Large Scale Synthesis of Native Turn and Helix Mimics Stabilized by a Generic Hydrogen Bond Surrogate

Short Turn and Helix mimics frequently represent molecular recognition surfaces perturbing bio-relevant protein-biomolecular interfaces. Generic methods that can stabilize short peptides into turns or helices by retaining all recognition elements, have tremendous applications in drug discovery. Here, a versatile modular synthetic protocol is presented for stabilizing turns and helices of different sizes by replacing their ring-closing hydrogen bonds with a generic three-carbon covalent surrogate. Two Fukuyama-Mitsunobu reactions insert the surrogate between desired residues in high yields and purity. Coupling with turn size-dependent oligopeptides containing both sterically restricted and non-coded residues, followed by macrolactamization yield a variety of stabilized turns. Short peptide extensions at the C-terminus of these turns yield stabilized 3 10 -helices and α-helices. This solution-phase synthetic approach provides combinatorial access to libraries of stabilized turns and helices with all their native residues retained, in >100 mmole scales, in about one week per stabilized mimic, to technicians with postgraduate level training.

The hydrogen bond surrogate (HBS) strategy, where the putative i+4ài peptide hydrogen bond is replaced by a covalent surrogate, was employed initially by Satterthwait and co-workers 43 and then by Arora and co-workers 44 .The former used a hydrazone surrogate while the latter used ole n, alkane, thioether and disul de surrogates.The ole n model has been most successful among these in mimicking the α-helix 44 and the π-helical turn 45 .In these models, the i+1 st residue of the turn is also replaced, apart from the hydrogen bond.Broussy and co-workers 46 , Alewood and co-workers 47 and Prabhakaran and co-workers 48 developed the propane, substituted-propane and an additional exocyclic sp² hybridization of i+1 st residue nitrogen respectively, as HBS models.Here, exclusively the hydrogen bond is replaced by the HBS model and all residues in the HBS-constrained turn are retained.The sp²-constrained HBS model, termed the 13 7 3 model 48 , has yielded the highest known α-helicities for single turns in tetrapeptides 48 .It has been used to decipher the presence of a synergetic guidance mechanism for ultra-fast helix growth during helix folding 49 , following the relatively slow helix nucleation step 50,51 .It is the only HBS model to mimic the 3 10 -helical turn 52 .Moreover, we have also shown that our HBS model has also been applied in studying dynamic β-sheet structures 53 .Thus, this HBS model is a generic replacement for H-bonds in a variety of turns (Figure 1).This protocol details the procedures for the large-scale solution-phase synthesis of β-turn, 3 10 -helical, αhelical and larger turn peptidomimetics constrained by the generic propyl HBS model, containing the exocyclic sp² hybridization.Two Fukuyama-Mitsunobu reactions 54 place all elements of the HBS between any two desired residues, with great facility and in high yields.Two salting-out processes isolate two crucial intermediates from triphenylphosphine (PPh 3 ) and triphenylphosphine oxide (TPPO) in high purity, without column chromatography.A macrolactamization process yields solution-phase synthetic access to any HBS-constrained turn structure.This follows our recent demonstration of a trimodular synthetic protocol for the large (>100 mmoles) scale access to single turn α-helices and their extended structures, in the laboratory 55 .Current protocol gives facile, generic, large-scale access to a variety of HBS turns, while retaining all residues in the turns.

Experimental design-
Our protocol has been demonstrated to replace the i+nài H-bonding interaction closing ordered turns, where n=2, 3, 4, but is potentially applicable for any value of n.These HBS turns employ inexpensive standard synthons and protected amino acids used for the solution-phase synthesis of peptides.Any HBS turn is divided into two modules, which are synthesized independently and coupled together.The rst module is common to all turns and has the propyl HBS linker connecting the nitrogens of two amino esters -one N-Boc and CO2Me protected and the other N-Moc and CO2Bn protected (7) (Figure 2).Each of the two N-alkylations is e ciently enabled through a Fukuyama-Mitsunobu (FM) reaction on N-onitrosulphonyl (Ns) amino esters (1 eq.) in the presence of diisopropyl azodicarboxylate (DIAD, 1.5 eq.), triphenylphosphine (PPh3, 1.5 eq.) and alcohol (2 eq.), in tetrahydrofuran (THF); followed by denosylation in the presence of benzene thiol (PhSH, 1.1 -1.5 eq) and potassium carbonate (K2CO3, 2.0 eq.) in acetonitrile (ACN); then acidi cation with 1N HCl; followed by washing of the PPh3 and triphenylphosphine oxide (TPPO) with diethyl ether (DEE) to get the ammonium chloride salt in high purity (HPLC); followed by protection of the amines as N-Boc (4) ( rst amine) and N-Moc (7) (the second).The ether wash is a salient feature that precludes cumbersome repetitive column chromatographic puri cations which are otherwise essential after every FM reaction, to remove PPh3 and TPPO.The second module is a simple N-Cbz-protected peptide carboxylic acid-containing n-1 residues, with basetolerant, acid-sensitive, but not hydrogenation-sensitive side-chain protecting groups.Synthesis of both these modules have been demonstrated in >100 mmole scale in our standard academic laboratory and is amenable to much larger industrial scales.
Boc deprotection of the HBS linked rst module (7) in the presence of tri uoroacetic acid (TFA) in dichloromethane (DCM), followed by peptide coupling with C-terminus of the second module in the presence of ethyl chloroformate (ECF, 1.1 eq.) N-methyl morpholine (NMM, 4.0 eq.) in THF to get the precursor for the macrolactamization (9), followed by reductive double deprotection of N-Cbz and O-Bn groups in the presence of 1% Palladium on Carbon (Pd/C) in methanol (MeOH), followed by macrolactamization cyclization of the resulting amino acid (10) in the presence of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), 1-hydroxy benzotriazole (HOBT), diisopropylethylamine (DIPEA) in 1 mM ACN, yields the desired HBS turn (11) in good yields (30-65%) depending on the ring size and sequence.Cyclization scales are limited only by the size of vessel available for 1 mM concentrations.Thus, a combinatorial library of HBS turns can be prepared from a variety of the two modules.
