Spps and side reactions in peptide synthesis

solid phase peptide synthesis

1. SPPS-SIDE REACTIONS IN PEPtIDE SYNTHESIS 1 Submitted by: K.KAVYA 1 st yr,M.pharmacy, Pharmaceutical chemistry.

2. CONTENTS: INTRODUCTION SOLID PHASE PEPTIDE SYNTHESIS HISTORY PLANNING OF SOLID PHASE SYNTHESIS APPLICATIONS ADVANTAGES DIS ADVANTAGES SIDE REACTION BY PROTON ABSTRACTION SIDE REACTION INITIATED BY PROTONATION SIDE REACTION BY OVERACTIVATION SIDE REACTIONS RELATED TO INDIVIDUAL AMINO ACID CONCLUSION REFERENCE 2

3. Abbreviations Boc - tert-butoxycarbonyl DMF- N,N-dimethylformamide Fmoc - 9-fluorenylmethoxycarbonyl HF-Hydrofluoric acid HHPSR- Hybrite Hydrophilic Polystyrene Resin SPPS - solid-phase peptide synthesis PEG - polystyrene glycol tBu -tert-butyl TFA-trifluoroacetic acid 3

4. INTRODUCTION Peptides are the sequence of amino acids which are formed by the condensation of two amino acids . In which a peptide bond is formed between a carbonyl group of one amino acid and the amino group of another amino acid . Peptides are synthesized in two ways they are 1.solid phase synthesis 2.solution phase synthesis 4

5. Solid Phase Synthesis Solution Phase Synthesis (Reagent) purification 5 Substrate (product) + (reagent ) (product )

6. SOLID PHASE PEPTIDE SYNTHESIS In chemistry solid-phase synthesis is a method in which molecules are bound on a bead and synthesized step-by-step in a reactant solution; compared [with] normal synthesis in a liquid state, it is easier to remove excess reactant or byproduct from the product. This method is used for the synthesis of peptides, deoxyribonucleic acid (DNA), and other molecules that need to be synthesized in a certain alignment. This method has also been used in drug discovery process. 6

7. Problems with Traditional Synthesis 1 chemist 1 molecule Can only make one molecule at a time Each synthesis very time consuming Purification of products very time-consuming between steps. Yields can be low and produces very few molecules at a time for testing 7

8. Solid Phase Peptide Synthesis Principle: 8

9. HISTORY 9 Solid phase synthesis was invented by Bruce Merrifield in 1963 and became very quickly the routine tool for preparation of peptides and later small proteins and earned him the Nobel Prize in 1984 .

10. Planning Of Solid Phase Synthesis The Resin (Solid Support) The Linkers The Protective groups The Reaction monitoring Purification Automation 10

11. Resin (Solid support) Resin act as a solid support for a solid phase synthesis The term solid support used to denote the matrix upon which chemical reaction is performed Solid support must be inert It always swell extensively in solvent. 11

12. Types of Resins Resins are two types they are: 1. Hydrophobic polystyrene resins 2. Hybrite Hydrophilic Polystyrene Resin (HHPSR) Hydrophobic polystyrene resins: Polystyrene resin beads under class Gelatinous solid support . Cross linked with 1-2% divinylbenzene. Particle size 90-200µm Used in large number of reaction sites. They are cheap & commercially available with any functional groups 12

13. Types of Hydrophobic polystyrene resins: 1. Benzylic halides – a) Merrifide resin. b) Trityl chloride resin. 2. Benzylic amines – a) Rink amide resin b) Amino Ethyle polystyrene 3. Benzylic alcohols – a) Wang resin 4. Aromatic aldehyde – a) Backbone amide linker b) Bal resin 13

14. Benzylic Halide Type Merrifield Resin Attachment - Carboxylic acid, Alcohol, Phenol, Cleavage - strong acidic condition Application - Peptide synthesis Trityl chloride Resin Attachment - Carboxylic acid, Alcohol, Amine. Cleavage - Mild acidic condition Application - Peptide synthesis, Alcohol & amine synthesis 14

15. 2.Benzylic Amine Type resin Attachment - Carboxylic acid , alcohol , Phenol. Cleavage – Mild acidic condition. Application - Peptide synthesis( Aryl & Alkyl carboxamide) 3. Benzylic alcohol Type Resin Attachment - Carboxylic acid. Cleavage – Acidic condition. Application - Peptide synthesis. 15 Rink amide resin Wang resin

16. 4.Aromatic Aldehyde Type Resin Attachment – Primary Amine. Cleavage – Mild acidic condition. Application - Peptide synthesis. 16 Bal resin

