fragments or therapeutic oligopeptide products can be prepared by solid-phase or liquid-phase synthesis, or by a mixture of both. The largest therapeutic synthetic oligopeptide manufactured to date is the HIV fusion inhibitor Fuzeon or enfuviritide, developed by Trimeris and Roche.

This totally synthetic peptide, comprising 36 amino acids, is manufactured in sections by a solid-phase synthesis, using the FMOC protecting group protocol, and the sections then combined using a liquid-phase synthesis.⁵The final product has the structure:

Ac - Tyr - Thr - Ser - Leu - Ile - His - Ser - Leu - Ile - Glu - Glu - Ser - Gln - Asn - Gln - Gln - Glu - Lys - Asn - Glu - Gln - Glu - Leu - Leu - Glu - Leu - Asp - Lys - Trp - Ala - Ser - Leu - Trp - Asn - Trp - Phe - NH₂.

Shorter oligopeptides can be conveniently manufactured using liquid-phase methods, where it is often advantageous to couple protected dipeptides or higher peptide fragments. These protected short-chain building blocks can be produced and purified economically on a large scale using cheaper coupling methods or protecting groups. An example of the liquid-phase synthesis of a decapeptide, performed at Degussa, is the manufacture of Cetrorelix, an LHRH-antagonist developed by Asta Medica and marketed under the trade name Cetrotide for in vitro fertilisation.

Cetrorelix contains five unnatural amino acids, which were manufactured separately in enantiomerically pure form using chemical and enzymatic processes, and coupled using a liquid-phase strategy.⁶ The complete decapeptide was assembled using two- and three-amino acid fragments in a convergent process, as shown in figure 5. A total of 26 separate stages were required to complete the synthesis.

Degussa is not only a leading manufacturer of amino acids and peptide building blocks, but also offers a variety of peptide coupling reagents for commercial applications. Examples of these are 1,1'-carbonyldiimidazole (CDI), produced by the use of phosgene chemistry and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT).

The latter, used in combination with a tertiary amine base such as 1,4-dimethylpiperazine, forms a novel peptide coupling system, which provides high yields, negligible racemization and less waste compared with the established CDMT/N-methylmorpholine system.⁷ For special cases, when water-soluble by-products are essential for the purification of the peptide, water-soluble carbodiimide (ethyldimethylaminopropylcarbodiimide hydrochloride, EDC x HCl) is also available, figure 6.

production scale

Degussa has, with the acquisition of Laporte, incorporated many years of experience in nucleoside and nucleotide chemistry. Raylo Chemicals (Edmonton, Canada) has had a long history in the preparation of nucleotide building blocks and has also devised production scale methods for the preparation of deoxy- and di-deoxy-nucleosides. Degussa moved further into the oligonucleotide area in 2001 by completing its purchase of Proligo (Boulder, Colorado), and in November 2002 began the development and production of therapeutic oligonucleotides for genetic medicines under cGMP at the Raylo Chemicals site. Together with the Proligo operations in Boulder and in Hamburg, Germany, Degussa is now able to offer the entire value chain in oligonucleotide chemistry.

anti-AIDS drugs

The nucleoside building blocks for DNA have traditionally been isolated from natural sources, but the increasing amounts required for manufacture of therapeutic oligonucleotide pharmaceuticals has led to the development of alternative chemical, enzymatic and fermentation processes.

Nucleosides such as thymidine are required in multi-tonne quantities, for example for the manufacture of the anti-AIDS drug zidovudine or AZT.⁸

This first reverse transcriptase inhibitor active against the HIV virus acts by stopping DNA chain extension, as it does not have a 3'-hydroxyl group, figure 7. A similar complex chemical route from natural nucleosides was used to make 2',3'-dideoxyadenosine, which was converted by an enzymatic transamination to the HIV reverse transcriptase inhibitor dideoxyinosine (DDI).⁹

Later generations of HIV reverse transcriptase inhibitors, such as lamivudine (Epivir) and abacavir (Ziagen) have modified sugar side chains that are manufactured completely synthetically and without employing natural nucleosides.

