The World of Peptides

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WELCOME TO THE WORLD OF PEPTIDES

Insights into the peptide chemistry of Bachem Insights into Peptide Chemistry

AN INTRODUCTION INTO THE WORLD OF AMINO ACIDS AND PEPTIDES presented by Monika Mergler, Ph.D., Bachem AG

Contents

1. The business areas of Bachem 03 2. What are peptides? 06 3. What are amino acids? 07 4. Manufacture of peptides 14 5. Analysis of amino acids and peptides 24 6. Modiﬁ cation of peptides 26

Abbreviations

ADS Analytical Datasheet API Active Pharmaceutical Ingredient (drug substance in medicines) CCD Counter Current Distribution (purifi cation method) CofA Certifi cate of Analysis DMF Dimethylformamide (solvent used in SPPS) GMP Good Manufacturing Practice* HPLC High Performance Liquid Chromatography (method of analysis and purifi cation) MS Mass Spectrometry (method of analysis) NCE New Chemical Entity (drug substance not yet licensed) NMP N-Methylpyrrolidone (solvent used in SPPS) RSLC Rapid Separation Liquid Chromatography (method of analysis) SPPS Solid-phase Peptide Synthesis TLC Thin Layer Chromatography (method of analysis) Xaa, Yaa any amino acid (3-letter code)

Symbols for chemical elements C Carbon HHydrogen N Nitrogen O Oxygen S Sulfur

* GMP = Good Manufacturing Practice, production guidelines that assist quality assurance. These guidelines must be followed when substances to be used in humans are manufactured. Their adherence is regularly checked by specialist authorities (such as Swiss- medic in Switzerland, FDA in the USA). Drug substances in medicines, ingredients in cosmetics and additives in foods are some of the items that sell under these guidelines.

2 The business areas of Bachem New Chemical Entities (NCEs) Contract synthesis The core activities of Bachem are the NCEs are patented peptides or organic contract manufacture of pharmaceutical compounds that Bachem manufactures active substances and the synthesis and exclusively for the patent holder or a sale of amino acid derivatives, peptides and licensee. Even during the ﬁ rst, small-scale ﬁ ne chemicals. Bachem produces peptides manufacture, the key point to remember as well as small molecule APIs as drug with NCEs is that the product may prove to substances for the pharmaceutical industry be pharmaceutically active and therefore under GMP conditions*. must be capable of manufacture in larger quantities and potentially under GMP conditions. The business areas of Bachem are: Bachem strives to develop a long-term partnership with such customers. This Research Chemicals begins with the initial synthesis of an NCE Catalog products, available from stock on a laboratory scale, moves on to the These amino acid derivatives, peptides and manufacture of larger quantities under other chemicals are not manufactured GMP conditions for the clinical phases and under GMP conditions and may only be continues right up to large-scale used for laboratory and research purposes. manufacture of the drug substance for the They may not be used in humans. approved medicine.

Custom Synthesis Generic Active Pharmaceutical Contract synthesis, exclusively for a Ingredients (Generic APIs) customer Pharmaceutically active compounds Mainly peptides, that are manufactured in Substances used as active ingredients in quantities ranging from a few milligrams up medicines have to be manufactured under to several grams. On request, the products GMP conditions. They are sold in large can be synthesized under GMP conditions. quantities.

GENERIC APIs CATALOG PRODUCTS • peptide and small molecule generics • more than 7500 products available online • supply of commercial quantities • peptides, amino acids, inhibitors, substrates • reputation for sustained quality & supply • fast & easy ordering @ shop.bachem.com • high-demand APIs available from stock • bulk quantities according to your needs Research • close and long term partnerships • excellent customer and technical support

CommercialSupply

ManufacturingContract NCEs CUSTOM SYNTHESIS • process development, optimization and validation • from discovery stages to early clinical candidates Development • comprehensive analytical services • strong focus on quality and timely execution • supply of cGMP material for all clinical phases • development of synthetic routes for scale up • sterile ﬁ ll & ﬁ nish for clinical trials (Clinalfa®) • best industry practice • cGMP-manufacturing of APIs for commercial supply • highly motivated and experienced team

Fig. 1: The Bachem 360º business model.

3 Insights into Peptide Chemistry

Generic Active Pharmaceutical Ingredients The patent protection of a drug substance (Generic APIs) generally expires after 18 years. Small quantities of the peptide can be sold for Generics are drug substances or medicines, research and development purposes even whose patent has expired. before the patent has expired (Bolar Ex- Medicine = “formulated” drug substance: emption). Some peptide APIs manufactured Depending on how medicines are admin- by Bachem and their indications are listed istered (as tablet, as injection etc.), various in Table 1. non-active additives or “excipients” are The structures of calcitonin, glucagon, go- mixed with the actual drug substance. This nadorelin (GnRH or LHRH) and process is called formulation and it can be secretin correspond to those of the body’s patented independent of the drug sub- own “endogenous” natural peptide stance patent. Suitable formulation can, for hormones; the other peptides listed in example, enable the drug substance to be the table are modiﬁ ed natural peptides. released only slowly (depot formulations), Through changes made to their structure, to be delivered as a nasal spray or absorbed they are more slowly broken down in the through the skin. The development and body than endogenous peptides. patenting of new formulations of generic drug substances offers the drug substance Triptorelin is available in the form of two manufacturer the possibility of good, long- different salts, acetate and pamoate. The term sales. pamoate is released more slowly and is therefore used in depot formulations. The discovery of other pharmacological The small molecule APIs sold by Bachem activities can lead to additional indica- are manufactured by our subsidiary in tions for a generic and thereby increase Vionnaz. the demand for the drug substance. This is Examples are propofol - used to induce another case when patents (usage patents) general anesthesia, or for sedation in can be granted. Generics are also used to artiﬁ cially ventilated adults during inten- diagnose diseases (i.e. diagnostic agents). sive care - and zolpidem, a most frequently A further market with great potential is the prescribed drug against insomnia. use of generics in veterinary medicine. If you are interested, more about our pep- Table 1: Example of some peptide APIs manu- tide and non-peptide generic APIs can be factured by Bachem and their indications found on our homepage www.bachem.com Peptide Indication You can download our generics catalog and Calcitonin Osteoporosis request a quote. Deslorelin Fertility control New Chemical Entities (veterinary medicine)

Desmopressin Diabetes insipidus During preclinical development, lead fi nding Glucagon Hypoglycemia (low blood and lead optimization require large panels sugar) in diabetes of peptides. These are generated as custom Goserelin Cancer synthesized molecules for customers around the world. Frequent consultation Gonadorelin Reproduction medicine with Bachem experts allows further refi ning Leuprolide Cancer of target compounds. As such, a clear part- Octreotide Acromegaly nering aspect is required to come up with Secretin Diagnosis pioneering concepts and molecules to bring (pancreas) into clinical development. There is a large pipeline of peptide drug Triptorelin Cancer candidates in clinical development. Also, over 50 peptides have been approved to treat various diseases. Bachem has the largest portfolio of projects for which we supply the peptide. 4 When clients have selected their lead diffi cult peptides. Other specialties include compound they commence clinical trials. multi-step organic transformations and It is a decade-long process to approval of peptidomimetic molecules. the drug. During this time, there is a close collaboration to learn more about the prod- Research Chemicals uct. Each production step is scrutinized „Catalog products“ and manufacturing reproducibility strived for. Scale-up and full control of the pro- These products are sold from stock in cess is targeted. Validation and control of various pack sizes or in larger quantities the process is the end result of an intense (minibulk and bulk). Most of them are man- partnership. ufactured by Bachem UK or Bachem AG at Bubendorf (CH). Clinalfa The catalog products cover a broad customer base. The products in the main Bachem sells peptides for clinical trials and catalog are divided into two categories. market-ready formulations of peptides as The fi rst section of the main catalog an additional service under the brand name contains amino acid derivatives and Clinalfa®. Two product lines are available: products for the synthesis of peptides, Clinalfa® basic products are sterile freeze- i.e. products mainly ordered by chemists. It dried or liquid ready-to-use formulations also contains biochemicals that are not exclusively for use in approved clinical amino acid derivatives or peptides. So this trials. They are manufactured and released section also serves customers involved in in accordance with the requirements biological and medical research. and relevant guidelines of the regulatory The second section is larger than the fi rst authorities and are available from stock. one. It lists the available peptides and is Under the brand name Clinalfa® plus, we addressed mainly to life science research. offer customized formulation as an addi- This division into two product categories tional service to the contract manufacture (amino acids, products for peptide synthe- of peptide drug substances. sis, and non-peptide biochemicals/peptides) is mirrored in the Online Shop (s. Fig. 3). The catalog business is also important for the acquisition of customers for our other services, such as contract synthesis. It is a simple way by which we can show potential customers the high product quality and excellent service offered by Bachem. Fig. 2: Clinalfa®-logo.