17. Hybrite Hydrophilic Polystyrene Resin (HHPSR): A major drawback of hydrophilic PS resin is poor swelling in protic solvent. Thus support is prepared by grafting hydrophilic monofunctional or bi-functional polystyrene glycol (PEG). Examples: ChemMatriex 1 It has chemical and thermal stability 2 Compatible with microwave 3 High degree of swelling in acetonitrile, DMF, TFA 4 Used for synthesis of difficult and long peptides. 17

18. THE LINKER Linker referred as a handle to attach the small molecule onto polymeric resin Linkers has many similarities to protecting group in solid phase synthesis. 18 Resin Linker X Attachment Resin Linker Molecule Resin Linker Molecule Cleavage Molecule

19. Properties of linkers which may assist Solid phase synthesis are: Stable to the reaction conditions Cleaved selectively at the end of synthesis Re-useable Facilitate reactions monitoring Sequential / Partial release Easy to prepared It should be highly selective to one or most small number of specific cleavage reagents/ conditions. 19

20. Types of Linkers Linkers are different types they are : 1 Acid-Cleavable Anchors and Linker: SPS of peptides was developed using this type of linkers 20 Acid labile, commercialy available SP Linker

21. 2.Base /Nucleophil-Liable linkers: The commonly used Fmoc peptide coupling protocols required Fmoc deprotection under basic conditions during synthesis. 21 Base/ Nucleophilic Laibale Linker

22. 3)Photo Labile Linkers: The light is used to break the bond between the intermediate and the linker and releases pure compound from the SPS into solution without interference from side produced . 22 photo labile SP Linker

23. 4.Safety-Catch Linkers: It is totally stable during the synthetic procedure & labile after a process known as activation. It is very popular and allows the support and release of many different functionalities. Examples : sulfonamide based safety catch linkers, phenol based safety linkers, acid labile safety catch linkers, imidazole safety linkers. 23 Slphonamide Based SCL Safety catch (SC) Linker

24. Examples of linkers: Ellman linker Trialkylsillyl chloride (or triflate ) generated from ethanophenylsane. Attachment of alcohols ketones 24 +anisole Function of linkers: 1) As a functional group 2) As a linker releases another functional group

25. PROTECTING GROUPS A species conjugated to a functional group that block the reactivity of that group. Good protective group are easily attached and removed using reaction. It mainly two types of protecting groups are used they are: Fmoc (9-fluorenylmethoxycarbonyl) t-Boc (tert-butoxycarbonyl) 25

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31. SPS procedure In this the stating molecule to an inert solid. Typically inert polymers or resin are used. These are commercially available. Solid are deep into solution containing subsequent reagent next they can be removed from the dip and placed into a ash solution.(Tea bags also used) After all reactions are done the product is still attached to the insoluble bead. Product can be washed in a reaction well, excess solvent is washed out . Finally, the product is cleaved from the bead and isolated. 31 Washing: After each synthetic step and prior to cleavage the solid support must be exhaustically washed with large vol. of solvent.

32. Purifications: purification is done by semi preprative HPLC utilizing new developments in column technology to cut down run times to less than 10 mins. 32

33. Solid phase synthesis strategy 33

34. Reaction monitoring Chromatography is the first monitor (i.e TLC). Non destructive methods such as infrared spectroscopy, Nuclear magnetic resonance are also used in reaction monitoring. 34

35. APPLICATION: Combinatorial synthesis of solid phase Peptide synthesis In DNA synthesis In various reactions Clainsan rearrangement Beckmann rearrangement Organic synthesis 35

36. ADVANTAGES Over all process is quick Purification of each product can be achieved in one step. Only purification technique is filtration. Purification in each step is possible. Synthetic intermediates don’t have to be isolated. Can be automated with Roberts. Use of solvent are minimized. Less health hazards and eco- friendly and hence follows the principle of GREEN CHEMISTRY. 36

37. DISADVANTAGES High chemical consume. Expensive. Low reaction rates. Special substances needed. It produces very few molecules at a time for testing. Solid phase synthesis without using any solvents. 37

38. SIDE REACTION IN PEPTIDE SYNTHESIS 38

39. SIDE REACTION BY PROTON ABSTRACTION Abstraction of acidic proton in presence of a base from carboxyl group results in carboxylate anion which prevents the formation of another anionic ester at α-carbon. Therefore, this anion prevents the elongation of peptide chain due to the absence of carboxyl group to form a peptide bond . This reaction in which glycine when treated by a base is taken as an example. 39

40. Racemization Mechanism of racemization was involved in different steps they are: a) Direct abstraction of α- proton: When an amino acid which is attached with a protecting group (Z), is treated with a base, proton abstraction occurs at α-carbon resulting in the formation of carbanion which can be attacked by any electrophile resulting in undesired reaction which changes the stereochemistry of the amino acid. 40 Racemization by direct abstraction of proton

