Macrocyclization of glucagon-like peptide (GLP-1) by thiol-ene reaction

Macrocyclization of peptides helps maintain a stable alpha helical structure using a conformational constraining staple. One such peptide, glucagon-like peptide-1 (GLP-1), is a 37 amino acid peptide agonist for the GLP-1 receptor and has the ability to lower blood glucose levels and is responsible for postprandial insulin release. Natural GLP-1 is quickly degraded by the proteases dipeptidyl peptidase-IV (DPP-IV) and endopeptidase (NEP) 24.11, and therefore, analogues have been researched for potential therapeutic treatmen ts of Type 2 Diabetes (T2D). Stapling peptides increases the stability of the alpha helical conformation, native to peptides such as GLP-1. Some stapling techniques include a ring-closing olefin metathesis (RCM) and formation of a lactam bridge between side chains or the peptide backbone in a i, i+4 manner. An alternative macrocyclization technique is the two component thiolene reaction. This reaction does not use unnatural amino acids or toxic metal catalysts, making this reaction useful under a variety of conditions. Additionally, since this process does not require post synthetic modification, the peptides has the potential to use a wide varieties of linkers to accomplish cyclization. This project determined and applied a new method to staple analogues in GLP-1 with cysteine residues to carry out the thiol-ene reaction for peptide cyclization. GLP-1 mono stapling was achieved for three GLP-1 cysteine substituted analogues and two staples were accomplished for one GLP-1 cysteine substituted analogue.

MACROCYCLIZATION OF GLUCAGON-LIKE PEPTIDE (GLP-1) BY THIOL-ENE REACTION by Rudi Zurbuchen A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Chemistry Approved: ______________________________ Danny Hung-Chieh Chou, Ph. D. Thesis Faculty Supervisor _____________________________ Dr. Cynthia Burrows Chair, Department of Chemistry _______________________________ Dr. Thomas Richmond Honors Faculty Advisor _____________________________ Sylvia D. Torti, PhD Dean, Honors College May 2016 Copyright © 2016 All Rights Reserved ABSTRACT Macrocyclization of peptides helps maintain a stable alpha helical structure using a conformational constraining staple. One such peptide, glucagon-like peptide-1 (GLP-1), is a 37 amino acid peptide agonist for the GLP-1 receptor and has the ability to lower blood glucose levels and is responsible for postprandial insulin release. Natural GLP-1 is quickly degraded by the proteases dipeptidyl peptidase-IV (DPP-IV) and endopeptidase (NEP) 24.11, and therefore, analogues have been researched for potential therapeutic treatments of Type 2 Diabetes (T2D). Stapling peptides increases the stability of the alpha helical conformation, native to peptides such as GLP-1. Some stapling techniques include a ring-closing olefin metathesis (RCM) and formation of a lactam bridge between side chains or the peptide backbone in a i, i+4 manner. An alternative macrocyclization technique is the two component thiol-ene reaction. This reaction does not use unnatural amino acids or toxic metal catalysts, making this reaction useful under a variety of conditions. Additionally, since this process does not require post synthetic modification, the peptides has the potential to use a wide varieties of linkers to accomplish cyclization. This project determined and applied a new method to staple analogues in GLP-1 with cysteine residues to carry out the thiol-ene reaction for peptide cyclization. GLP-1 2 mono stapling was achieved for three GLP-1 cysteine substituted analogues and two staples were accomplished for one GLP-1 cysteine substituted analogue. 