Custom Synthesis

In addition to our research grade products listed in our catalog and available from stock, Bachem offers an effi cient and high quality custom synthesis service for peptides and amino acids derivatives. Our peptide chemists can prepare compounds according to your specifi cations in any scale you need - from milligram to kilogram and ton quantities. We are experts in producing fl uorescently labeled peptides, peptides Fig. 3: with multiple disulfi de bridges, peptides Website entry containing stable isotopes, and long and page of the Bachem Online Shop.

5 Insights into Peptide Chemistry

What are peptides ? Biologically active peptides are, for instance, hormones or toxins (see p. 23). Peptides are small proteins. The majority of our hormones are peptides Peptides and proteins differ only in their of widely varying length: length: TRH (thyreotrophin-releasing hormone) is a Proteins >100 amino acids tripeptide (it consists of 3 amino acids). Peptides 2-100 amino acids LHRH (also known as GnRH (gonadotro- phin-releasing hormone) is a peptide made Peptides and proteins are macromole- from 10 amino acids (decapeptide). cules, i.e. long molecules made from small Calcitonin consists of 32 amino acids. subunits. They can be imagined as chains pTH (parathormone), with its 84 amino of pearls, composed of differently shaped acids, is almost a protein. beads. The “pearls” are the 20 proteinogenic Insulin consists of 2 chains of peptides, one amino acids (amino acids that are used in composed of 30 amino acids and one of 21 the cell to build peptides and proteins). They that are linked to each other via disulfi de are linked together in any combination, i.e. bridges (see p. 23). the number of possible combinations is virtually infi nite. The amino acids may be In general, peptide hormones are produced repeated in any frequency, so for example, by specialized cells, released into the blood glycine (Gly) in the protein collagen is found and transported to the target organ. The in every third position in the chain: cells to be stimulated have receptors that …-Xaa (mostly Pro (L-proline)-Yaa-Gly-… recognize the hormones and bind them specifi cally. These receptors are special- Apart from water, our body consists mainly ized proteins that are embedded in the cell of proteins. Although sharing the same membrane. The binding of the hormone structural principle, they have a huge acts as a signal for the cell and the desired variety of functions (see Table 2). Proteins effect is triggered. are also a vitally important constituent of our diet. Short peptides • 2 amino acids = dipeptide What makes this variety possible? • 3 amino acids = tripeptide Through variation in the amino acid building • 4 amino acids = tetrapeptide blocks and in length, macromolecules can • 5 amino acids = pentapeptide etc. be “constructed” with the most diverse • a few amino acids (2-20) = oligopeptide properties imaginable!

Table 2: Endogenous proteins and their functions

Examples Occurrence of the protein Requirements/function Myosin, actin Muscle tissue Flexible molecule, contractible Collagen Connective tissue, tendons, skin Molecule stable in shape, stretch-resistant (the most common protein in the body) Hemoglobin, albumines Blood Transporter molecule, soluble Enzymes such as the digestive en- Throughout the body Enzymes = catalysts zyme trypsin in the small intestine (trypsin is a “protease”, which splits peptides and proteins) Hormones such as thyreotrophin Throughout the body Hormones = messengers hormone (TSH) (stimulates the thyroid glands) Antibodies (immunoglobulins) Blood Immune defense

6 With the 20 amino acids that can occur proteins of the cell are made. They differ several times in the peptide, even with short solely in the side chain R. In addition many peptides many combinations are possible, other α-amino acids are found in nature, e.g. approx. 3.2 million different pentapep- either free, as products of metabolism or tides! incorporated into peptides/proteins. Exam- Even dipeptides can be biologically active: ples are L-hydroxyproline (Hyp) in collagen, Leu-Trp* and other dipeptides lower blood or L-ornithine (Orn) free in the urine. Other pressure. α-amino acids, e.g. L-norleucine (Nle) have Acetyl-Asp-Glu (NAAG) is an important so far only been obtained through chemical neurotransmitter, a substance that medi- synthesis. Bachem calls non-proteinogenic ates the transmission of signals between amino acids such as Hyp and Nle “unusual nerve cells. amino acids”. Often, they are designated as The peptides found in the body are usually “unnatural amino acids” irrespective of their obtained by enzymatic splitting or occurrence in nature. “cleavage” of proteins. Amino acids (whether proteinogenic or not) can be biologically active, e.g. L-tryptophan, What are amino acids? L-glutamic acid.

Structure of an α-amino acid The side chain R Four different substituents** are attached • can be a hydrogen atom: to a central carbon atom (the α-C atom): glycine, the simplest α-amino acid • the acid group • can carry an additional acid group (COOH): (carboxylic acid group = carboxyl group, aspartic acid, glutamic acid or a modiﬁ ed

COOH, which Bachem abbreviates to –OH in acid group (an amide, CONH2): asparagine, the three-letter code*) glutamine • a basic group • can carry an additional basic group:

(amino group, NH2, abbreviated H- in the arginine (strong base), lysine, histidine three-letter code) (weak base) • the side chain • can carry a polar group: (R can vary greatly and thereby determines serine, threonine the properties of the peptide) • can be a hydrocarbon (non-polar): • a hydrogen atom alanine (R = methyl), phenylalanine (R = (H) benzyl), valine (R = isopropyl) Accordingly, the α-C atom carries 4 groups • can contain sulfur: with different chemical properties that play cysteine, methionine a very large role in what follows (Fig. 4). In addition to the α-amino acids from which H NH peptides and proteins are built, Bachem 2 has many other amino acids on offer, including those in which the amino group is C bound to another carbon atom.

R COOH L- und D-amino acids The four different substituents of the α-car- Fig. 4: α-amino acid, the α-C atom with the bon atom are not arranged in one plane, but four different substituents. lie at four corners of a tetrahedron with the α-C atom in the center (Fig. 5). As already mentioned, there are 20 different Therefore two forms of the amino acid mol- “proteinogenic” amino acids from which ecule are possible that behave like mirror

*See Tab. 4 on p.9 for the meaning of the abbreviations and codes. **Substituents are atoms or groups of atoms, that are bound to a central carbon atom (which is, in case of α-amino acids, the α-C atom) instead of a hydrogen atom.

