Peptide Chemistries. Judit Tulla-Puche. Chemical Process Development Course February 23, 2012

Peptide Chemistries Judit Tulla-Puche Chemical Process Development Course February 23, 2012

Peptide ynthesis General aspects Protection and activation. Peptide bond formation. olid phase synthesis. ynthetic strategies. Main protection schemes in solid phase synthesis. Coupling reagents. Epimerization. elected side reactions. ynthesis of Cyclic peptides. rthogonality. Deprotection and cleavage.

Peptide synthesis is about protection & activation Making a specified dipeptide sequence from two amino acids takes 5-6 formal chemical steps: 2 1 C 1 P r ot C 2 P r ot -protection 2 C 2 1 3 C-protection C-activation 2 A c t 2 1 -P ro t 4 Coupling (peptide bond formation) 2 1 2 C 5-6 & C-deprotection P r ot 1 2 -P ro t Despite apparent simplicity, this strategy involves considerable effort and often turn out to be frustrating, due to the (unpredictable) insolubility of intermediates.

olid phase peptide synthesis: the basics anchor X new peptide bond formed 1 2 1 Prot Prot n times 1+2 deblock -term 2 couple new residue 1* 1 2 Act cleavage & deprotection 2 Prot Fully protected peptide resin *Depending on the protection scheme, an additional neutralization step may be required (e.g., in Boc chemistry) free peptide Purification Characterization

olid phase peptide synthesis (PP) A method that allows to produce peptides, proteins and a wide range of analogs efficiently and in substantial amounts cleavage & deprotection chain growth solid support (insoluble polymer matrix) free peptide side chains of trifunctional residues protected C-terminal amino acid Chemical synthesis often competitive with recombinant DA techniques up to 50-60 residues. In addition: allows site-specific labelling (radioactive, fluorescent, etc.) chemically synthesized peptides are chemically well defined, thus easier to register and patent than most biotechnological products can be easily automated

The main requirement of PP: quantitative couplings Quantitative reactions are required for homogeneous products. Thus, assuming that we are interested in the sequence but obtain repeated non-quantitative ( 95%) incorporation yields: 95% 5% ~90% ~5% 95% 95% Final product: ~5% defective sequences Target sequence (Deletion of ) (Deletion of ) (Deletion of ) (Deletion of ) (Deletion of ) etc. ~85% 95% defective sequences Yield in target sequence: 0.95 5 = 0.77 (77%) For a 11-mer sequence, 0.95 10 = 0.46 (46%) Yield gets worse exponentially!! Deletions complicate very much product purification

Quantitative couplings in PP To avoid heterogeneity in the final product, excess of the incoming (acylating) species must be used at every coupling stage. If necessary, couplings have to repeated until quantitative incorporation of the residue is ensured. 95% 5% 100% 95% excess analytical control (on-polymer) of the extent of coupling reaction 95% 100% etc 100% 95% 5% excess homogeneous final product analytical control (on-polymer) of the extent of coupling reaction

Experimental setups in PP manual mode rxn vessel automated mode PP syringe with porous PE frit aa cartridges solvents/ reagents multiple (parallel) mode vacuum line reagent rack 48 individual (syringe) reactors

The Boc/benzyl strategy of PP Labile to medium acid: TFA/C 2 Cl 2 a c b Labile to strong acid: F, TFMA a: -terminal urethane deprotection 1. -C 2 2. + 3 b: C-terminal deprotection (cleavage) c: ide chain (benzyl-type) removal C 2 C 2 F - FC 2 carbocation scavenger

The Boc/benzyl strategy of PP Typical side chain protections in Boc chemistry benzyl ether (er, Thr) 4-methylbenzyl thioether (Cys) cyclohexyl ester (Asp, Glu) -tosyl guanidine (Arg) Cl Br 2 2 2-bromobenzyloxy carbonate (Tyr) 2-chlorobenzyloxy carbamate (Lys) τ -2,4-dinitrophenyl imidazole (is) in -formyl (Trp)

The Boc/benzyl strategy of PP Pam (phenylacetamidomethyl) anchoring for peptide acids 1 Improved acid stability (>100x) over conventional benzyl ester Benzhydrylamine anchoring for peptide amides Very useful, as many synthetic peptides are obtained as C-terminal carboxamides

The Fmoc/t-butyl strategy of PP b: C-terminal benzyl ester cleavage Labile to base a C c b Labile to acid C 2 C 2 a: -terminal urethane deprotection p-alkoxy group enhances acid sensitivity; TFA cleavage (no F) c: butyl side chain deprotection : -C 2 2 - : Fmp adduct carbocation scavenger

The Fmoc/t-butyl strategy of PP Typical side chain protections in Fmoc chemistry Ph Ph Ph Ph Ph Ph t-butyl ether (er, Thr, Tyr) trityl thioether (Cys) t-butyl ester (Asp, Glu) -trityl amide (Asn, Gln) Ph Ph Ph Boc urethane (Lys) Pmc (tosyl-type) guanidine (Arg) Boc or trityl imidazole (is) im -Boc (Trp)

The Fmoc/t-butyl strategy of PP elected handles for peptide acids in Fmoc chemistry C 4-hydroxymethylphenoxyacetic acid (labile to 95% TFA) andle: a bifunctional spacer carrying a peptide attachment site on one end and a functional group (often a C) on the other end that can be coupled to a solid (amino-type) support. C 4-hydroxymethylphenoxypropionic acid (labile to 95% TFA) AA 1 ADLE ADLE 3 C C MPB (labile to 1% TFA) PEPTIDE ADLE C 3 AA 1 ADLE 3 C C AL (labile to dil. TFA, Ac) AA 1 ADLE AA 1 ADLE PEPTIDE ADLE