3 TABLE OF CONTENTS ABSTRACT 2 INTRODUCTION 5 GLUCAGON-LIKE PEPTIDE (GLP-1) 9 STAPLING TECHNIQUES 9 THIOL-ENE REACTION 11 METHODS 14 PEPTIDE SYNTHESIS 14 PURIFICATION 15 THIOL-ENE REACTION 16 RESULTS 18 DISCUSSION AND CONCLUSION 25 REFERENCES 28 4 INTRODUCTION A. GLUCAGON-LIKE PEPTIDE-1 (GLP-1) Glucagon-like peptide-1 (GLP-1) is a 37 amino acid endogenous incretin secreted from L cells naturally found in the human body. This peptide plays a crucial role in glucose homeostasis and has been shown to have antidiabetic functions1, as it plays a central role for controlling postprandial blood sugar levels2. GLP-1 promotes insulin secretion, in effect, lowering the blood glucose levels as well as stimulating glycogenolysis and gluconeogenesis3. The negative attribute of GLP-1 is short lived activity, inhibiting its full usefulness as a therapeutic agent for type 2 diabetes (T2D). The protease dipeptidyl peptidase IV (DPP-IV) cleaves GLP-1 between Ala8-Glu9. Neutral endopeptidase 24.11 (NEP 24.11), a membrane-bound zinc metallopeptidase4, cleaves GLP-1 between Asp15Val16, Ser18-Tyr19, Tyr19-Leu20, Glu27-Phe28, Phe28-Ile29, Trp31-Leu32. All of these cleavages cause a large decrease in agonistic activity1. GLP-1 and the similar GIP are both hormones, which together, are responsible for up to 60% of postprandial insulin release, an important factor for diseases such as diabetes2. They both exhibit the incretin effect where beta cells increase secretory response2 and have similar glucose turnover rate5. GLP-1 specifically is active in vivo and the two truncated products, GLP-1(7-37) and GLP-1(7-36)amide, are recognized by GLP-1 receptors in pancreatic beta cells. Both of these factors are the reason behind 5 research for therapeutics for T2D as GLP-1 plays a central role for controlling postprandial blood sugar levels2. Peptides Peptide Sequence GLP-1 HAEGTFTSDVSSYCLEGQAAKEFIAWLVKGR Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT Exendin-4 HGEGTFTSDLSKQMEEAVRLFIEWLKNGGPSSGAPPPS GIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Table 1: Sequences of GLP-1, Glucagon, Exendin-4, and GIP with the similar amino acid residues to GLP-1 in blue. Type 2 diabetes is commonly attributed to a steady decline of beta cell function when there is high glucotoxicity, or high exposure to glucose. The development of T2D is possibly secondary to loss of the insulinotropic effect5, and there is little to no incretin effect6. For glucose turnover rates, the effects are similar for GLP-1 and GIP, however, GIP is less potent than GLP-1. When T2D patients were exposed to near normalized blood glucose levels for 4 weeks, the beta cell responsiveness increased by a factor of 3 to 45. There are two important aspects of the amino acid sequence: the receptor binding and the receptor activating. Manandahar and Ahn found that residues His7, Gly10, Phe12, Thr13, Asp15, Phe28, and Ile29 were crucial for receptor binding and substitution of these residues with Ala resulted in loss of affinity1. His7, Gly10, Asp15, and Phe28 were 6 important for receptor activation. For activation to the receptor, His at position 1 is critical and sensitive to stimulation3. The structure of GLP-1 has often been compared to exendin-4 as the two ligands are 50% identical, which is shown in Table 1. Both GLP-1 and exendin-4 have a larger N-terminal extracellular ligand binding domain7 and definite alpha helicity from residue 7 to 288. GLP-1 has a glycine at residue 16, giving the peptide increased flexibility but is also destabilizing. An NMR study yielded a distortion in the alpha helix at residue 15 to 17, consistent with the glycine residue8. The glucagon-like peptide-1 receptor is a class B receptor. These type of receptors are seven membrane-spanning alpha helices9. The C-terminal domain is intracellular and interacts with the G-protein. The larger N-terminal domain is the extracellular domain (ECD) and is involved in ligand binding. The specific GLP-1 receptor has been shown to ligand bind with both GLP-1 and GIP. When these ligands bind, the alpha helical structure is more defined. This is because the receptors share a common secretion family recognition fold, which helps bind ligands and facilitate the fold into an alpha helix9. For GLP-1 specifically, the face interactions with the ECD is conserved for both the agonist and antagonist bound forms. The hydrophobic interactions are with Ala 24, Ala25, Phe28, Ile29, Leu 32, Val 33. Additionally, the C-terminus is hydrophobic and the Nterminus is amphiphilic7. Currently research for diabetic therapeutics involves long-acting GLP-1. Native GLP-1 would require 24-hour administration due to the short duration of the active 7 forms. In order to decrease the amount of injections a patient with T2D would need to take, current medications involving long acting GLP-1 receptor agonists are required. This drug