7 Insights into Peptide Chemistry

Table 3: The functional groups important for amino acids

Name Structure Name Structure

Amino group NH2 Carboxyl group COOH

Hydroxyl group OH Amide group CONH2 (Carboxamide group)

Thiol or mercapto group SH Guanidino group NH-C(=NH)-NH2 (in cysteine) (in arginine)

images”, the D-amino acids, occur much less often in nature. D stands for dexter (Latin for “right”). To reiterate this point, L or D denote the arrangement of the four different substituents on the α-C atom, i.e. the respective enantiomer (see Fig. 6). The direction of rotation cannot be deduced Fig. 5: Arrangement of the substituents from the designation L or D. In the case of around the α-carbon atom (gray). L-amino acids, it can be positive or negative (see below), with the correspond- images of each other and are known as ing D-amino acids, it is always in the stereoisomers or enantiomers: opposite direction. The two enantiomers do not differ in their The values of rotation are often stated as physical properties, except that in solution, [α] on the ADS (see p. 24) of amino ac- they rotate the plane of polarized light. ids and their derivatives, since they are characteristic for the particular compound. Compounds that do this are called “optically Values of rotation are often also measured active”. Optical activity is found very widely for peptides.

mirror plane Fig. 6: Absolute conﬁ guration of the L- and D-isomers of amino acids.

in nature. Apart from glycine (R = H, image Some examples: and mirror image identical), all proteino- L-alanine: + 14.3° genic amino acids show this phenomenon. D-alanine: -13.9° Glucose (sugar) also rotates the light and L-tryptophan: - 31.8° this property is used to determine the D-tryptophan: + 30.7° concentration of glucose solutions, as does (the discrepancies between the absolute DNA and its building blocks. values for the L- and corresponding All proteinogenic amino acids (apart from D-enantiomer lie within the range of Gly) are L-enantiomers (L stands for laevus, accuracy of the method) Latin for “left”), e.g. L-alanine. Their “mirror

8 Table 4: Abbreviations of the proteinogenic amino acids (L-form, except Gly)

Name in peptide* 3-letter 1-letter Name in peptide 3-letter 1-letter Alanine alanyl Ala A Leucine leucyl Leu L Arginine arginyl Arg R Lysine lysyl Lys K Asparagine asparaginyl Asn N Methionine methionyl Met M Aspartic acid aspartyl Asp D Phenylalanine phenylalanyl Phe F Cysteine cysteyl Cys C Proline prolyl Pro P Glutamine glutaminyl Gln Q Serine seryl Ser S Glutamic acid glutamyl Glu E Threonine threonyl Thr T Glycine glycyl Gly G Tryptophan tryptophyl Trp W Histidine histidyl His H Tyrosine tyrosinyl Tyr Y Isoleucine isoleucyl Ile I Valine valyl Val V *s.Table 5 on p. 15

A 1:1 mixture of the two enantiomers is The abbreviations of the 20 proteinogenic called a racemate. In this case, the rotations amino acids are given in Table 4. of the L- and D- form compensate each The one-letter code is only used for the 20 other. proteinogenic amino acids, for D-amino In the Bachem notation, the L-form is not acids, the usual practice is to use small explicitly shown because of its ubiquity letters, e.g. f = D-Phe. (e.g. L-alanine = H-Ala-OH). Only the less On the other hand, for many of the non-pro- common D-form (D-alanine = H-D-Ala-OH) teinogenic amino acids there is often also and the racemate, which is shown as DL, an obligatory three-letter code, e.g. Hyp are designated as such. (L-trans-hydroxyproline), Nle (L-norleucine), Orn (L-ornithine). Not all abbreviations Notation for amino acids usually found in the literature are also used If the name of an amino acid is written out by Bachem. For some amino acids, the in full, the enantiomeric form is shown as non-abbreviated name is used, e.g. well: L-alanine and D-alanine. L-thiazolidine-4-carboxylic acid (otherwise In the case of a racemate - as is common Thz). in organic chemistry - the DL is routinely omitted. Brief portraits of the 20 proteinogenic Amino acids and peptides are represented amino acids by their three-letter codes (usually the ﬁ rst An organism such as the human body must three letters of the name). have all 20 proteinogenic amino acids avail- In addition, there is also a one-letter code able in order to synthesize proteins. They all that is preferred, especially for longer pep- need to be in the L-form (except Gly). tides and proteins A distinction is drawn between essential Examples: and non-essential amino acids. L-alanine → Ala, L-Ala, H-Ala-OH (3-letter); Non-essential amino acids can be synthe- A (1-letter) sized by the body itself; essential cannot, L-arginine → Arg, L-Arg, H-Arg-OH (3-letter); i.e. they must be supplied in the diet. R (1-letter) Amino acids can also be subdivided Bachem notation: according to the side chain (see p. 7) that H-Ala-OH, H-Arg-OH determines their properties. There are The notation signiﬁ es that it is the L-form acidic, basic, polar and non-polar amino and that the amino and carboxyl groups are acids. They are distinguished by different free. colors in the "Periodic Chart of Amino Acids" (see p. 13).

9 Insights into Peptide Chemistry

The properties of peptides and proteins are Aspartic acid (Asp, D) determined by the side chains of the amino acids from which they are constructed.

Glycine (Gly, G)

is an acidic amino acid because it contains is the simplest amino acid and one of the a further acid group in the side chain. This most frequently encountered in peptides/ persists when Asp is incorporated into a proteins. Gly is non-essential. Glycine owes peptide. Aspartic acid was discovered in its name to its sweet taste. asparagus, which helped give it its name. Asp is non-essential for mammals. Alanine (Ala, A) Cysteine (Cys, C)

is the simplest optically active amino acid Oxidation of and the second most common in proteins. cysteine gives The L-form shown here of this non-essen- rise to cystine tial amino acid is incorporated into proteins by the body, but D-alanine is also found quite frequently in nature in peptides. Ala- nine is one of the non-polar amino acids.

Arginine (Arg, R) is a rare amino acid, although it is extremely important for the structure of peptides and proteins. The non-essential Cys contains sulfur in the side chain, as a slightly acidic thiol group. Thiol groups are readily oxidized, with the formation of a disulﬁ de

is a strongly basic amino acid. The nitro- bond. One molecule of cystine (Cyt or (Cys)2) gen-containing guanidino group in the side is formed from two molecules of cysteine. In chain is such a strong base that it forms peptides that contain a Cys, two chains are a salt with virtually any acid. It owes its linked together. If there are two Cys in the name to the fact that it was isolated as the peptide, then a ring can be produced, as we silver salt. Arg is semi-essential (the body will see again later. can produce it, but often in insufﬁ cient amounts). Arg-containing peptides are Glutamine (Gln, Q) highly polar and readily water-soluble. is actually a derivative (the amide) of glutamic acid. Glutamine is a polar amino Asparagine (Asn, N) acid that occurs as the free form in large is actually a derivative of aspartic acid (the quantities in the human body. Glutamine at amide) and like the latter, was discovered in the start of a peptide chain forms pyroglu- asparagus. Asn is a polar amino acid and is tamic acid spontaneously, or with the help relatively labile in peptides and proteins. of an enzyme.

10 Glutamic acid (Glu, E) The name leucine comes from the white (Greek: leucos) leaves in which it crystall- izes. Further stereoisomers are possible with isoleucine (allo-Ile).

Lysine (Lys, K) N-terminal in peptide

is a basic amino acid, that carries a second amino group in its side chain. It is an α,ε- is an acidic amino acid. Glu is non-essen- diamino acid. Lysine is essential. tial. The salts of glutamic acid are called glutamates and the sodium salt (mono- Methionine (Met, M) sodium glutamate, MSG) is known as a taste-enhancer. Glu has great physiological importance in our nervous system as a neurotransmitter. Glu forms pyroglutamic acid much less readily than glutamine.

Histidine (His, H) is, like cysteine, a sulfur-containing amino acid. The sulfur is present as methyl thioether, as reﬂ ected in the name “Me-thio”. Thioethers are sensitive to oxidation, which should be heeded when handling Met- containing peptides. This essential amino acid is one of the non-polar amino acids. is a weakly basic, polar amino acid that is Phenylalanine (Phe, F) essential for humans. The name comes from “histos” (Greek for tissue). The imid- azole ring that it carries in the side chain, contains two nitrogen atoms. Imidazoles catalyze many reactions and therefore His (in combination with Cys, Ser or Thr) is often to be found in the active center of enzymes.

Leucine (Leu, L) and Isoleucine (Ile, I) is an essential, non-polar amino acid. Together with His, Tyr (that can be consid- ered as a phenylalanine derivate) and Trp, it is one of the aromatic amino acids.