A C-terminal exocyclic methyl ester group (-CO2Me) is presented in all these HBS turns (10), for extension of sequence beyond the HBS turn, through methyl ester deprotection in the presence of LiOH in MeOH:H2O, followed by acidi cation with 1 N HCl and coupling with the desired peptide.The C-terminal extension peptide can be synthesized independently and its coupling with the HBS turns can be achieved in either solution or solid phase.The N-Moc protecting group remarkably remains unperturbed under standard (acidic, basic and hydrogenation) deprotection conditions but can be cleaved in 40% HBr in acetic acid.The conditions, reagents and their quantities in each step are optimized for fast, cheap, largescale synthesis and facile isolation of the desired intermediates and products and to eliminate competing undesired byproduct formation.Together, isolated HBS turns and C-terminal extended HBS turns are both accessed with high e ciency and functional group tolerance through this trimodular synthetic protocol.

Applications of the method
Current HBS design is such that all native residues within H-bond closed turns, and hence their molecular recognition elements, are uniquely retained in the HBS turn analogues.The presence of sp2 hybridization at N-Moc nitrogen, in addition to two sp2 hybridized atoms presented per peptide bond in the constrained turn sequence, provides su cient Thorpe-Ingold effect for e cient cyclization of even 16-membered rings.HBS turns containing unnatural and sterically hindered residues (D-amino acid, Aib = αaminoisobutyric acid) and tertiary amide bonds (proline), have also been synthesized with high e ciency, which shows potential for accessing even larger HBS turns.Delineation of the HBS turn from the Cterminal extension sequence during synthesis allows combinatorial access to variants of both HBS turn and their extended analogues.The highest known thermal and chemical stabilities and ellipticities have been achieved for a variety of these HBS turns48, 52, 55.Both these are important features with applications during the identi cation of drug leads.EQUIPMENT SETUP Analytical HPLC Analytical reversed-phase HPLC was performed on a Shimadzu (Shimadzu Scienti c Instruments, Inc., USA) instrument using an analytical column (Supelco, 250 × 4.6 mm, 5 μm).The two solvent systems water (A) (0.06% TFA) and acetonitrile (B) (0.05% TFA) were used to elute all the peptides.The ow rates were xed at 1.0 mL/min (analytical).The corresponding peptide loading was set up to a maximum of 5 mg/injection.The HPLC chromatograms were acquired at a xed wavelength of 214 nm for all the peptides.
2. Seal with rubber septum and wrap with para lm.Evacuate it by high vacuum using an oil pump for 2 min via an inlet needle (0.8 x 25 mm).
3. Back ll the ask with N 2 balloon through a needle (0.8 x 25 mm).
∆ CRITICAL STEP The dry THF should be freshly prepared.We found that the reaction is moisture sensitive, so dryness should be maintained.Keep the N 2 balloon connected to the RBF through a needle (0.8 x 25 mm).
5. Stir the reaction mixture at -15 o C for the next 1:30 h.∆ CRITICAL STEP We strongly recommend that the temperature should be maintained at -15 o C because it stabilizes the in situ generated mixed anhydride.
6. Subsequently, seal the RBF with rubber septum and wrap with para lm of H 2 N-N(Trt)-OMe and repeat step 2 and 3 twice.Make a solution by addition of 10 ml of dry THF via syringe equipped with aspiration needle (20 Gaze x 12 Inch).
10. Immediately add 2.6 ml of NMM (23.6 mmol, 2.5 eq.) in a disposable syringe equipped with a stainless-steel needle (0.55 x 25 mm) over a period of 30 sec.Let the reaction mixture stir at -15 o C for 1.5 h.
∆ CRITICAL STEP We advise the checking of TLC (50% vol/vol ethyl acetate : pet.Ether (Petroleum ether)) for the full consumption of acid in the form of corresponding ester.The formation of mixed anhydride is very fast and generally, it takes 3 -4 min.
∆ CRITICAL STEP The addition of NMM is exothermic in nature.Maintain the temperature throughout the reaction course at -15 o C. We recommend monitor the reaction progress by TLC (70% vol/vol ethyl acetate : pet.ether) and HRMS.R f of Cbz-S(tBu)N(Trt)-OMe = 0.4 in EtOAc/pet.ether (70% (vol/vol)), R f of Cbz-S(tBu)-OH = 0.0 in EtOAc/pet.ether (70% (vol/vol)).For HRMS, take 1mg of the crude product in 1.5 ml Eppendorf vial and submit for mass spectrum.Reaction is complete when TLC and HRMS show no traces of Cbz-S(tBu)-OH.
11. Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).
12. Quench the reaction by the addition of few drops of double-distilled water, the solution turns from turbid to clear.
13. Remove the THF by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C. 14.Next transfer the reaction mixture after dissolving in 20-30 ml ethylacetate (EtOAc) in 100 ml separatory funnel and add 1N HCl (20 ml) slowly (10ml / 30 sec).Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more EtOAc until there is (~10 ml) clear separation.
15. separate the aqueous layer and add saturated sodium bicarbonate (10 ml) to the organic layer.Seal the separatory funnel again with polyethylene cap, shake the reaction mixture and settle down for 2 min until two layers are separated out.16. separate the aqueous layer and pass the organic layer through the crystalline sodium sulphate to remove the residual amount of water and collect in 100 ml one neck RBF. 17. Repeat step 14 -16 twice with EtOAc (2 x 15 ml) and discard the aqueous layer.
∆ CRITICAL STEP It is essential to remove all the NMM.HCl salt and unreacted NMM, otherwise it interferes with the compound during the column.saturated sodium bicarbonate removes the unreacted Cbz-S(tBu)-N(Trt)-OH.