class lowers the risk of hypoglycemia in a glucose-dependent mechanism, as well as leads to weight loss and reduced cardiovascular risk10. One current analog is Liraglutide, whose sequence is 97% similar to GLP-1; the difference highlighted in Figure 1 with an Arg34 and a Glu residue at Lys26 allowing for the palmitoyl group to attach. Liraglutide, a currently stable FDA-approved GLP-1 agonist10, remains active longer than 24-hours, maintaining the affinity for the GLP-1 receptor. After injection, the palmitoyl group binds to albumin non-covalently11 allowing Liraglutide to escape glomerular filtration and increases stability6. In vivo, the peptide is still cleaved by DPPIV and NEP 24.11, however, when albumin binds, the cleavage sites are less accessible and a majority of the peptide is intact in plasma11. 8 Figure: 1 The sequences of GLP-1 (7-37) and Liraglutide with differences at Lys34 and the Glu and C-16 fatty acid branch off Lys26 . In addition to diabetic therapeutics, highly stable GLP-1 analogues would be useful in developing molecular probes to image pancreatic 𝛽𝛽-cells10. Manandhar and Ahn found GLP-1 analogues aided in the restoration of pancreatic 𝛽𝛽-cell mass and functions in rodent, but was not ready for consideration of human clinical trials1. B. STAPLING TECHNIQUES Synthesized peptides in an alpha helical conformation can acquire additional stability through the introduction of a cross link or staple. Staples typically occur by 9 reacting with the amino acids on the peptide chain in the i, i+4 positions, which constitutes one turn of the helix, with another smaller peptide12. These staples can be covalent as well as noncovalent. Blackwell and Grubbs is one such research group which successfully used a ring-closing olefin metathesis (RCM) to increase the alpha helix stability12. Other macrocyclization techniques have been implemented such as the 1,3-dipolar cycloaddition between azide and alkyne and leucine rich tetra-, penta-, hexa-, and heptapeptides. Other techniques use metals ions as a catalyst, such as lithium salts, sodium ions, and cesium ions to achieve the stapled product. Catalysis by copper (I) under mild conditions give regioselectivity and a 1,4-disubstitution pattern. The nucleophilic catalyst imidazole has been used for aminolysis of the peptide to yield a thioester. Another stapling technique by Crich and Saski involves using an SNAr reaction with Sanger’s reagent, giving a reactive thioester which is attacked by the amino group on the N-terminus13. Besides additional stability, an advantage of stapling the peptide is to increase protein-protein interaction (PPI). Constrained peptides, whether through the use of salt bridges, chelating metal ion clips, or covalent linkages, produce a more pronounced helical shape of peptides, which is beneficial for PPI14 and the cellular functions they control. Another advantage of stapling is to produce high affinity for receptor binding for the desired protein in vitro15. 10 C. THIOL-ENE REACTION The thiol-ene reaction has been around for a century. Yet, recently, it is being considered a reaction in “click chemistry” because of the wide scope use potential, quantitative yields, ability to change the starting material, and the byproducts of the reaction can easily be removed. The thiol-ene reaction can exist in many conditions, such as strong acids and bases, as well as oxidizing and reducing reagents, and usually reacts very quickly16. There are two mechanisms for thiol-ene reaction: one uses a free-radical addition and the other is a catalyze thiol Michael addition17. The radical mechanism is photochemically and thermally induced and this reaction has orthogonality with many functional groups. After subjection of the reaction solution to ultraviolet light, the reaction yields a cross linking product of multifunctional thiols18. The high quality of the thiol-ene reaction makes it appealing for research in any biomedical fields. Figure 2: General thiol-ene reaction using a free-radical addition. Another successful and well known click chemical reaction is the Copper (I)catalyzed azide-alkyne cycloaddition (CuAAC)18. The advantage of the thiol-ene reaction 11 is it does not use a transition-metal catalyst, such as copper, which can be toxic and harmful. For