Proline (Pro, P)

are isomeric α-amino acids. Isomeric compounds have the same molecular formula and the same molecular weight, but differ is the only cyclic proteinogenic amino acid, in their structure. Both are non-polar mole- a ring of 5 atoms that contains an α-amino cules. Leu and Ile are essential for humans. acid group and an α-C. Pro is non-polar and

11 Insights into Peptide Chemistry

non-essential. Due to its special structure, Amino acid derivatives Pro has a huge infl uence on the spatial Derivatives in organic chemistry are under- structure of peptides and proteins. stood to mean descendants of a compound. A derivative of an amino acid is obtained Serine (Ser, S) by modifi cation (chemical alteration) of the is a polar amino acid due to the hydroxyl amino group and/or the carboxyl group and/ group in the side chain. The name of this or the side chain. If this modifi cation can be non-essential amino acid is derived from reversed under certain conditions that do the Latin (sericum) for silk. not alter the amino acid, it can serve as a protecting group. Such derivatives are suit- Threonine (Thr, T) able building blocks for peptide synthesis.

Parent Derivative Compound H-Ala-OH →Ac-Ala-OH (amino group blocked)

→ H-Ala-NH2 like serine, contains a hydroxyl group in (carboxyl group blocked) the side chain, but is an essential amino → Fmoc-Ala-OH acid. Threonine, in common with isoleucine, (amino group blocked) contains a second asymmetric carbon atom However Fmoc can with four different substituents, so other be selectively split off stereoisomers are possible (allo-Thr). (“cleaved”) under mild conditions, i.e. is a suit- Tryptophan (Trp, W) able protecting group for peptide synthesis.

is a non-polar essential amino acid, an indole derivative. The free amino acid has an antidepressant effect and is a precursor of serotonin. Tryptophan ﬂ uoresces in the UV range (308-350 nm).

Tyrosine (Tyr, T) since it can be formed in the body from phenylalanine, tyrosine is a non-essential amino acid. The relatively non-polar amino acid was ﬁ rst isolated from cheese (Greek: tyros).

Valine (Val, V)

is a non-polar essential amino acid.

12 o 3-Letter Amin 3-Letter Acid Code Chemical Structure Chemical Name Glu Threonine Thr Asp Aspartic Acid 4 4 3 Glutamic Acid O Serine 1 NO NO N NO 7 9 9 .1

H H H

Ser 4 5 4 29 147.13 129.11 C D E 1 C T 119.12 101.10 C 133.10 115.09 C 3 e ne NO 7 si H 3 min S 105.09 87.08 C C a ro t u Tyrosine Ty l Tyr Gln 0 Glutamine G 2 – H 3

3 r O Relative Relative 2 Formula M N NO Acid Code Molecular 10 11 H H 5 9 1-Letter Amino 1-Letter Q 146.15 128.13 C C Y 181.19 163.17 C C Molecular Mass Serine Glycine Gly Ser 2 3 Basic Non-polar (hydrophobic) uncharged Polar, Acidic Copyright by Global Marketing Bachem Group. Reproduction forbidden without permission forbidden Reproduction Group. Bachem Global Marketing by Copyright NO NO 5 7 Periodic Chart Periodic of Amino Acids © H H 2 3 G 75.07 57.05 C S 105.09 87.08 C Valine Cysteine Val Cys Asn Asparagine S 3 2 2 O 2 NO NO N 7 8 11 H H H 3 4 5 C 121.16 103.14 C N 132.12 114.10 C V 117.15 99.13 C Proline Alanine Ala Pro Met Methionine S 2 2 2 NO NO NO 7 11 9 H H H 3 5 5 A 89.09 71.08 C C M 149.21 131.20 C P 115.13 97.12 C C han p to Leucine p Trp Leu Phe Tryptophan Try 2 O 2 2 2 Phenylalanine O N NO NO N 1 3 12 18 11 1 13 1 . H H H 9 6 11 9 6 11 F 165.19 147.18 147 C C L 131.18 113.16 C C W 204.23 186.21 C Lysine Ile Arginine Histidine His Isoleucine Lys Arg 2 2 2 2 O O O 4 2 3 9 N N NO N 9 14 14 13 .1 H H H H 6 6 6 6 56 H 155.16 137.14 C R 174.20 156.19 1 C C K 146.19 128.17 128.17 C C I 131.18 113.16 C C

13 Insights into Peptide Chemistry

Chemical Manufacture of Peptides H-Ala-Ala-OH H-Ala-OH H-Ala-Phe-OH How is a peptide synthesized? + H-Phe-Ala-OH Why synthesize peptides at all? H-Phe-OH Can’t nature do it better? H-Phe-Phe-OH

Chemical Synthesis has advantages as: In order to actually obtain the desired dipeptide • Any scale is possible H-Ala-Phe-OH and not a mixture, two protect- • It is simpler than biological methods for ing groups are needed. They must not be split off shorter peptides under the conditions of the coupling reaction, • The product is BSE/TSE-free but be readily cleaved in a separate step after and additional possibilities: coupling has taken place. • A change of the amino acid sequence of The amino group of alanine must be blocked: known peptides is feasible H-Ala-OH → X-Ala-OH • Modiﬁ cations of peptides can be achieved and the acid group of phenylalanine: (see p. 26) H-Phe-OH → H-Phe-OY Now only the acid group of alanine The aim is to improve on nature, i.e. to modi- fy the biological activity of the peptide: can react with the amino group of the • Increase the desired activity (biologically phenylalanine: active peptides usually show several X-Ala-OH + H-Phe-OY → only X-Ala-Phe-OY different activities) Finally, the protecting groups are removed: • Optimize stability X-Ala-Phe-OY → H-Ala-Phe-OH “not better, but longer” i.e. X and Y are removed under the same condi- (peptides cannot be taken orally and are rapidly broken down in the body, which is tions. not favorable for a drug) Now let us choose X and Y so that X is removed • Minimize side effects under conditions in which OY is retained. This • Elucidate structure-activity relationships allows us to synthesize longer peptides: • Synthesize peptides that do not occur X-Ala-Phe-OY naturally, and many more. Removal of X

Synthesis of a dipeptide from two different amino acids H-Ala-Phe-OY Example: Coupling X-Leu-OH L-Alanyl-L-phenylalanine (H-Ala-Phe-OH)

X-Leu-Ala-Phe-OY H-Ala-OH + H-Phe-OH  H-Ala-Phe-OH + H2O Cleavage X, coupling X-Gly-OH Two amino acids react with each other to form the dipeptide, with separation of water. This X-Gly-Leu-Ala-Phe-OY does not happen spontaneously, so they have to be activated for the reaction to occur. For this Cleavage of X and Y (two steps) purpose “coupling reagents” have been developed. They generate more reactive derivatives of H-Gly-Leu-Ala-Phe-OH the amino acids and remove the water which is the desired peptide formed concomitantly from the system. However, if a coupling reagent is simply added to a solution of alanine and phenylalanine, it results in a mixture of the 4 possible dipeptides; tri- and longer peptides can be produced as well:

14 In this way, a peptide can be elongated to any Many Boc or Z-amino acid derivatives are avail- desired length. X is called a temporary protect- able as DCHA or CHA salts ((Di)cyclohexyl- ing group, because it is only used for the ammonium salts). Salt formation improves the coupling step. Y is a permanent protecting stability on storage of acid-sensitive derivatives, group. It must be stable enough to endure all because amino acids are quite strong acids, the coupling and X-cleavage steps. However, stronger than acetic acid. Some Boc or it must be removable in the fi nal step, without Z-derivatives are oils; they can only be obtained damaging the peptide. A peptide always has two in solid, crystalline form as salts. Solid products different groups at each end, and thereby a are much easier to handle than oils, e.g. when “direction”. The amino group at the end of a weighing. peptide is known as the N-terminus, the car- Benzyloxycarbonyl (Z or Cbz) is the oldest boxyl group at the other end, the C-terminus. In usable Nα-protecting group for amino acids chemical synthesis, the peptide is constructed (M.Bergmann & L.Zervas 1932). The develop- from the C-terminus in the direction of the ment of Z brought about the start of modern N-terminus and therefore the terminal carboxyl peptide synthesis and hence also the abbrevia- group must be protected throughout the entire tion Z in honor of Leonidas Zervas. The Z group synthesis. Irrespective of the method of nota- is often today still used to protect the amino tion, when representing a peptide sequence, group, also in organic synthesis. one starts at the N-terminus, see Table 5. Protecting groups for carboxyl moieties Table 5: Notation of peptides In contrast to the α-amino group, the terminal acid group must be protected throughout the Notation Explanation entire synthesis. The most commonly used H-Gly-Leu-Ala- Bachem notation, here protecting groups are listed in Table 7. Phe-OH the N- and C-terminals Examples of esters of amino acids: are easily recognized, H-Ala-OtBu · HCl both are “free”, not modi- H-Val-OMe · HCl fi ed H-Glu(OBzl)-OBzl · p-tosylate Gly-Leu-Ala- Terminal groups are Esters of amino acids are only stable on storage Phe only explicitly stated if in the form of their salts with strong acids such they are not free as hydrochloric acid (hydrochloride) or p-tolu- ene sulfonic acid (p-tosylate). These salts are GLAF 1-letter code. Terminal also more readily available in crystalline form groups are only explicitly than the free amino acid esters. stated if they are not free Glycyl-L- “written out in full”, The choice of amino and carboxyl protecting leucyl-L-ala- especially common for group also depends on the method of synthesis. nyl-L-phenyl- shorter peptides The two most important methods are described alanine in the next section. In the case of solid-phase synthesis (SPPS), the C-terminal protecting group is an insoluble polymer. Amino protecting groups Table 6 lists the protecting groups most com- Side chain protecting groups monly used by Bachem for the α-amino group. The almost infi nite variety of the side chains They are cleaved with a variety of methods. of amino acids and their great signifi cance for Examples of Nα-protected amino acid the properties of the peptide has already been derivatives: mentioned. During peptide synthesis, some side Z-Leu-OH chain groups must be permanently protected. Boc-Ala-OH Fmoc-Phe-OH

15 Insights into Peptide Chemistry

Table 6: Common temporary amino protecting groups

Abbr. Chemical name Cleavage reagent Fmoc 9-Fluorenylmethoxycarbonyl Piperidine Boc t-Butoxycarbonyl Triﬂ uoroacetic acid Z Benzyloxycarbonyl Catalytic hydrogenation Hydrogen / palladium

Table 7: Common permanent acid protecting groups for the C-terminus and the side chain of Asp and Glu

Abbr. Chemical name Cleavage reagent OtBu t-Butylester Triﬂ uoroacetic acid OBzl Benzylester Catalytic hydrogenation Hydrogen / palladium OMe Methylester Bases (OMe used only for C-terminus)

Table 8: Examples of amino acid derivatives from the Bachem catalog

Derivative Use Standard derivative of arginine for Fmoc-SPPS, must be Fmoc-Arg(Pbf)-OH activated Standard derivative for Fmoc-SPPS, already activated Fmoc-Asn(Trt)-OPfp (“active ester”), can be used directly Z-Glu(OtBu)-OSu Derivative for solution synthesis, already activated ONLY for N-terminal serine! Nα and side chain protection Boc-Ser(tBu)-OH are cleaved under the same conditions

Table 9: Common side chain protecting groups Amino acid Protecting group Main use Arg Pbf, Pmc Fmoc-SPPS Arg (salt formation) Solution synthesis Asp, Glu OtBu Fmoc-SPPS, solution synthesis Asp, Glu OBzl Solution synthesis Asn, Gln Trt, Mtt Fmoc-SPPS Cys Trt Fmoc-SPPS, solution synthesis Cys Acm SPPS, solution synthesis His Trt Fmoc-SPPS Lys Boc Fmoc-SPPS, solution synthesis Lys Z Solution synthesis Ser tBu Fmoc-SPPS, solution synthesis Ser Bzl Solution synthesis Thr, Tyr tBu Fmoc-SPPS Trp Boc Fmoc-SPPS

16 Table 10: Meaning of the abbreviations for the side chain protecting groups

Abbr. Meaning Main use Acm Acetamidomethyl Cys, Fmoc-SPPS, solution synthesis Boc t-Butyloxycarbonyl Lys & Trp, Fmoc-SPPS tBu t-Butyl Ser, Thr & Tyr, Fmoc-SPPS Bzl Benzyl Ser (Thr, Tyr) solution synthesis OtBu t-Butyl ester Asp & Glu, Fmoc-SPPS OBzl Benzyl ester Asp & Glu, solution synthesis Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl Arg, Fmoc-SPPS Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl Arg, Fmoc-SPPS Trt Trityl (Triphenylmethyl) Cys, His, Asn & Gln, Fmoc-SPPS Z Benzyloxycarbonyl Lys, solution synthesis

Table 11: Comparison of methods – solution (or solution-phase) synthesis versus SPPS

Solution synthesis Solid-phase synthesis (SPPS) Reaction medium Solution Gel (swollen insoluble polymer) Batch size Any (especially large) Any (also very small) Rapidity of synthesis Slow Fast Automation Diffi cult to achieve Semi-automated or commercially available fully automated Synthesis strategy Generally convergent* Generally stepwise Temporary protecting Generally Boc or Z Fmoc (Fmoc-SPPS), group Boc (Boc-SPPS) Side chain protection Minimum Maximum Consumption of materials Moderate High (amino acid derivatives) Optimization Possible Possible Purifi cation + analysis of Usual Not possible intermediates Final purifi cation Relatively simple Laborious *convergent synthesis of longer peptides: fi rst of all, several fragments are synthesized that are combined at the end to the fi nal product. Convergent synthesis proceeds faster than a stepwise approach, because several fragments can be constructed at the same time. The combination “SPPS of protected fragments followed by coupling in solution” is also possible (e.g. synthesis of enfuvirtide).

Let us now look at the 20 proteinogenic amino Other side reactions occur with the side chain acid in more detail: functions of Ala, Leu and Phe have side chains that do not Cys react under the conditions of peptide synthesis. Ser, Thr, Tyr Likewise Ile, Pro and Val. Arg Other side chain functions can be modiﬁ ed His during peptide synthesis, if they are not blocked. Asn, Gln The carboxyl groups of Asp and Glu and the Trp amino group of Lys can take part in the coupling. Met (protection only in special cases) This produces a peptide with an incorrect link or a branched peptide.

17 Insights into Peptide Chemistry

The side chain functions of lysine and cysteine • Solution (or solution-phase) synthesis must be permanently blocked during peptide (“classic peptide synthesis”) assembly. For the rest of the amino acids carry- Table 11 shows the similarities and differences ing a third functionality the need for permanent of the two methods. side-chain protection depends on the method of synthesis and the peptide sequence. Solid-phase synthesis (SPPS) The most common side chain protecting groups Most peptides sold by Bachem or manufac- are listed in Table 9 and the abbreviations are tured as contract synthesis, are produced by explained in Table 10. During solution synthesis, SPPS. Solution synthesis is still chosen for the the side chains of Asn, Gln, His, Thr, Trp and Tyr synthesis of some of our peptide generics, for do not necessarily have to be protected. In the very short peptides (e.g. dipeptides) and for case of Arg, salt formation with a strong acid peptides modiﬁ ed at the C-terminus. such as HCl can sufﬁ ce. Solution synthesis The essential advantages of the solid-phase also does not make such high demands on the synthesis over the solution synthesis are its stability of protecting groups as SPPS. rapidity and ease of automation. As a general rule: However, it is often said that the larger the Amino acid derivatives for peptide synthesis batch, the slower the SPPS proceeds, because should be available in microcrystalline form the “manual work” increases. (no amorphous mass or oil) and thus readily Small and medium quantities of peptides can soluble. The Fmoc-derivatives should dissolve be manufactured in fully automated “synthe- readily and rapidly in DMF (or NMP); this is sizers” (Fig. 7). Very large quantities (several kg of important for fully automated Fmoc-SPPS. raw peptide) are produced in semi-automated machines (only the washing program still runs Methods of peptide synthesis automatically) or manually. Automation is possible, because the peptide is As already mentioned, there are two standard constructed in steps from the C-terminus to the methods of peptide synthesis: N-terminus. One amino acid is coupled after • Solid-phase synthesis (SPPS) another.

Fig. 7: Synthesizer, fully automated machine for solid phase peptide synthesis. The left picture shows reactors in which synthesis is already proceeding.