! CAUTION The HCl fume is very corrosive and irritates eyes and throats.Carry out this step inside fume hood.
18. Remove the organic portion in rotary evaporator to obtain a transparent liquid compound, set the water bath temperature to 45 o C and vacuum pressure gradient 400 -800 mbar.
21. Directly transfer the dry slurry to the column using a plastic funnel (D -55 mm).
23. Collect the fraction in 25 x 150 mm test tubes.Using the TLC, identify the fractions containing the desired product and visualize it with UV lamp (254 nm).
24. Combine the fractions containing the desired product in 500 ml RBF and remove the solvent by rotary evaporator, set the water bath temperature to 48 o C. Dry the compound using oil vacuum pump (10 mbar) to obtain a white solid compound in 65% yield (4.0 g).
□ PAUSE POINT This compound (Cbz-S(tBu)N(Trt)-OMe) is bench stable at room temperature for several months without appearance of any noticeable impurity on TLC. 17. separate the aqueous layer and add saturated sodium bicarbonate (5 ml) to the organic layer.Seal the separatory funnel again with polyethylene cap, shake the reaction mixture and settle down for 2 min until two layers are separated out.
18. separate the aqueous layer and pass the organic layer through the crystalline sodium sulphate to remove the residual amount of water and collect in 100 ml one neck RBF.Remove the DCM in high vacuum (100 mbar) to obtain a dry slurry.
23. Directly transfer the dry slurry to the column using a plastic funnel (D -55 mm).
25. Collect the fraction in 18 x 125 mm test tubes.Using the TLC, identify the fractions containing the desired product and visualize it with UV lamp (254 nm).
26. Combine the fractions containing the desired product in 100 ml RBF and remove the solvent by rotary evaporator, set the water bath temperature to 48 o C. Dry the compound using oil vacuum pump (10 mbar) to obtain a white solid compound in 85% yield (0.89 g).
□ PAUSE POINT This compound (Boc-AR(Z 2 )-IPr) is bench stable at room temperature for several months without the appearance of any noticeable impurity on TLC.BOX 3. Colorimetric detection of synthesized molecules at various steps.
Here we describe the general procedures for the qualitative estimation of the starting material and the product at different steps.2): Transfer 10 g of Ns-G-OMe (36.5 mmol, 1 eq.) and 14.3 g of triphenylphosphine (PPh 3 ) (54.7 mmol, 1.5 eq.) in oven-dried 250 ml RBF containing a Te on-coated magnetic stir bar.
2. Seal with rubber septum and wrap with para lm.Evacuate it by high vacuum using an oil pump for 2 min via an inlet needle (0.8 x 25 mm).
3. Back ll the ask with N 2 balloon through a needle (0.8 x 25 mm).
∆ CRITICAL STEP The dry THF should be freshly prepared.We found that the reaction is very moisture sensitive, so dryness should be maintained properly.5. Put the RBF into the ice bath to maintain the temperature of the reaction mixture 0 o C for the next 1 h and allow the reaction mixture to stir until everything gets dissolved.
∆ CRITICAL STEP The diol amount should be exactly two equivalents to suppress the dialkylation formation.More than 2 eq.doesn't give full consumption of starting material.7. Add 10.8 ml of diisopropyl azodicarboxylate (DIAD) (54.7 mmol, 1.5 eq.) in a disposable syringe equipped with a stainless-steel needle (0.55 x 25 mm) dropwise over a period of 5 min.Let the reaction mixture be stirred at room temperature for 1 h.
∆ CRITICAL STEP The addition of DIAD is exothermic.Maintain the temperature throughout the reaction course.We recommend monitoring the reaction progress by TLC (50% vol/vol ethyl acetate : pet.ether) and HRMS.R f of Ns-G-OMe = 0.6 in EtOAc/pet.ether (50% (vol/vol)), R f of N-(Ns-propyl alcohol)-G-OMe (2) = 0.1 in EtOAc/pet.ether (50% (vol/vol)).For HRMS, take 1mg of the crude product in 1.5 ml Eppendorf vial and submit for mass spectroscopic spectrum.Reaction is complete when TLC and HRMS show no traces of Ns-G-OMe.!CAUTION The HCl fume is very corrosive and irritates eyes and throats.Carry out this step inside a fume hood.
∆ CRITICAL STEP HCl forms ammonium salt of denosylated product and makes it water-soluble.18.Next transfer the reaction mixture after dissolving in 10 ml diethyl ether (Et 2 O) in 100 ml separatory funnel.Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more Et 2 O until there is (~10 ml) clear separation (Figure 5).∆ CRITICAL STEP Et 2 O dissolves undesired organic impurities (triphenylphosphine oxide, excess triphenylphosphine, and excess 1,3-propanediol), the desired compound remains dissolved in the aqueous layer.
19. Separate the aqueous layer and discard the organic layer.Repeat step 18 several times until TLC (ethyl acetate/pet.ether (80% (vol/vol))) con rms the complete removal of the impurities.
∆ CRITICAL STEP Use TLC (ethyl acetate/pet.ether (80% (vol/vol))) and 1 H NMR to check the purity of the product.20.Afterward collect the aqueous portion containing 3 in 250 ml RBF, keep it in ice bath (0 o C) and basify using sodium bicarbonate until pH reaches 11 -12.Check the pH using pH paper.21.Subsequently, take 9.2 ml of Di-tert-butyl decarbonate ((Boc) 2 O) using 20 ml disposable syringe equipped with aspiration needle (20 Gaze x 12 Inch) in another 100 ml pear shape.Pour 60 ml of THF using 100 ml measuring cylinder.25.Next transfer the reaction mixture after dissolving in 30 ml EtOAc in 100 ml separatory funnel.Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more EtOAc until there is (~10 ml) clear separation.26.Separate the aqueous layer and pass the organic layer through the crystalline sodium sulphate to remove the residual amount of water and collect in 100 ml one neck RBF.Extract the aqueous part twice with EtOAc (2 x 20 ml) and discard the aqueous layer.