this reason, the thiol-ene click chemistry reaction is currently being used to make biomaterials in medicine and dentistry, and other bioorganic chemistry research, such as a new anti-tumor vaccination18. Macrocyclization of peptides were successfully accomplished by Aimetti and coworkers on-resin. They incorporated the natural amino acid cysteine into the peptide sequence so there were no post-synthetic modifications that were needed. They found the reaction to be versatile and rapid, while having the possibility to use different alkenes. Additionally, this reaction had very high yield and allowed for site-specific conjugation19. The limit for previous thiol-ene coupling with unnatural amino acids is the type of linker which can be used. Since native proteins typically have few, if any, cysteines, substituting them into a peptide sequence provides excellent selectivity. Previously in Danny Chou’s lab, a successful two-component thiol-ene stapling was achieved for unprotected peptides20. Using seven different dienes with the same peptide, a thiol-ene i, i+4 staple was achieved with high yields and similar structure as macrocyclization using the RCM reaction. Similar yields were also accomplished with staples of i, i+7 which the stapled peptide had a significant increase in helical structures20. 12 Figure 3: The two component thiol-ene cyclization of unprotected peptides and seven different linkers20. Initially, the reaction was run in the solvent DMF, however the conversion was 65%. The solvent was switched to NMP, which increased the conversion of the reaction to 90%. This reaction also had functional groups with high tolerance and were chemioselective to the thiol group, producing the desired staple. Additionally, this reaction did not require any unnatural amino acid, which require solid-phase peptide synthesis and recombinant DNA when there are more than 50 amino acids in the peptide chain20. Due to the successful conversion rates of this two-component thiol-ene stapling for different length of peptides with different linkers, this reaction has the potential to apply to other peptide chains, such as GLP-1, as a way for macrocyclization. 13 14 METHODS Ahn and coworkers were able to synthesize a conformationally constrained GLP1 analogue through the introduction of lactam bridges of Lysi-Glui+4 and Glui-Lysi+4 in both the N and C terminal region to create additional stability in environments with proteases. This stapling not only enhanced the stability of the alpha helical structure but also showed resistance to NEP 24.11, giving the GLP-1 analogue a half life of more than 96 hours10. An advantage of peptide synthesis for the thiol-ene reaction only involves Cys rather than both Lys and Glu, enhancing the selectivity for the reaction. Additionally, peptides have Lys and Glu in their original sequence, whereas Cys are not common in peptide sequences, such as GLP-1, which contains none. This project synthesized all needed peptides, including wild type GLP-1, GLP-1 analogues, and linking peptide. Purification of these peptides was obtained by High Performance Liquid Chromatography (HPLC) and monitoring the progression of the thiol-ene reaction and characterization of the products was done by Liquid Chromatography-Mass Spectrum (LC/MS). A. PEPTIDE SYNTHESIS The peptides were synthesized on a Rink amide ChemMatrix resin on a 0.100 mmol scale. Alstra, Biotage, Inc. peptide synthesizer was used to synthesize the peptides in a 10 mL reaction vial. The chain was made using Fmoc amino acids and diluting in DMF to give a concentration of 0.4 M. The peptide chain was deprotected with 595.2 mL 15 of 20% piperidine in DMF. The chain was coupled with 44.2 mL of 0.4 M HATU and 37.0 mL of 1M DIEA. HATU, DIEA, and piperidine were mixed with the amino acid for 5 minutes at 75˚C, except for cysteine and histidine, which were mixed for 10 minutes at 50˚C. Double coupling of the arginine amino acid was applied to all peptides. At the final amino acid, deprotection with piperidine was performed again and a pre cleavage wash with DCM was applied. The finished peptide and resin were washed with DCM. They were dried with vacuum for 20 minutes20. The sequences for GLP-1 wild type and the alpha-Cys substitutions can be found in Table 