Modified nucleosides have also been applied in other therapeutic fields. One example is the anticancer agent cytarabine, where the ribose sugar group of cytidine has been replaced by the isomeric arabinose.

The only oligonucleotide pharmaceutical active ingredient to be approved to date is fomivirsen (Vitravene), launched by ISIS Pharmaceuticals and Novartis in 1998 for the treatment of cytomegalovirus retinitis in AIDS patients. It is available in Europe on a named patient basis.

phosphate chemistry

Fomivirsen is a 21-base phosphorthioated oligonucleotide with the structure d(P-thio)(GCGTT TGCTC TTCTT CTTGCG). The required therapeutic amounts of this drug are low because of the specialised indication. There is more interest, therefore, in a series of compounds in late clinical trials for indications such as cancer or eye disease, where there is the potential for the application of the product to a larger patient group. The manufacture of launch quantities and the continuing production requirements of these products will require the construction of large dedicated facilities for the manufacture of the oligonucleotide active compounds at a molar or multi-molar scale.

Two phosphorthioate products are in Phase III clinical trials: alicaforsen sodium (Isis/Boehringer Ingelheim, Crohn's disease), and aprinocarsen sodium (Affinitak, Isis/Lilly, non-small cell lung cancer).

binding RNA

Antisense oligonucleotides are single strands of DNA, which are complementary to the messenger RNA that encodes for the targeted protein.

They bind to the mRNA, preventing it from being translated into the protein by the ribosome, and inducing mechanisms that result in destruction of the RNA-DNA duplex.

As single-stranded DNA is relatively unstable in vivo because it is rapidly hydrolysed by cellular nucleases, these oligonucleotide products are produced as phosphorthioates, where one of the non-bridging oxygen atoms in all the phosphate groups of the natural oligomer is replaced by a sulfur atom.

Phosphorthioates retain important features of the unmodified oligomers, such as the recruitment of the enzyme RNAase in vivo, which cleaves RNA bound to the antisense oligonucleotide, but they are much more resistant to cellular nucleases than their natural counterparts. All the phosphorthioates are present as the sodium salts of the corresponding acids at neutral pH.

Another product in Phase III trials, edifoligide (Corgentech, peripheral vascular disease), is also a phosphorthioated oligonucleotide, but it consists of two 14-base strands of DNA, which overlap by 10 bases. This product acts as a duplex decoy to the transcription factor E2F, binding to the transcription factor and preventing it from activating genes involved in vascular cell growth. Edifoligide is being applied in clinical trials by Corgentech to prevent blocking and failure of vein grafts used in by-pass surgery.

A further product in late clinical trials, pegaptanib (Macugen, Eyetech/ Pfizer) differs from the products mentioned above in that it is an RNA, rather than a DNA, oligonucleotide.

It is an aptamer, which is a class of oligonucleotide that acts like an antibody by binding to a specific protein. It is being developed by Eyetech Pharmaceuticals and Pfizer to block Vascular Endothelial Growth Factor (VEGF) in the eyes of patients affected with age-related macular degeneration and diabetic macular oedema.

The structure of this 28-base aptamer includes several non-natural nucleosides, for example 2'-fluorouridine, and two 20 kDalton PEG groups conjugated to its 5' terminus via a spacer. Partial structures of these different types of oligonucleotide products are shown in figure 8.

Because of the growth in oligonucleotide pharmaceuticals in recent years, several companies, including Degussa, have announced the construction of manufacturing facilities for oligonucleotide active ingredients. The major pharmaceutical manufacturers are involved in the development and launch of these products. Aventis, Lilly, and Pfizer have all signed deals with oligonucleotide development companies. This continued interest and growth in therapeutic oligonucleotides is likely to continue.¹⁰

building blocks

Degussa has, through its Proligo subsidiary and through its Raylo Chemicals manufacturing site, acquired a full range of competencies in all stages of oligonucleotide manufacture.

At Proligo Hamburg, the DNA and RNA nucleoside building blocks are converted into activated phosphoramidite derivatives for oligonucleotide manufacture. This process is shown in figure 9.