27. Evaporate the organic portion in rotary evaporator to obtain a transparent liquid compound, set the water bath temperature to 45 o C and vacuum pressure gradient 400 -800 mbar.
28. Dissolve the residue in a minimum amount of DCM (5 ml) and add 10 -15 g silica gel (100 -200 mesh).Remove the DCM in high vacuum (100 mbar) to obtain a dry slurry.
29. Pack the chromatography column (L -300 mm, D -43 mm) with dry silica gel (100 -200 mesh).30.Directly transfer the dry slurry to the column using a plastic funnel (D -55 mm) 31.Elute the desired product using EtOAc/ pet.ether (40% vol/vol) by gravity column (rate -400 ml/1h) (Table 3).32.Combine all the fractions in 25 x 150 mm test tubes.Using the TLC, identify the fractions containing the desired product and visualize by ninhydrin test.33.Combine the fractions containing the desired product in 500 ml RBF and remove the solvent by rotary evaporator, set the water bath temperature to 48 o C. Dry the compound using oil vacuum pump (10 mbar)   to obtain the compound as yellowish oil in 58% yield (5.2 g).
□ PAUSE POINT This compound (N-(Boc-propyl alcohol)-G-OMe, 4) is stable at room temperature, can be stored in a pear-shaped ask wrapped with para lm for several months without appearance of any noticeable impurity on TLC.
36.Back ll the ask with N 2 balloon through a needle (0.8 x 25 mm).Repeat steps 35 and 36 twice.
∆ CRITICAL STEP The dry THF should be freshly prepared.We found that the reaction is very moisture sensitive, so dryness should be maintained properly.38.Put the round RBF into the ice bath to maintain the temperature of the reaction mixture -15 o C for the next 30 min and allow the reaction mixture to stir until everything gets dissolved.
39. Add 3.91 ml of diisopropyl azodicarboxylate (DIAD) (19.8 mmol, 1.5 eq.) in a disposable syringe equipped with a stainless-steel needle (0.55 x 25 mm) dropwise over a period of 3 min.Let the reaction mixture be stirred at room temperature for 45 min.
∆ CRITICAL STEP The addition of DIAD is exothermic in nature.Maintain the temperature throughout the reaction course.We recommend monitoring the reaction progress by TLC (50% vol/vol ethyl acetate : pet.ether) and HRMS.R f of N-(Boc-propyl alcohol)-G-OMe (4) = 0.5 in EtOAc/pet.ether (50% (vol/vol)), R f of N-(Boc-propyl alcohol)-G-OMe-N-(Ns-propyl alcohol)-A-OBn (5) = 0.4 in EtOAc/pet.ether (50% (vol/vol)).For HRMS, take 1mg of the crude product in 1.5 ml Eppendorf vial and submit for mass spectrum.Reaction is complete when TLC and HRMS show no traces of N-(Boc-propyl alcohol)-G-OMe (4).51.Separate the aqueous layer and discard the organic layer.Extract the aqueous portion with diethyl ether (each time 10 ml) several times until TLC (ethyl acetate/pet.ether (80% (vol/vol))) con rms the complete removal of the impurities.
∆ CRITICAL STEP Use TLC (ethyl acetate/pet.ether (80% (vol/vol))) and 1 H NMR to check the purity of the compound.Purity is important for the next step.52.Afterward collect the aqueous portion in 250 ml RBF, keep it in ice bath (0 o C) and basify using sodium bicarbonate until pH reaches 11 -12.Check the pH using pH paper.53.Subsequently, take 2.56 ml of methoxycarbonyl chloride using 20 ml disposable syringe equipped with an aspiration needle (20 Gaze x 12 Inch) in another 100 ml pear shape.Pour 20 ml of THF.62. Directly transfer the dry slurry to the column using a plastic funnel (D -55 mm).
64. Combine all the fractions in 25 x 150 mm test tubes.Using the TLC, identify the fractions containing the desired product and visualize by ninhydrin test.
65. Combine the fractions containing the desired product in 500 ml RBF and remove the solvent by rotary evaporator, set the water bath temperature to 48 o C. Dry the compound using oil vacuum pump (10 mbar) to obtain the compound as yellowish oil in 90% yield (5.5 g).