41. b)By forming azlactones: The keto group of the amide bond undergoes ketoenol tautomerism to form a hydroxyl group which upon treatment with a base, abstracts a proton from the hydroxyl group resulting in formation of negatively charged oxygen. This initiates the activating group (X) to leave the carboxylic end. As a result, the carbon holding the keto group gets positive charge on it. the carbon now has deficient of electrons, and the oxygen with excess of electrons, a bond is formed between the carbon and the oxygen, and therefore forms an azlactone ring. 41 Racemization by forming of azlactone

42. Due to presence of unsaturation in the azlactone, and upon treating with a base leads to the abstraction of proton at α-carbon, which results in a total of three resonating structures . Therefore, the resonance stabilized structure forms the carbanion . Formation of azlactones is better explained by considering Benzoyl L-leucine-p-Nitrophenol, in which, the carboxyl and amino groups are protected by p-Nitrophenol and Benzoyl group respectively. 42 Resonating structures of azlactones

43. This when treated with a basic solvent (tertiary amine), it results in formation of azlactone. 43 Formation of azlactone in Benzoyl L-leucine-p-Nitrophenol

44. Most of the racemization occurs through azlactones only and rarely by direct abstraction . The Factors that affect racemization through azlactones are: i. Nature of amino acid involved ii. Solvent used in reaction iii. Presence of tertiary amine. When stability aspect of anion produced by proton abstraction through azlactones is considered, it is enhanced by electron withdrawing effects in acyl groups . methyl azlactone is less stable when compared to phenyl azlactone. This is so, as aryl azlactone has more resonating structures than alkyl azlactone. 44 Order of stability of azlactones

45. Cyclization: During peptide synthesis (solid phase), presence of benzyl ester can cause premature cleavage of the chain from insoluble support. The esters formed upon cleavage, undergoes cyclization to form diketopiperzines . This cyclic compound, when subjected to hydrolysis, leads to amide bond cleavage, as a result, the dipeptide is obtained with a different stereochemistry making it inactive 45

46. O – Acylation When an amino acid is treated with a base such as tertiary amine, it abstracts the proton and converts alcohols or phenols to alcoholates or phenolates. The formed alcoholate/phenolate, then reacts with an acylating agent and facilitates acylation at the electron rich oxygen atom. Since the acylation occurs at the nucleophile (O-), the reaction is named as OAcylation. 46

47. p-Hydroxy alanine (tyrosine) is treated with a tertiary amine which acts as proton abstractor, and also with p-Nitrophenyl ester, as a result, the acylated product p-Acyl oxy phenyl alanine (Acyl tyrosine) is formed along with pNitrophenol. 47 (a) Formation of tyrosine phenolate (b) Formation of carbocation (c) Acylation of Tyrosine

48. It can also occur in coupling reactions mediated by carbonyldiimidazole with alcohols when tertiary amine is absent. Since, imidazole acts as proton abstractor; it mediates the abstraction of proton from alcohols. 48

49. SIDE REACTION INITIATED BY PROTONATION Racemization : It is an acid catalyzed reaction involving protonation of carbonyl oxygen resulting in the formation of a carbocation . Proton abstraction then occurs at the adjacent carbon next to carbocation and therefore forms a double bond by sharing the electrons. This produces enolized products which do not retain their stereochemistry and lose their chiral purity . It requires a strong acid as protonation does not occur with weak acids. Racemization by protonation occurs during the process of deprotection of the groups using strong acids like HB or HF resulting in loss of chirality . 49

50. Racemization through protonation Cyclization: The products obtained by cyclization via protonation are same as that of products obtained by cyclization via proton abstraction. The only difference is that, former occurs in presence of acids where as latter occurs in the presence of the base . The mechanism is explained by taking dipeptide (Aspartyl glycine) of which carboxylic acid end of aspartic acid is protected by oxy benzyl group. In the resulting products, the protecting group leaves as hydroxy toluene and the dipeptide forms a cyclic molecule which is a succinamide derivative 50

51. 51 Cyclization by protonation

52. Alkylation: Formation of carbocation is the general step during the removal of protecting groups from amino acids in presence of an acid . These carbocations then act as alkylating agent to any nucleophilic centers and undergo intramolecular rearrangement to form the alkylated amino acid. 52

53. Sometimes, the carbocations formed also react with the solvent surrounding them and form a better alkylating agent and act by electrophilic substitution reaction. Alkylation in tyrosine occurs only at -ortho position to hydroxyl group and not at -meta position due to steric hindrance by the bulkiness of the amino acid skeleton . 53 Alkylation by electrophilic substitution

54. Chain Fragmentation: The amide bonds linking amino acids to each other to create the backbone of a peptide chains are stable enough to withstand the usual rigors of peptide synthesis. Under the influence of strong acids, an acyl group attached to the nitrogen atom of a serine residue migrates to its hydroxyl oxygen. Such an N O shift takes place also when the acyl group is a part of a peptide chain. This reaction, which in all likelihood proceeds via cyclic intermediates, is easily reversed by treating the product with aqueous sodium bicarbonate but partial hydrolysis of the sensitive ester bond will lead to fragmentation of the chain . 54