18 Table 12: Protecting groups for Fmoc-SPPS

Group Protecting group Cleavage reagent Stable against Nα Fmoc Piperidine Triﬂ uoroacetic acid Side chains Boc, tBu, OtBu, Triﬂ uoroacetic acid Piperidine Trt, Pbf (also released from the resin)

The process always involves four repeated steps or N-methylpyrrolidone (NMP), the standard 1. Cleavage of the temporary protecting group solvents of SPPS. 2. Washing The better the swelling, the more gel-like the 3. Coupling of the next protected amino acid beads and the faster the reaction occurs in the 4. Washing resin beads. One disadvantage of SPPS is that purifi cation Depending on the nature of the temporary Nα can only take place at the end of synthesis, after protecting group, a distinction is drawn between release of the peptide from the resin. In solution Fmoc- and Boc-SPPS. A switch Fmoc <-> Boc synthesis, partial sequences are fi rst construct- during synthesis is not possible. Boc- and ed and put together during the fi nal steps (see Fmoc-SPPS also differ in the choice of the side p. 17 and 21). chain protecting group. Cross-linked polystyrene is usually employed as Due to its great importance, only Fmoc-SPPS support material (resin) in SPPS. The polysty- will be discussed in detail here. Because of the rene is obtained in the form of beads. Bachem milder reaction conditions and hence better only uses polystyrene cross-linked with 1% divi- quality of the raw peptide, Fmoc-SPPS has nylbenzene (polystyrene-co-1% divinylbenzene) prevailed over the cheaper Boc-variant. as support. Two particle sizes are available for Wang resin (4-alkoxybenzyl alcohol resin) selection: and 2-chlorotrityl resin (2-chlorotrityl chlo- 200 – 400 mesh = 38 – 75 mm ride resin) are the resins most commonly 100 – 200 mesh = 75 – 150 mm used for the Fmoc-SPPS of peptides with (mesh: the beads resulting from polymeriza- a free C-terminus. Resin loading with the tion are separated by sieving into fractions of a carboxy-terminal Fmoc amino acid is not a certain particle size). simple matter and therefore Bachem offers Due to the cross-linking, the polystyrene beads a wide choice of loaded resins. The Fmoc are not soluble in any solvent, but in some, they group is split off (when using loaded swell to form a gel (see Fig. 8). What is wanted Wang resin) and the next Fmoc amino acid is good swelling in dimethylformamide (DMF) coupled. Completion of the coupling is

Fig. 8: Swollen resin beads under the Fig. 9: Incomplete coupling. microscope. The color shows unconverted amino groups.

19 Insights into Peptide Chemistry

Fmoc-Ser(tBu)-Wang-Resin

1. Piperidine/DMF Cleavage of Fmoc 2. Washing steps Piperidine must be completely removed

H-Ser(tBu)-Wang-Resin

3. Fmoc-Asp(OtBu)-OH Added in excess TBTU/DIPEA/DMF 4. Washing steps Complete ﬂ ushing out of the coupling reagents

Fmoc-Asp(OtBu)-Ser(tBu)-Wang-Resin

1. and 2. as above

H-Asp(OtBu)-Ser(tBu)-Wang-Resin

3. Fmoc-Gly-OH TBTU/DIPEA/DMF 4. Washing steps

Fmoc-Gly-Asp(OtBu)-Ser(tBu)-Wang-Resin

1. and 2. as above 3. Fmoc-Arg(Pbf)-OH TBTU/DIPEA/DMF 4. Washing steps

Fmoc-Arg(Pbf)-Gly-Asp(OtBu)-Ser(tBu)-Wang-Resin

1. and 2. as above 3. Fmoc-Asp(OtBu)-OH TBTU/DIPEA/DMF 4. Washing steps

Fmoc-Asp(OtBu)-Arg(Pbf)-Gly-Asp(OtBu)-Ser(tBu)-Wang-Resin

1. and 2. as above 3. Additional washing steps, drying

H-Asp(OtBu)-Arg(Pbf)-Gly-Asp(OtBu)-Ser(tBu)-Wang-Resin

Final cleavage 95% TFA

H-Asp-Arg-Gly-Asp-Ser-OH crude peptide

Fig. 10: Synthesis of H-3532 H-Asp-Arg-Gly-Asp-Ser-OH (DRGDS). TBTU (Product No. Q-1665) is a common coupling reagent. To activate the Fmoc amino acid with TBTU, an equivalent amount of base (DIPEA, diisopropylethylamine) must be added.

20 determined with color tests (positive: free Solution synthesis amino groups are “dyed”; negative: color- A solution synthesis requires careful plan- less, because there are no more free amino ning. There is a standard protocol for Fmoc- groups). SPPS that works for the synthesis of most Fig. 9 shows how the test appears under the peptides (although the quality of the raw microscope in the case of incomplete peptide can be poor), but no such protocol coupling. Since the peptide is cleaved from exists for solution synthesis. There is a far the resin with trifl uoroacetic acid at the greater variety with solution synthesis - not end of synthesis, the side chain protecting only for the possible protecting groups - but groups must be labile against this acid. The also of coupling methods, the solvents used protecting groups used are listed in Table and the preparative methods. 12. Not every conceivable protecting group combination works. Fig. 10 gives an example of an SPPS of an If an α-amino protecting group has been “RGD” peptide. specifi ed (Z, Boc, Fmoc…), then this also restricts the choice of side chain protecting A different resin such as Ramage resin groups, especially if one wants to remove all D-2200 or Rink amide AM-resin D-1675 is of them in one step at the end of synthesis. needed if a C-terminal peptidamide is to be The protecting group combination Fmoc/ synthesized. After cleavage of Fmoc from tBu is particularly effi cient and favored in the resin, the C-terminal Fmoc amino acid SPPS, because the groups can be selec- is coupled to it and then the procedure is tively cleaved and in any order. A compa- continued as described above. rable combination in solution synthesis is Resins such as 2-chlorotrityl resin or Boc/Bzl or Z/tBu, as shown in Table 13. SASRIN enable even fully protected peptide An example of a solution synthesis of a pep- fragments to be synthesized. tide modifi ed at the C-terminus (a “peptide

Boc-Ala-OH + H-Ala-Pro-OBzl

1. Coupling with DCC (a coupling reagent) 2. Removal of Bzl (hydrogenation)

Boc-Ala-Ala-Pro-OH

1. Coupling with DCC/base + H-Arg-pNA ∙2 HCl 2. Cleavage of Boc (Salt formation also acts as (with hydrochloric acid) side chain protection)

H-Ala-Ala-Pro-Arg-pNA · HCl

Reaction with succinic anhydride

Suc-Ala-Ala-Pro-Arg-pNA (sold as acetate salt)

Fig. 11: Synthesis of L-1720 Suc-Ala-Ala-Pro-Arg-pNA in solution.