□ PAUSE POINT This compound (N-(Boc-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OBn, 7
) is stable at room temperature, can be stored in a pear-shaped ask wrapped with para lm for several months without appearance of any noticeable impurity on TLC.!CAUTION As TFA is very corrosive, rotary evaporator is avoided to remove the TFA.Use a short path distillation set up to remove the TFA (Figure 6).∆ CRITICAL STEP The dry THF should be freshly prepared.We found that the reaction is very moisture sensitive, so dryness should be maintained properly.Keep the N 2 balloon attach to the RBF.74.Subsequently take 6.72 g of Cbz-S(tBu)N(Trt)-OH (10.3 mmol, 1.2 eq.) in oven-dried 50 ml RBF.75.Seal the RBF with rubber septum and wrap with para lm.Evacuate it by high vacuum using an oil pump for 5 min via an inlet needle (0.8 x 25 mm).76.Back ll the ask with N 2 balloon through a needle (0.8 x 25 mm).Repeat steps 74 and 75 twice.77.Add 20 ml dry THF via 20 ml syringe equipped with aspiration needle (20 Gaze x 12 Inch).78.Stir the reaction mixture at -15 o C for the next 1 h.∆ CRITICAL STEP We strongly recommend that the temperature should be maintained at -15oC because it stabilizes the in situ generated mixed anhydride.79.In the solution of Cbz-S(tBu)N(Trt)-OH, add 12.9 ml of N-methyl morpholine (NMM) (12.9 mmol, 1.5 eq.) in a disposable syringe equipped with a stainless-steel needle (0.55 x 25 mm).80. Next add 0.9 ml of Ethyl chloroformate (ECF) (9.46 mmol, 1.1 eq.) in a disposable syringe equipped with a stainless-steel needle (0.55 x 25 mm).Reaction mixture turns clear to turbid.85.Next transfer the reaction mixture after dissolving in 20-30 ml ethylacetate (EtOAc) in 100 ml separatory funnel and add 1N HCl (20 ml) slowly (10ml / 30 sec).Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more EtOAc until there is (~10 ml) clear separation.86.Separate the aqueous layer and add saturated sodium bicarbonate (10 ml) to the organic layer.Seal the separatory funnel again with polyethylene cap, shake the reaction mixture and settle down for 2 min until two layers are separated out.87.Separate the aqueous layer and pass the organic layer through the crystalline sodium sulphate to remove the residual amount of water and collect in 100 ml one neck RBF.92.Directly transfer the dry slurry to the column using a plastic funnel (D -55 mm).93.Elute the desired product using EtOAc/pet.ether (60% vol/vol) ether by gravity column (rate -400 ml/1h) (Table 5).119.Directly transfer the dry slurry to the column using a plastic funnel (D -55 mm).120.Elute the desired product using MeOH/EtOAc (2% vol/vol) by gravity column (rate -400 ml/1h) (Table 6).129.Next transfer the reaction mixture along with 20 ml DCM in 50 ml separatory funnel.Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more DCM until there is (~10 ml) clear separation.130. Separate the aqueous layer and pass the organic layer through the crystalline sodium sulphate to remove the residual amount of water and collect in 50 ml one neck RBF.152.Directly transfer the dry slurry to the column using a plastic funnel (D -55 mm).

19 .
Repeat steps 16 -18 twice with EtOAc (2 x 10 ml) and discard the aqueous layer.∆ CRITICAL STEP It is essential to remove all the NMM.HCl salt and unreacted NMM, otherwise it interferes with the compound during the column.saturated sodium bicarbonate removes the unreacted Boc-A-OH.!CAUTION The HCl fume is very corrosive and irritates eyes and throats.Carry out this step inside a fume hood.20.Remove the organic portion in rotary evaporator to obtain a white solid compound, set the water bath temperature to 48 o C and vacuum pressure gradient 400 -800 mbar.21.Dissolve the residue in minimum amount of DCM (2 ml) and add 1 g silica gel (100 -200 mesh).
(A) Ninhydrin test Reagents Dissolve 50 mg of ninhydrin in 500 ml of ethanol (1% w/v) Procedure (i) Perform analytical TLC on pre-coated TLC plates with silica gel 60 F 254 .(ii) Spray the 1% ninhydrin solution on the TLC plate.(iii) Heat the TLC plate on a hot plate (120 o C) for 30 sec !CAUTION Ninhydrin forms brown mark upon exposure to skin.Wear gloves during the test.(B) Iodine test Reagents Put solid I 2 in a wide-mouth glass jar with a lid.Procedure (i) Perform analytical TLC on pre-coated TLC plates with silica gel 60 F 254 .(ii) Heat the TLC plate on a hot plate (120 o C) for 30 sec (iii) Keep the TLC plate into iodine chamber for 5 mins !CAUTION I 2 is allergic to skin, causes burns.Keep the I 2 chamber in the well-vented fume hood, wear gloves during the experiment.(C) UV absorption test Procedure (i) Perform analytical TLC on pre-coated TLC plates with silica gel 60 F 254 .(ii) visualize the TLC plate under the UV (254 nm) chamber.!CAUTION Do not expose hands on the UV chamber for a long time (usually check the TLC within 15 -20 sec).Wear UV-protected glass during the test.Synthesis of N-(Boc-propyl alcohol)-G-OMe (4) • TIMING 15 h 1. Synthesis of N-(Ns-propyl alcohol)-G-OMe (

8 .
Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).9. Quench the reaction by the addition of few drops of double-distilled water.10.Remove the THF by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C. 11.Keep the RBF in the rotary evaporator for an extra 10 -15 min to remove the residual amount of water.∆ CRITICAL STEP We advise doing the next denosylation reaction without any further column puri cation.At this step, column puri cation is tedious because triphenylphosphine oxide (TPPO) eluates with the desired compound as an impurity.During the denosylation reaction, triphenylphosphine oxide doesn't interfere.12. Synthesis of N-(HCl-propyl alcohol)-G-OMe (3): Add 125 ml of ACN, start stirring at room temperature.13.Add 10.1 g of potassium carbonate (73 mmol, 1 eq.)14.Then add 5.6 ml of thiophenol (54.8 mmol, 1.5 eq.) and let the reaction mixture be stirred for 18 h.The colour of the solution turns transparent to yellow-orange turbid.∆ CRITICAL STEP Monitor the reaction progress by TLC (80% vol/vol ethyl acetate : pet.ether).R f of N-(Ns-propyl alcohol)-G-OMe (2) = 0.5 in EtOAc/pet.ether (80% (vol/vol)), R f of N-(HCl-propyl alcohol)-G-OMe (3) = 0.1 in EtOAc/pet.ether (80% (vol/vol)).Reaction is complete when TLC shows no traces of N-(Ns-propyl alcohol)-G-OMe (2).15.Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).16.Remove the acetonitrile by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C. 17. Afterward acidify the reaction mixture under ice bath (0 o C) with 3N HCl until the pH reaches 2 -3.

22 .
Transfer the (Boc) 2 O solution to the reaction mixture of step -13 using Pasteur pipette while stirring the reaction mixture.After 30 min remove the ice bath and allow the reaction mixture to stir for 12 h.∆ CRITICAL STEP Monitor the reaction progress by TLC (50% vol/vol ethyl acetate : pet.ether) and HRMS.R f of N-(HCl-propyl alcohol)-G-OMe (3) = 0.0 in EtOAc/pet.ether (50% (vol/vol)), R f of N-(Bocpropyl alcohol)-G-OMe (4) = 0.5 in EtOAc/pet.ether (50% (vol/vol)).For HRMS, take 1mg of the crude product in 1.5 ml Eppendorf vial and submit for mass spectrum.Reaction is complete when TLC and HRMS show no traces of N-(HCl-propyl alcohol)-G-OMe (3).23.Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).24.Upon completion, remove the THF by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C.