2. Peptide Name Sequence 1 GLP-1 (7-37)-NH2 H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 2 [C18,C22]GLP-1 (737)-NH2 H-HAEGTFTSDVSCYLECQAAKEFIAWLVKGRG-NH2 3 [C22,C26]GLP-1 (737)-NH2 H-HAEGTFTSDVSSYLECQAACEFIAWLVKGRG-NH2 4 [C30,C34]GLP-1 (737)-NH2 H-HAEGTFTSDVSSYLEGQAAKEFICWLVCGRG-NH2 5 [C18,C20,C30,C34]GLP1 (7-37)-NH2 H-HAEGTFTSDVSCYLECQAAKEFICWLVCGRG-NH2 Table 2: GLP-1 wild type and GLP-1 analogue sequences with Cys substitutions in red. B. PEPTIDE CLEAVAGE The peptide resin was split evenly and transferred to two cleavage vessels. A cleavage cocktail was made with 400µL TIS, 400 µL water, 400 µL EDT, and 14.8 mL 16 TFA for all peptides containing cysteine. For peptides without cysteine, the cocktail was 400µL TIS, 400 µL water, and 14.8 mL TFA. 8 mL of the cleavage cocktail was added to each of the cleavage vessels in the hood and allowed to cleave in a rotator for 3 hours. After 3 hours, the solution and peptide was filtered into ~40-45 mL of ice-color ethyl ether. A white precipitation formed and the ethyl ether and precipitant were transferred to a freezer (-20˚C) overnight. The two vessels were transferred to a centrifuge and spun down for 10 minutes at 3225 RCF. The ethyl ether and other liquids were removed, leaving a white solid pellet. The pellet was washed with ~10-15 mL of ice cold ethyl ether and was transferred to centrifuge to spin. The pellet was washed twice. After the final wash, the Falcon tubes were covered with the Kimtech kimwipe and secured with a rubber band. The tube was dried in a vacuum for >3 hours. The dried peptide was analyzed by LC/MS20. C. PURIFICATION The crude peptides were purified in by HPLC Agilent. 50 mg of the dried peptide were dissolved in 5 mL of DI water and gas was removed by centrifugation. The peptide was injected at a flow rate of 3.0 mL/min with a 0.1% TFA in water/acetonitrile gradient. All fractions containing the purified peptides were characterized by LC/MS. Fractions containing the desired product were collected, frozen to solid, and lyophilized. D. THIOL-ENE REACTION 17 For the reaction, 5 mg of an alpha-Cys substituted peptide (2-5), 5 equivalents of DPAP and 3 equivalents of DTT were added and dissolved in 400 µL of NMP. 3 equivalents of Octa-1,7-diene was added to the mixture and subjected to 365 nm UV light and stirred for 15 minutes20. For purification of the thiol-ene product, the solution was diluted with 3 mL of distilled deionized water and washed once with 5 mL of ethyl acetate. The solution was centrifuged for 5 minutes and the organic phase was removed. The aqueous phase was submitted for reverse-phase HPLC under the same conditions for peptide purification. The product was characterized by LC/MS, frozen to solid, and lyophilized. 18 RESULTS Since GLP-1 does not contain any natural cysteines, the synthesized proteins were mutated in specific residues to yield thiols for the reaction. Two and four cysteines were successfully introduced for the thiol-ene reaction to give one and two thiol ether bridges, respectfully. Completion of the thiol-ene reaction was monitored by the use of mass spectroscopy/liquid chromatography (MS/LC). The sequences for GLP-1 and synthesized analogues are as follows: GLP-1 (7-37)-NH2: H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 [C18,C22]GLP-1 (7-37)-NH2: H-HAEGTFTSDVSCYLECQAAKEFIAWLVKGRG-NH2 [C22,C26]GLP-1 (7-37)-NH2: H-HAEGTFTSDVSSYLECQAACEFIAWLVKGRG-NH2 [C30,C34]GLP-1 (7-37)-NH2: H-HAEGTFTSDVSSYLEGQAAKEFICWLVCGRG-NH2 [C18,C20,C30,C34]GLP-1 (7-37)-NH2: H-HAEGTFTSDVSCYLECQAAKEFICWLVCGRG-NH2 The corresponding MS, LC, and HPLC spectrum for each peptide, both unstapled and stapled, are depicted in Figure 4-Figure 26. Figure 4: GLP-1 wild type with title peaks of 839.5 (M+4H)4+, 1119.0 (M+3H)3+, and 1678.3 (M+2H)2+. 19 Figure 5: GLP-1 wild type with retention time of 6.219 minutes. Figure 6: GLP-1 (18,22) Thiol-ene Product MS with title peaks of 882.5 (M+4H)4+ and 1176.5 (M+3H)3+. Figure 7: GLP-1 (18,22) Thiol-ene Product LC. 20 Figure 8: GLP-1 (22,26) MS with title peaks of 844.8 (M+4H)4+, 1125.9 (M+3H)3+, and 1688.8 (M+2H)2+. Figure 9: GLP-1 (22,26) LC with a retention time of 6.457 minutes. Figure 10: GLP-1 (22,26) Thiol-ene Product MS with title peaks of 872.2 (M+4H)4+, 1162.7 (M+3H)3+, and 1743.8 (M+2H)2+. Figure 11: GLP-1 (22,26) Thiol-ene Product LC with a retention time of 6.747. 