The 5'-position of the nucleoside is protected with the dimethoxytrityl group, and the 3'-position is activated by reaction with the key reagent bis(diisopropylamino)-(2-cyanoethoxy) phosphine, which is prepared at Hamburg to an exacting quality specification. For RNA synthesis, protection of the 2'-OH position is required in addition.

The individual phosphoramidite building blocks are assembled on a solid support, using the method originally introduced by Beaucage and Caruthers¹¹ and improved by Koester.¹². Proligo operates the Koester patents for the phosphoramidite process under licence.

Various supports are used in oligonucleotide manufacture, for example, a variety of polymer supports are used on a column. The Proligo method uses controlled pore glass (CPG) beads, which are prepared in-house. Using CPG beads, the oligonucleotides can be manufactured in a stirred batch reactor, and problems associated with swelling of the support are avoided.

late phase

In oligonucleotide synthesis, the first nucleoside is attached to the solid support at the 3'-position, and sequential coupling at the 5'-position, oxidation, capping to remove any unreacted precursor, and deprotection is used to add each subsequent nucleoside. At the end of the oligonucleotide synthesis, the final product is removed from the support, all the protecting groups (including those on the base nitrogens) are cleaved, and the final product is purified by HPLC to the required pharmaceutical specification, figure 10.

At Raylo Chemicals, two suites of a GMP oligos unit have been constructed for the manufacture of therapeutic oligonucleotide active ingredients to cGMP, and a third is under construction. This facility will allow production of oligonucleotide products for late phase clinical trials and commercial quantities. In addition, new development facilities are available for the manufacture of oligonucleotides for early phase clinical trials.

Besides the antisense and aptamer approaches described above, a whole range of methods in drug development uses modified nucleic acid-based products. Only a few of these can be described in this article. Isis Pharmaceuticals has patented a 'second generation' antisense technology using 2'-O-methoxyethyl substituents on the sugar.

degradation resistant

These oligonucleotides containing both DNA and RNA units (see figure 11) are said to be more resistant to degradation and to have a higher affinity for their RNA targets. AVI Biopharma has developed a 'third generation' technology, where the sugars are replaced with a morpholine backbone. Its lead product, Resten-NG, is entering Phase III clinical trials for the treatment of restenosis, figure 11. Peptide nucleic acids are even more unusual, in that they contain neither ribose nor phosphate linkers.

The individual purine and pyrimidine bases are linked by a repeating N-(2-aminoethyl)-glycine chain, and the resulting oligo PNA does not have a charged backbone. This means that it not only binds to single stranded RNA or DNA, but also to double-stranded DNA, forming a triple helix.

Proligo, at its site in Boulder, Colorado, has developed a wide range of specialised oligonucleotide products for use in drug design and development, and in diagnostics.¹³. Locked Nucleic Acid (LNA) oligonucleotides contain LNA units to provide superior hybridisation characteristics and enhanced biostability compared with conventional DNA oligonucleotides, figure 11. Proligo has a licence to use the LNA technology from Exiqon.¹⁴ LNA-LNA duplexes form very strong Watson-Crick bonds, and LNA-RNA and LNA-DNA duplexes are also more stable than RNA-DNA and DNA-DNA structures. Proligo offers oligo-nucleotides containing one or more LNA nucleosides made to order for a variety of genomic applications.

Proligo also manufactures small interfering RNA (siRNA) oligonucleotides under licence from the Massachusetts Institute of Technology. These are short RNA oligonucleotides, with 19 to 23 bases in single-strand or duplex form, which bind to complementary strands of mRNA in the cell.

The cell recognises the double-stranded mRNA as foreign, and a dsRNA-specific endonuclease enzyme called Dicer destroys the dsRNA after transcription. When small interfering RNA oligos of fewer than 30 bases are used, the Dicer enzyme splits the RNA into small pieces, which are destroyed without inducing cell apoptosis.

This is an exciting new approach to oligonucleotide chemistry, which has the potential for wide application in therapeutics, functional genomics, and diagnostics.¹⁵ Nucleoside and oligo-nucleotide chemistry will continue to have much to offer to the pharmaceutical industry in the future.