40 .
Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).41.Quench the reaction by the addition of few drops of double-distilled water.42.Upon completion, remove the THF by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C. 43.Keep the RBF in the rotary evaporator for an extra 10 -15 min to remove the residual amount of water.44.Add 44 ml of ACN, start stirring at room temperature.∆ CRITICAL STEP We advise doing the next denosylation reaction without any further column puri cation.At this step, column puri cation is tedious because of TPPO eluates with the desired compound as an impurity.During the denosylation reaction, triphenylphosphine oxide doesn't interfere.45.Add 10.1 g of potassium carbonate (26.4 mmol, 2 eq.) 46.Then add 2.03 ml of thiophenol (19.8 mmol, 1.5 eq.) and let the reaction mixture be stirred for 8 h.The colour of the solution turns transparent to yellow-orange turbid.∆ CRITICAL STEP Monitor the reaction progress by TLC (80% vol/vol ethyl acetate : pet.ether).R f of N-(Boc-propyl alcohol)-G-OMe-N-(Ns-propyl alcohol)-A-OBn (5) = 0.4 in EtOAc/pet.ether (50% (vol/vol)), R f of N-(Boc-propyl alcohol)-G-OMe-N-(H-propyl alcohol)-A-OBn (6) = 0.1 in EtOAc/pet.ether (50% (vol/vol)).

47 .
Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).48.Upon completion, remove the acetonitrile by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C. 49.Afterward acidify the reaction mixture under ice bath (0 o C) with 1N HCl until the pH reaches 2 -3.!CAUTION The HCl fume is very corrosive and irritates eyes and throats.Carry out this step inside the fume hood.∆ CRITICAL STEP HCl forms ammonium salt of denosylated compound and makes it water-soluble.50.Next transfer the reaction mixture after dissolving in 10 ml diethyl ether (Et 2 O) in 100 ml separatory funnel.Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more Et 2 O until there is (~10 ml) clear separation.∆ CRITICAL STEP Et 2 O dissolves undesired organic impurities, the desired compound remains dissolved in the organic layer.

54 .
Transfer the methoxycarbonyl chloride solution to the reaction mixture of step -52 using Pasteur pipette while stirring the reaction mixture.After 30 min remove the ice bath and allow the reaction mixture to stir for 10 h.∆ CRITICAL STEP Monitor the reaction progress by TLC (50% vol/vol ethyl acetate : pet.ether) and HRMS.R f of N-(Boc-propyl alcohol)-G-OMe-N-(HCl-propyl alcohol)-A-OBn (6) = 0.1 in EtOAc/pet.ether (50% (vol/vol)), R f of N-(Boc-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OBn (7) = 0.7 in EtOAc/pet.ether (50% (vol/vol)).For HRMS, take 1mg of the crude product in 1.5 ml Eppendorf vial and submit for mass spectrum.Reaction is complete when TLC and HRMS show no traces of N-(Boc-propyl alcohol)-G-OMe-N-(HCl-propyl alcohol)-A-OBn (6).55.Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).56.Upon completion, remove the THF by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C. 57.Next transfer the reaction mixture after dissolving in 20 ml EtOAc in 100 ml separatory funnel.Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more EtOAc until there is (~10 ml) clear separation.58.Separate the aqueous layer and pass the organic layer through the crystalline sodium sulphate to remove the residual amount of water and collect in 100 ml one neck RBF.Extract the aqueous part twice with EtOAc (2 x 20 ml) and discard the aqueous layer.59.Evaporate the organic portion in rotary evaporator to obtain a transparent liquid compound, set the water bath temperature to 45 o C and vacuum pressure gradient 400 -800 mbar.60.Dissolve the residue in minimum amount of DCM (5 ml) and add 10 -15 g silica gel (100 -200 mesh).Remove the DCM in high vacuum (100 mbar) to obtain a dry slurry.61.Pack the chromatography column (L -300 mm, D -43 mm) with dry silica gel (100 -200 mesh).
Cyclisation • TIMING 8 h 66. Weigh out 4 g of N-(Boc-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OBn (7) (8.6 mmol, 1 eq.) in an oven-dried 50 ml RBF containing a Te on-coated magnetic stir bar and stopper it with polyethylene cap.67.Add 24 ml of DCM.68.Put the RBF into the ice bath to maintain the temperature of the reaction mixture 0 o C for next 10 min.Take 6 ml tri uoroacetic acid (TFA) (20 % (vol/vol) of DCM) in a disposable syringe equipped with a stainless steel needle (0.55 x 25 mm) dropwise over a period of 1 min while stirring the reaction mixture with Te on-coated magnetic stir bar.Remove the ice bath after 10 min, and let the reaction mixture be stirred at room temperature for 2.5 h.!CAUTION TFA is corrosive and its vapors cause skin burns and eye damage.Open the bottle inside the well-vented hood.After addition neutralize it with saturated sodium bicarbonate solution.∆ CRITICAL STEP We recommend monitor the reaction progress in every 1 h by TLC (50% vol/vol ethyl acetate : pet.ether).R f of N-(Boc-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OBn (7) = 0.7 in EtOAc/pet.ether (50% (vol/vol)), R f of N-(TFA-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OBn (8) = 0.0 in EtOAc/pet.ether (50% (vol/vol)).69.Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).70.Upon completion, remove excess TFA and DCM using short path distillation set up (Figure 6).Dry the compound using oil vacuum pump (10 mbar) to obtain 4.13 g (8.6 mmol) of N-(TFA-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OBn (8) as reddish oil in a quantitative yield.∆ CRITICAL STEP Do the next step without any further column puri cation.