21 Figure 12: GLP-1 (30,34) MS with title peaks of 841.4 (M+4H)4+, 1121.3 (M+3H)3+, 1680.0 (M+2H)2+. Figure 13: GLP-1 (30, 34) LC with a retention time of 6.443. Figure 14: GLP-1 (30, 34) Thiol-ene Product MS with title peaks of 868.6 (M+4H)4+, 1168 (M+3H)3+, 1736.8 (M+2H)2+. 22 Figure 15: GLP-1 (30, 34) Thiol-ene Product LC with a retention time of 6.564. Figure 16: GLP-1 (18, 22, 30, 34) MS with title peaks of 1141.9 (M+3H)3+ and 1712.3 (M+2H)2+. Figure 17 : GLP-1 (18, 22, 30, 34) LC with a retention time of 6.579 minutes. 23 Figure 18: GLP-1 (18, 22, 30, 34) Thiol-ene Product MS with title peaks of 912.0 (M+4H)4+, 1215.6 (M+3H)3+, 1822.8 (M+2H)2+, demonstrating two staples were achieved. All reactions successfully yielded a stapled product. The purification technique once the peptide was synthesized or the thiol-ene product was formed, was HPLC. Figure 19: GLP-1 Wild Type HPLC with a retention time of 17 and 18 minutes. Figure 20: GLP-1 (18,22) HPLC with a retention time of 21 and 22 minutes. Figure 21: GLP-1 (18,22) Thiol-ene Product HPLC with a retention time of 25 minutes. Figure 22: GLP-1 (22, 26) HPLC with a retention time of 17 and 18 minutes. 24 Figure 23: GLP-1 (22,26) Thiol-ene Product HPLC with a retention time of 24 minutes. Figure 24: GLP-1(30-34) HPLC with a retention time of 15 and 16 minutes. Figure 25: GLP-1(30-34) Thiol-ene Product HPLC with a retention time of 20 minutes. Figure 26: GLP-1 (18, 22, 30, 34) HPLC with a retention time of 20 minutes. 25 DISCUSSION AND CONCLUSION The radical thiol-ene reaction successfully stapled octa-1,7-diene to peptides containing both two cysteines as well as four cysteines. The conditions for optimal conversion of the stapled product resulted when 5 equivalents of the catalyst DPAP was added to the reaction instead of 3 equivalents. Additionally, 15 minutes in UV light of 365 nm was sufficient for maximum conversion and minimal side products. Figure 27: Thiol-ene coupling reaction example between the GLP-1 (18,22) Cys analogue and Octa-1,7-diene. Complications arose in obtaining enough solid product after two purifications, one for the peptide after synthesis and another after the thiol-ene reaction. To avoid this, the thiol-ene reaction was run without purification of newly synthesized peptide and the conversion rate was low. Therefore, both purification steps were necessary. 26 In conclusion, a new methodology of using the thiol-ene reaction for stapling and macrocyclization of glucagon-like peptide-1 (GLP-1) was successful. Additionally, only cysteine mutants of GLP-1 were used as starting material and therefore no unnatural amino acids were needed. Two staples on the same tetra-cysteine mutant of GLP-1 were successfully achieved. Since Cys is in low abundance in naturally occurring peptides, they are preferred for amino acid substitution. For future research, a GLP-1 receptor activation assay will be performed in order to determine the activity of the GLP-1 analogues and the thiol-ene products, as well as duration of activity. Since GLP-1 enhances cyclic AMP (cAMP), the accumulated cAMP will be isolated and the EC50 value will be calculated to determine activity. Since the short half life of GLP-1 due to the degradation by DPP-IV and NEP 24.11, each GLP-1 analogue and thiol-ene product should subsequently be tested for protease resistance. The peptides will be incubated with recombinant human DPP-IV enzyme and NEP 24.11 enzyme and following incubations will be analyzed via HPLC to obtain peak areas necessary for determination of intact and degraded peptides alike. 27 Figure 28: The thiol-ene coupling reaction in aqueous solvent with the linker 1,3-di(but3-en-1-yl)urea20. Since this reaction can be accomplished in NMP, different linkers should be reacted with the GLP-1 analogues to determine which linker can be carried out in water. Successful stapling of unprotected peptides with substituted cysteines were accomplished in an aqueous solvent by using the linker 1,3-di(but-3-en-1-yl)urea as shown in Figure 28. This technique should be used to approach thiol-ene coupling with GLP-1 analogues. 28 REFERENCES 1. Manandhar, B.; Ahn, J. J. Med. 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