55. The reaction of the fragmentation in which a dipeptide (serine and alanine) forms cyclic intermediate in presence of acid followed by acyl group attached to the nitrogen atom of serine residue shift to its hydroxyl oxygen and its hydrolysis to form two different amino acids. 55

56. SIDE REACTION BY OVERACTIVATION Overactivation occurs in the process of acylation of amino acid where the carboxyl component is too powerful to be acylated. Therefore, acylation occurs primarily at the amino group which is exposed for peptide bond formation followed by acylation of hydroxyl group of the carboxylic component. Sometimes, during coupling of amino acids, using a coupling agent like N, N’- disubstituted carbodiimide, subtle intermediates are formed such as O-Acylisourea which give rise to symmetrical anhydrides and azlactones and can also undergo rearrangement to N-acylurea derivatives. 56

57. Reaction showing complete overactivation 57

58. Imidazole containing amino acids such as tryptophan react with carbodiimide and forms substituted guanidine and similar is the case with that of histidine. 58 Formation of substituted guanidine in (a) Tryptophan (b) Histidine

59. However, the O-Acylation or substituted guanidine side reaction that occurred can be revered by acid catalyzed methanolysis. 59

60. SIDE REACTIONS RELATED TO INDIVIDUAL AMINO ACID Amino acids with no functional side chains are not involved in side reactions which can be possible only in case of alanine and leucine as there is no side chain in alanine whereas in leucine, branching is at γ carbon which is far away from the α-carbon to undergo a side reaction. In case of valine and isoleucine, branching at β-carbon atom leads to steric hindrance which lowers the rate of coupling reaction and therefore, cause an increase in the extent of unimolecular side reactions. The condensation reaction with carbodiimide where isoleucine undergo a unimolecular side reaction (higher rate of reaction) by forming acylated intermediate (O-Acylisourea) followed by ureides. 60

61. 61 Formation of ureides (left) and peptide bond (right) from O - Acylisourea

62. β - Carbon branching also interferes with other reactions such as alkaline hydrolysis and hydrazinolysis of alkyl esters. Alkylcarbonic mixed anhydride, which is a second acylation product (urethane), is formed as a result of coupling of valine or isoleucine since the nucleophile has better chance to attack on the undesired carbonyl group. 62

63. This causes the reaction to occur at other position rather than the desired position. when isoleucine is treated with trimethyl acetyl chloride, alkylcarbonic mixed anhydride is formed which upon treatment with a primary amine, undergo hydrolysis at the undesired carbonyl group rather than the desired carbonyl group. This is because of the bulkiness of isoleucine that prevents the hydrolysis at first carbonyl carbon. 63

64. Despite forming alcoholates in presence of a base, alcoholic hydroxyls, under neutral conditions are reactive to undergo intramolecular reactions to get acylated at the carbodiimide activated carboxyl group and produce lactone which upon further treatment with another amino acid forms a peptide bond. Such reactions are seen in amino acids containing a hydroxyl group such as serine and threonine. 64

65. the reaction of threonine forming lactone followed by a peptide bond which is similar for serine as well. 65 In case of tyrosine, the phenolate ion formed after proton abstraction acts as an excellent nucleophile and gets acylated easily to form esters which can be later deacylated by treating with ammonia, hydrazine or hydroxylamine.

66. CONCLUSION Peptide synthesis involves different robust techniques . In sps is used in the peptide, DNA and combinatiorial synthesis. Solid phase synthesis is green chemistry. In spps temporary and permanent protecting groups are used. The side chains in the peptides are prone to side reactions which can degrade the amino acid or stop the peptide synthesis. 66

67. REFERENCE: 1.Introduction to medicinal chemistry 3 rd edition by Patric. 2.Solid phase synthesis & combinatorial technologics P.Seneci,Wiley 2000. 3.Organic synthesis on solid phase,F.Z.Dorwald Wiley-VCH2000. 4.Solid phase organic synthesis, A.R.Vanio, K.D.Janda, J.Comb. Chem. 2000. 5.Combinatorial peptide and non peptide libraries-A Handbook, Jung, G.Ed.VCH Weinheim,1996. 6. R.B. Merriffield , G. Barany, W.L. Cosand, M. Engelhard and S. Mojsov, pept. Am pept. Symp. 5 th edittion 1997, page no 48-55. 7. Bodanszky M, Kwei JZ. Side reactions in peptide synthesis. Chemical Biology & Drug Design. 1978; 12(2):69-74. 8.www.google.com 67

68. Thank You! 68