□
PAUSE POINT This compound (N-(TFA-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OBn, 8) is stable in a refrigerator at 0-4 o C for few days.71.Seal the RBF with rubber septum and wrap with para lm.Evacuate it by high vacuum using an oil pump for 5 min via an inlet needle (0.8 x 25 mm).72.Back ll the ask with N 2 balloon through a needle (0.8 x 25 mm).Repeat steps 71 and 72 twice.73.Add 10 ml dry THF via 20 ml syringe equipped with aspiration needle (20 Gaze x 12 Inch).
88. Repeat steps 85 -87 twice with EtOAc (2 x 20 ml) and discard the aqueous layer.∆ CRITICAL STEP It is essential to remove all the NMM.HCl salt and unreacted NMM, otherwise it interferes with the compound during the column.saturated sodium bicarbonate removes the unreacted Cbz-S(tBu)-N(Trt)-OH. !CAUTION The HCl fume is very corrosive and irritates eyes and throats.Carry out this step inside the fume hood.89.Remove the organic portion in rotary evaporator to obtain a transparent liquid compound, set the water bath temperature to 45 o C and vacuum pressure gradient 400 -800 mbar.90.Dissolve the residue in minimum amount of DCM (2 ml) and add 10 g silica gel (100 -200 mesh).Remove the DCM in high vacuum (100 mbar) to obtain dry slurry.91.Pack the chromatography column (L -400 mm, D -30 mm) with dry silica gel (100 -200 mesh).
102.Upon completion, remove the MeOH by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 45 o C. 103.Dry the compound using oil vacuum pump (10 mbar) to obtain 4.13 g (8.6 mmol) of NH 2 -S(tBu)-N(Trt)-N-(Boc-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OH (10) as a white solid in a quantitative yield.∆ CRITICAL STEP Do the next step without any further column puri cation.□ PAUSE POINT This compound (NH 2 -S(tBu)-N(Trt)-N(Boc-propyl alcohol)-G-OMe-N(Moc-propyl alcohol)-A-OH, 10) is stable in refrigerator at 0-4 o C for few days.104.Add 478 mg of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl) (2.5 mmol, 5 eq.) and 205 mg of 1-hydroxybenzotriazole (HOBt) (1.52 mmol, 3 eq.).Seal the RBF with rubber septum and wrap with para lm.105.Evacuate it by high vacuum using an oil pump for 5 min via an inlet needle (0.8 x 25 mm).106.Back ll the ask with N 2 balloon through a needle (0.8 x 25 mm).Repeat steps 105 and 106 twice.Keep the N 2 balloon to maintain the inert atmosphere throughout the reaction process.107.Add 500 ml dry ACN (1 mM concentration).∆ CRITICAL STEP The cyclization should be done at high dilution to avoid the dimer/polymer formation.108.Stir the reaction mixture at 0 o C for next 15 min.109.Add 0.44 ml of N,N-Diisopropylethylamine (DIPEA) (2.5 mmol, 5 eq.) in a disposable syringe equipped with a stainless-steel needle (0.55 x 25 mm), remove the ice and allow the reaction to stir for 8 h.∆ CRITICAL STEP We recommend monitoring the reaction progress by HRMS.For HRMS, take 1mg of the crude product in 1.5 ml Eppendorf vial and submit for mass spectrum.Reaction is complete HRMS shows no traces of NH 2 -S(tBu)-N(Trt)-N-(Boc-propyl alcohol)-G-OMe-N-(Moc-propyl alcohol)-A-OH (10).110.Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).111.Upon completion, remove the acetonitrile by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C.112.Next transfer the reaction mixture after dissolving in 20-30 ml DCM in 50 ml separatory funnel and add 1N HCl (10 ml) slowly (10ml / 30 sec).Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more DCM until there is (~10 ml) clear separation.113.separate the aqueous layer and add saturated sodium bicarbonate (5 ml) to the organic layer.Seal the separatory funnel again with polyethylene cap, shake the reaction mixture and settle down for 2 min until two layers are separated out.114.Separate the aqueous layer and pass the organic layer through the crystalline sodium sulphate to absorb the residual amount of water and collect in 100 ml one neck RBF.115.Repeat steps 112 -114 twice with DCM (2 x 20 ml) and discard the aqueous layer.∆ CRITICAL STEP Saturated sodium bicarbonate removes the unreacted HOBt and HCl removes the unreacted DIPEA.!CAUTION The HCl fume is very corrosive and irritates eyes and throats.Carry out this step inside a fume hood.116.Remove the organic portion in rotary evaporator to obtain a transparent liquid compound, set the water bath temperature to 45 o C and vacuum pressure gradient 400 -800 mbar.117.Dissolve the residue in minimum amount of DCM (2 ml) and add 5 g silica gel (100 -200 mesh).Remove the DCM in high vacuum (100 mbar) to obtain dry slurry.118.Pack the chromatography column (L -400 mm, D -20 mm) with dry silica gel (100 -200 mesh).