21 Insights into Peptide Chemistry

Table 13: Protecting groups for solution synthesis Group Protecting group Cleavage reagent Stable against Nα Boc Trifl uoroacetic acid catalytic hydrogenation Side chains, possibly Bzl, OBzl, Z catalytic Trifl uoroacetic acid* C-terminal hydrogenation Nα Z catalytic Trifl uoroacetic acid* hydrogenation Side chains, possibly tBu, OtBu, Boc Trifl uoroacetic acid catalytic C-terminal hydrogenation C-terminal OMe** Bases Trifl uoroacetic acid, catalytic hydrogenation *Not completely stable against trifl uoroacetic acid, but suffi ciently stable for use in solution synthesis. **The C-terminal methyl ester is often used for moderate-length fragments. On treatment with the base hydrazine hydrate, the hydrazide is obtained from the methyl ester (…-Xaa-OMe →…Xaa-

NHNH2). This derivative can be “activated” (as azide) and coupled to another fragment with free N- terminus (“azide coupling”).

substrate”) is illustrated in Fig. 11. mostly used only for pre-purifi cation. The example also shows the benefi ts of the In the example in Fig. 11, Boc was used as modular principle: temporary Nα protecting group. Boc or Z The fragment Boc-Ala-Ala-Pro-OH is also are generally used in solution synthesis for used in the synthesis of other substrates temporary protection. such as L-1180 (Boc-Ala-Ala-Pro-Ala-pNA) Reactive “active esters” of protected amino and inhibitors such as N-1280 (MeOSuc- acids are also often used instead of a Ala-Ala-Pro-Ala-chloromethyl ketone). coupling reagent. Hydroxysuccinimide All intermediate steps are isolated, char- esters are popularly used in solution acterized and purifi ed if necessary. This synthesis (Bachem: OSu, Fig. 12). naturally takes time. However, frequently-needed fragments can Purifi cation of the raw product be synthesized in large quantities and held If the quality is poor, purifi cation of the raw in stock, so saving time again. peptide by preparative HPLC can be very Peptides that have been synthesized in time-consuming. Particularly with large solution are often purifi ed by countercur- quantities of peptide, the purifi cation can rent distribution (CCD). This method allows prove a bottleneck. to purify very large quantities, but it is With CCD and also with preparative HPLC,

Boc-Ala-OSu + H-Glu(OBzl)-OBzl

Boc-Ala-Glu(OBzl)-OBzl + HOSu insoluble in water, soluble in readily water-soluble organic solvents

EXTRACTION Cleavage of Boc...

Fig. 12: Solution synthesis, coupling with active ester.

22 the purifi ed peptide is obtained as a solution. The solvent is removed without stress- ing the product by freeze-drying (lyophilization) and a fi ne white powder is usually obtained. Water cannot ever be completely removed, but this is neither necessary nor worthwhile. If a peptide contains basic groups (N-terminus, Arg, Lys, His) it binds trifl uoroacetic acid (peptides obtained by Fmoc-SPPS) or acetic acid as salt. This cannot be removed. With few exceptions, our catalog peptides are available as trifl uoroacetate or acetate. Fig. 13: omega-Conotoxin MVIIA. Disulfi de bridges Source: PDB 1OMG Many peptides in the catalog have the ad- Kohno, T., Kim, J. I., Kobayashi, K., Kodera, Y., dition “Disulfi de bond” in the name. Such a Maeda, T. and Sato, K. bond is formed by the oxidation of the thiol Three-dimensional structure in solution of groups of two cysteines in the peptide the calcium channel blocker omega-conoto- chain. In the case of Fmoc-SPPS, this xin MVIIA. oxidation is carried out after cleavage from Biochemistry 34, 10256-10265 (1995) the resin, during which Trt is split off from Cys. Acm groups are cleaved with iodine and simultaneously oxidized yielding disulfi de bonds. A peptide can contain several disulfi de bridges. Disulfi de bridging and the correct linkage of the cysteine pairs (if there are several bridges) are extremely important for the biological activity of a peptide. Peptides do not exist as elongated chains, instead they form three-dimensional structures that are stabilized by disulfi de bridges. Examples are shown in Fig. 13 and Fig. 14.

• One disulﬁ de bridge can be found in Fig. 14: Human relaxin (a hormone, has a e.g. calcitonin, somatostatin, vasopressin, similar structure to insulin). oxytocin (natural peptide hormones); oct- Source: PDB 6RLX reotide, desmopressin (hormone analogs, Eigenbrot, C., Randal, M., Quan, C., Burnier, J., APIs) O‘Connell, L., Rinderknecht, E. and Kossia- koff, A. A. • Several disulﬁ de bridges are present in X-ray structure of human relaxin at 1.5 A. e.g. endothelin-1 (2), conotoxin (3), GaTx1, Comparison to insulin and implications for hepcidin-25 (4) (peptide hormones and receptor binding determinants. toxins) J. Mol. Biol. 221, 15-21 (1991)

• Two peptide chains, linked by disulﬁ de bridges: insulin, relaxins (peptide hormones)

23 Insights into Peptide Chemistry

Fig. 15: ADS of octreotide, a peptide also manufactured by Bachem as an API.

Fig. 16: ADS of Fmoc-Phe- OSu, an amino acid derivative.

Analysis of amino acids and peptides the product: an amino acid, a very short peptide or a peptide. SPPS, puriﬁ cation and lyophilization produce a white powder. This must now under- Identity go a detailed analysis (Quality Control, QC) Is it the desired product? to ﬁ nd answers to the following questions: If so, a measured value corresponds • Is it, in fact, the desired product? with that in the literature, or the value • How pure is the product? obtained with a reference. • What by-products does it contain? The following parameters are used to • How high is the product content? demonstrate identity: Our QC departments have a huge arsenal • Melting point of equipment and methods available to • Optical rotation [α] (see p.8)

analyze our products. The choice of analyt- • The Rf value is determined by thin layer ical method also depends on the nature of chromatography (TLC):

24 Fig. 17: Water content by Karl Fischer titration ≤ 7.0% Acetic acid content by HPLC 3.0 - 8.0% Assay by HPLC 95.0 - 105.0% Specifi cations (water- and acetic acid-free substance) Related substances by HPLC ≤ 0.5% D-Ser4-Triptorelin ≤ 0.5% D-Tyr5-Triptorelin and for the triptorelin GENERIC APIs Triptorelin free acid (combined peak) ≤ 0.5% D-Leu7-Triptorelin ≤ 0.5% each individual unknown acetate sold by report each individual unknown ≥ 0.10% ≤ 1.5% total . Heavy metals by ICP-MS ≤ 1 mg/kg palladium Bachem Residual organic solvents by GC ≤ 880 mg/kg DMF ≤ 5000 mg/kg n-butanol Bacterial endotoxins (Ph. Eur. 2.6.14, USP <85>) ≤ 10 IU/mg Microbial limit test (Ph. Eur. 2.6.12, USP <61>) Total aerobic microbial count (TAMC) ≤ 103 CFU/g (0.5 g tested) Total yeasts and moulds count (TYMC) ≤ 102 CFU/g (0.5 g tested) TRIPTORELIN ACETATE DMF Further Information Additional solubility information 1% in water: clear, colorless solution H-4075-GMP, 4008442 5% in water: clear, colorless solution 1% in 1% acetic acid: clear, colorless solution Triptorelin Acetate is a synthetic decapeptide LHRH agonist. Continuous administration results in a sustained decrease 5% in 1% acetic acid: clear, colorless solution of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion followed by a marked reduction of testicular 1% in PBS-buffer pH 7.2: not soluble and ovarian steroidogenesis. The suppressive effect on gonadal steroid concentrations is indicated for the treatment of 5% in PBS-buffer pH 7.2: not soluble hormone-dependent prostate cancer, endometriosis, central precocious puberty, and assisted conception. Handling and storage The product remains stable for at least 36 months when stored at ≤ -15°C or 2-8°C. Prolonged exposure to elevated temperatures (> 25°C) and light should be avoided. Amino acid sequence Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2, acetate salt International non-proprietary names Triptorelin Acetate (USAN) Active substance Triptorelin Acetate is a synthetic decapeptide analog of lutein- Triptorelin (INN; BAN) izing hormone-releasing hormone (LHRH or GnRH) with a higher (D-Trp6)-LHRH, acetate salt potency than the naturally occurring LHRH. Continuous administration of Triptorelin Acetate results, after a Molecular formula, relative molecular mass C64H82N18O13 · C2H4O2 1311.5 (net) + 60.1 (acetate) = 1371.6 (1) short initial stimulation, in a sustained decrease in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion with CAS-Number [140194-24-7] a consequent reduction of ovarian and testicular steroidogenesis. Bachem regulatory documentation Drug Master File (CTD) These effects are usually reversible after cessation of therapy. Fields of application Tests Specifications Treatment of prostate carcinoma Administration of Triptorelin Acetate leads to suppression of go- Appearance white to off-white powder nadotropins and a subsequent fall in peripheral circulating levels Appearance of solution clear, colorless to slightly colored solution in water of testosterone and dihydrotestosterone in male. In hormone- (10 mg/ml) dependent prostate carcinoma, a tumor remission or retardation of tumor progression is frequently observed after treatment with Identification by TLC complies Triptorelin Acetate. This is accompanied by an improvement of complies Identification by HPLC function and objective symptoms. Identification by amino acid analysis Arg 0.9 - 1.1 His 0.9 - 1.1 Ser 0.7 - 1.0 Management of endometriosis Continued administration of Triptorelin Acetate suppresses Glx 0.9 - 1.1 Leu 0.9 - 1.1 Trp detected estrogen secretion which results in a shrinking of the uterine and Gly 0.9 - 1.1 Pro 0.9 - 1.1 Tyr 0.9 - 1.1 ectopic endometrial tissue and in the relief from the symptoms of [α] 20 (1% in 1% acetic acid) = - 69.0 ± 3.0° Specific optical rotation D abdominal pain, dysmenorrhoea, and menorrhagia. (corrected for net assay) Treatment of central precocious puberty Triptorelin Acetate reduces the premature stimulation of gonado- tropin secretion in children with central precocious puberty and allows normal physiological growth and development. Normal pu- (1) Triptorelin Acetate exists essentially as the monoacetate equivalent to 4.4% acetic acid. The acetic acid is mainly bound to the arginine bertal development is resumed after discontinuation of Triptorelin residue and, to a lesser degree, bound to the histidine residue. Its level depends on the final freeze-drying process. Acetate therapy.