121.Collect the fraction in 15 x 125 mm test tubes.Using the TLC, identify the fractions containing the desired product and visualize it with UV lamp (254 nm).122.Combine the fractions containing the desired product in 250 ml RBF and remove the solvent by rotary evaporator, set the water bath temperature to 48 o C. Dry the compound using oil vacuum pump (10 mbar) to obtain a transparent solid compound in 45% yield (0.225 g).□ PAUSE POINT This compound (Moc-[AS(tBu)N(Trt)]G-OMe, 11) is bench stable at room temperature for several months without appearance of any noticeable impurity on TLC.Synthesis of Moc-[ASN]GAR-Ipr (C-terminal extended TIMING 12 h 123.Synthesis of Moc-[AS(tBu)N(Trt)]G-OH. Weigh out 200 mg of Moc-[AS(tBu)N(Trt)]G-OMe (11) in an oven-dried one-neck 10 ml RBF containing a Te on-coated magnetic stir bar.124.Add 0.5 ml of MeOH.125.Add aqueous solution (0.5 ml) of 17 mg of LiOH (0.4 mmol, 1.5 eq.) and keep the reaction mixture stirring for 30 min.∆ CRITICAL STEP We recommend monitor the reaction progress by TLC (100% vol/vol ethyl acetate/pet.ether).R f of Moc-[AS(tBu)N(Trt)]G-OMe = 0.3 in EtOAc/pet.ether (100% (vol/vol)), R f of Moc-[AS(tBu)N(Trt)]G-OMe = 0.0 in EtOAc/pet.ether (100% (vol/vol)).126.Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).127.Upon completion, remove the MeOH by rotary evaporator under a low-pressure gradient (400 -800 mbar) and with water bath temperature set at 48 o C. 128.Keep the RBF into the ice bath (0 o C), acidify with 1N HCl until it reaches pH 2 -3.!CAUTION The HCl fume is very corrosive and irritates eyes and throats.Carry out this step inside fume hood.
131.Repeat steps 129, 130 twice with DCM (2 x 10 ml) and discard the aqueous layer.132.Remove the organic portion in rotary evaporator to obtain a transparent liquid compound, set the water bath temperature to 42 o C and vacuum pressure gradient 600 -800 mbar.133.Dry the compound using oil vacuum pump(10 mbar) to obtain 0.19 g (8.6 mmol) of Moc-[AS(tBu)N(Trt)]G-OH as a white solid in 97% yield.∆ CRITICAL STEP Do the next step without any further column puri cation.□ PAUSE POINT This compound (Moc-[AS(tBu)N(Trt)]G-OH) is stable in refrigerator at 0-4 o C for few days.134.Coupling between Moc-[AS(tBu)N(Trt)]G-OH and TFA-AR(Z 2 )-Ipr.Take 0.19 g (0.26 mmol) of Moc-[AS(tBu)N(Trt)]G-OH in a 25 ml RBF Te on-coated magnetic stir bar.135.Add 0.099 g of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl) (0.52 mmol, 2 eq.) and 0.07 g of 1-hydroxybenzotriazole (HOBt) (0.52 mmol, 2 eq.).Seal the RBF with rubber septum and wrap with para lm.136.Evacuate it by high vacuum using an oil pump for 5 min via an inlet needle (0.8 x 25 mm).137.Back ll the ask with N 2 balloon through a needle (0.8 x 25 mm).Repeat steps 136 and 137 twice.Keep the N 2 balloon to maintain the inert atmosphere throughout the reaction process.138.Add 2 ml dry ACN via syringe equipped with aspiration needle (20 Gaze x 12 Inch).139.Stir the reaction mixture at 0 o C for next 15 min.140.Subsequently, Seal the RBF containing 0.23 g of TFA-AR(Z 2 )-Ipr (0.32 mmol, 1.5 eq.) with rubber septum and wrap with para lm of step 133 and repeat step 136 and 137 twice.Make a solution by adding 2 ml of dry ACN via syringe.141.Transfer the TFA-AR(Z 2 )-Ipr solution via syringe equipped with an aspiration needle.142.Add 0.18 ml of N,N-Diisopropylethylamine (DIPEA) (1.04 mmol, 4 eq.) in a 250 μl Hamilton microsyringe, remove the ice and allow the reaction stir for 10 h.∆ CRITICAL STEP We recommend monitor the reaction progress by TLC (100% vol/vol ethyl acetate/pet.ether) and HRMS.R f of Moc-[AS(tBu)N(Trt)]G-OH = 0.0 in EtOAc/pet.ether (100% (vol/vol)), R f of Moc-[AS(tBu)N(Trt)]G-AR(Z 2 )-Ipr (12) = 0.2 in EtOAc/pet.ether (100% (vol/vol)).For HRMS, take 1mg of the crude product in 1.5 ml Eppendorf vial and submit for mass spectrum.Reaction is complete when TLC and HRMS show no traces of Moc-[AS(tBu)N(Trt)]G-OH. 143.Do the qualitative estimation of the starting material and the product during the reaction by performing steps A, B, C (mentioned in BOX3).144. the reaction and remove the acetonitrile in rotary evaporator, set the water bath temperature 48 o C and pressure gradient 400 -800 mbar.145.Next transfer the reaction mixture after dissolving in 20 ml DCM in 50 ml separatory funnel and add 1N HCl (10 ml) slowly (10ml / 30 sec).Seal the separatory funnel with polyethylene cap, shake the reaction mixture and let the contents settle down for 2 min until two layers separated out.If there is no clear separation, then add more DCM until there is (~10 ml) clear separation.146.Separate the aqueous layer and add saturated sodium bicarbonate (5 ml) to the organic layer.Seal the separatory funnel again with polyethylene cap, shake the reaction mixture and settle down for 2 min until two layers are separated out.147.Separate the aqueous layer and pass the organic layer through the crystalline sodium sulphate to absorb the residual amount of water and collect in 50 ml one neck RBF.148.Repeat steps 145 -147 twice with DCM (2 x 10 ml) and discard the aqueous layer.∆ CRITICAL STEP Saturated sodium bicarbonate removes the unreacted HOBt and HCl removes the unreacted DIPEA.!CAUTION The HCl fume is very corrosive and irritates eyes and throats.Carry out this step inside fume hood.149.Remove the organic portion in rotary evaporator to obtain a transparent liquid compound, set the water bath temperature to 42 o C and vacuum pressure gradient 400 -800 mbar.150.Dissolve the residue in minimum amount of DCM (2 ml) and add 3 g silica gel (100 -200 mesh).Remove the DCM in high vacuum (100 mbar) to obtain dry slurry.151.Pack the chromatography column (L -400 mm, D -20 mm) with dry silica gel (100 -200 mesh). Figures