Rf value = ratio of the distance travelled by Purity the substance spot/solvent front. It Purity is determined by: depends on the solvent (mobile phase), • Thin layer chromatography (TLC) for amino

Rf always <1. acid derivatives, very short peptides, and • Co-elution with reference (TLC): biochemicals: Mixture of product with reference gives a TLC is often a valuable method, because single spot (and not two). reference material of potential impurities • The molecular weight is determined by e.g. the educts (reactants) can also be run at mass spectrometry. the same time and a large choice of detec- • The chemical composition of the com- tion methods is available. pound is determined by elemental analysis: • Analytical high performance liquid chro- The content of carbon, hydrogen and nitro- matography (analytical HPLC or the faster gen is measured and compared with the variant RSLC): values that can be calculated from the standard method for peptides.

molecular formula CxHyNzOwSv… Oxygen is • “Optical purity”: content of incorrect only determined in exceptional cases. Sulfur enantiomers (see p. 7). can also be determined in-house. An important value for amino acid deriva- • Spectroscopic methods: tives and loaded resins. IR, NMR, UV spectroscopy, for amino acid derivatives. Product content • Co-elution with reference (analytical In addition to impurities arising during HPLC): the synthesis, products may contain trace Mainly with peptides. amounts of residual solvents and/or water. • Amino acid composition: Peptides may also contain trifl uoroacetic Amino acid analysis (AAA), the peptide is acid or acetic acid that is fi rmly bound as cleaved into the individual amino acids with salt. strong acid, the resulting mixture is These compounds are not measured with separated by chromatography and the the methods used to determine the purity amino acids quantifi ed. The correctness of the desired product. In order to calculate of the amino acid sequence cannot be a reaction mixture for example, parameters checked with this method. such as the water content must be known.

25 Insights into Peptide Chemistry

• Water content: determined with Karl phosphorylation of Ser, Thr or Tyr) others are Fischer titration (KF-titration). “undesired”, or even indicators of patho- • Residual solvents: determined by gas logical processes. chromatography. Modifi cations such as N-terminal acetyl- • Acetic acid content: by HPLC or ion chro- ation or pyroglutamine formation and matography. C-terminal amidation also serve in nature • Residual trifl uoroacetic acid, for our API to stabilize peptides. “Blocked” peptides are peptides that are sold as acetate: by ion less rapidly broken down by enzymes. chromatography. Natural peptide hormones are often mod- • Titration with solutions of acids or bases: ifi ed at the end groups, e.g. gonadorelin, for products that are bases or acids. TRH. • Halide determination: the chloride or bro- The C-terminal amide group is generated by mide content can be determined by titration enzymatic degradation of a glycine residue, with silver nitrate solution for products that another enzyme catalyzes the cyclization of are sold as the corresponding salts. Gln to pyroglutamate (Pyr): • Nitrogen content (from elemental analysis): for peptides, this is a measure of H-Gln-Xaa-Yaa-…-Zaa-Gly-OH

the peptide content. → Pyr-Xaa-Yaa-…-Zaa-NH2

A selection of the results obtained can be Since the oxidation of methionine (see p. found on the ADS (Analytical Data Sheet) or 11) in peptides often leads to deactivation, CofA (Certifi cate of Analysis), that is made biologically active peptides are stabilized by available to the customer who is buying the the replacement of Met with the analo- product (see Figs. 15 and 16). gously structured, but non-oxidizable In the case of GMP products, microbiologi- norleucine (Nle). cal analyses are added that are carried out Chemical modifi cations also allow to fi x or in a specialized laboratory at Bachem (see change the spatial structure of a peptide. Fig. 17). The chemical synthesis of peptides offers For many of our generics, the analyses to many possibilities for modifi cation. The be undertaken are specifi ed by the Europe- N-terminus can be altered most easily: it an Pharmacopoeia*. “Ph. Eur.” in the name only requires one additional step in SPPS. signifi es that the drug substances conform Important N-terminal modifi cations to the specifi cations of the pharmacopoeia, • Acetylation e.g. Desmopressin Acetate Ph. Eur. • Biotinylation – biotin (= Vitamin B7) (many examples of acetylated and biotinylated peptides can be found in the cata- Modifi cation of peptides log). Biotinylated peptides bind specifi cally to the protein avidin. The modifi cation of a peptide means a per- • “Fluorophore” – Mca, Abz, FITC manent chemical alteration of the molecule, (The modifi ed peptides fl uoresce and are in contrast to the protecting groups that are detectable in the minutest amounts). removed after synthesis. The side chains of Cys and Lys can also be Peptides can be modifi ed in a great variety easily modifi ed and examples can be found of ways. in the catalog. In nature too, one fi nds a large number of H-Lys(biotinyl)-Lys-Glu-Asp-Val-Val-Abu-

peptide/protein modiﬁ cations. Some are Cys-Ser-Abu-Ser-Tyr-Lys-Lys-NH2 (M-2130) important for the bioactivity or function of the molecule (e.g. Pro in collagen is oxidized The peptide “backbone” can be modiﬁ ed by by enzymes to hydroxyproline; side chain the incorporation of special amino acids:

*A pharmacopoeia is a compilation of recognized pharmaceutical rules concerning the quality, purity, storage and naming of pharmaceuticals and the substances, materials and methods used in their manufacture and testing. The European Pharmacopoeia applies in Switzerland.

26 • D-amino acids An example of modifi cations: • N-methyl amino acids • Non-proteinogenic amino acids natural peptide (unusual amino acids) Leu-Enkephalin (Product No. H-2740) • Replacement of the peptide bond by other H-Tyr-Gly-Gly-Phe-Leu-OH bonds (e.g. reduced peptides) • Ring closure, e.g. disulfi de bridges slightly modifi ed (see p. 23) (3,5-Dibromo-Tyr1)-Leu-Enkephalin (Product No. H-2575) Peptidamides, the most important modi- H-3,5-Dibromo-Tyr-Gly-Gly-Phe-Leu-OH fi cation of the C-terminal carboxyl group Leu-Enkephalin amide (Product No. H-2745) are readily accessible by SPPS. They are H-Tyr-Gly-Gly-Phe-Leu-NH synthesized on specially developed resins 2 such as Ramage resin (D-2200). strongly modifi ed Much effort is required for other C-terminal DAMGO (Product No. H-2535) modifi cations. H-Tyr-D-Ala-Gly-N-Me-Phe-glycinol (D-Pen2,p-chloro-Phe4,D-Pen5)-Enkephalin (Product No. H-8875) H-Tyr-D-Pen-Gly-p-chloro-Phe-D-Pen-OH (disulfi de bond)

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restricted in certain countries. www.bachem. com shop.bachem.com December 2016 Group, Bachem Global Marketing, by 2011767 Published