The Fmoc/butyl strategy of PP Typical resins for peptide acids in Fmoc chemistry C 3 Wang resin, labile 50% TFA 3 C ink resin, labile to 1% TFA 3 C Cl Cl asrin resin, labile to 1% TFA tandard esterification protocol: Fmoc-aa + DIPCDI + DMAP (10 eq each) in DMF. epeat coupling if possible. Also: Fmoc-aa + MeIm + MT 2-chlorotrityl chloride resin, labile to very dilute TFA, Ac (for partially protected peptides)

The Fmoc/butyl strategy of PP elected resins and handles for peptide amides in Fmoc chemistry Me 2 Fmoc- Me Fmoc Me ink amide resin Me ink amide linker Me 4-(4 -methoxybenzhydryl)phenoxyacetic acid Fmoc Me Me Fmoc- C PAL linker XAL linker

olid upport: General Features An optimal solid support should have the following characteristics: 1. Mechanically obust: Batchwise and Flow-Continous Modes 2. table to Variation in Temperatures 3. Mobile, Well-olvated, and eagent Accessible ites 4. Acceptable Loadings 5. Good welling in Broad ange of olvents 6. omogeneous Bead izes 7. table in Acidic, Basic, educing, and xidizing Conditions 8. Biocompatible and welling in Aqueous Buffers (iff applicable) ubstitution level: 1. igh ( 1 mmol/g): better yield/g resin industrial production 2. Low ( 0.5 mmol/g): faster reactions, purer products long peptides 3. Extra-low (<0.5 mmol/g): intramolecular cyclizations, dendrimers

Polymeric upport: Polystyrene + Polymerization tyrene Divinylbenzene Physical Characterization of beads - 100-200 ( 75-150 µm) or - 200-400 (75-38 µm) mesh - 1 or 2 % crosslinking - Mechanically robust - > 99% of the pendant functional groups are located within the matrix olvent Compatibility - DCM - DMF - TF - Toluene - TFA ecommended Loadings 0.5-0.8 mmol/g for PP 1-2 mmol/g for P

Polyethylene Glycol-Polystyrene (PEG-P) PEG-P, Applied Biosystems (a) Champion I, II, Bioresearch Technologies (a) Dendrogel, Bioreserach Technologies (a) ArgoGel, Argonaut Technologies (a) TentaGel, app Polymer (b) (a) By reaction of preformed oligooxyethylenes with aminomethyl P resins (b) By graft polymerization olvent Compatibility - DCM - DMF - TF - Toluene - TFA - Me - AC - Ac Loadings - 0.15-0.4 mmol/g

Amphiphilic (PEG) upports ChemMatrix VersaMatrix f: 0.5-1.0 mmol/g easiness of handling ready to use dried powder! shorter swelling time P PEG-P Graft esin ChemMatrix

Coupling chemistries Classical carbodiimide coupling a (through enol) '-=C=-' '= Chx, ipr 5(4)-oxazolone (active) a b ' -acyl isourea (active) 1 extra equivalent ' Protected symmetric anhydride (active) b ' -acylurea (inactive) '

Coupling chemistries Bt as an additive ' ' Bt ester (active)

Coupling chemistries Phosphonium and aminium/uronium salts X X Me 2 Me 2 Me 2 Me 2 PF 6 PF BF 6 4 P X X Bt (X=C) At (X=) PyBP(X=C) PyAP (X=) BTU (X=C) ATU (X=) TBTU (X=C) TATU (X=) C PF 6 C xyma CMU

ide reactions: epimerization Its rate depends on the type of catalyzing base, the solvent and especially the electronic effects of the and C substituents. For W= -C and X=,, risk is minimal. ome risk exists during activation, when X= good leaving group W- C-X Tips PP, minimal risk Fragment condensations: Gly or Pro at C- terminal preferred Minimize DMAP exposure during anchoring steps! L C acemization at a C-terminal Cys residue is a risk in Fmoc synthesis for peptide acid-type anchorings. Extent of racemization varies with the protecting group (- t Bu > Trt > Acm > t Bu) _ C + + C C :B base?? Possible racemization via reentry not ruled out Piperidinyl-Ala usually detected ( m=51 Da)

Cysteine acemization -Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly- 2 Fmoc/tBu. Fmoc-Cys(Trt)-, DMF DIPCDI/Bt, 5-min preactivation DIPCDI/Bt, BP/Bt/DIEA, BP/Bt/DIEA, DIPCDI/Bt BP/Bt/DIEA

Aminium alts: ide eactions Guanidinium Formation (XTU) Cl -Phe-Fm + ATU DIEA (2 eq) (C 3) 2. (C 3 ) 2 C C 2 C

ide reactions: Diketopiperazine formation Diketopiperazine Formation Diketopiperazine (DKP) formation is especially serious when Gly, Pro, D- or other residues such as Me aa s can adopt an amide bond in the cis configuration, either at the n (C-terminal) or n-1 positions. In Boc chemistry, the problem appears during the neutralization step. In Fmoc chemistry, the problem appears during deprotection. ' 2 Fmoc D-Val L-Pro Wang-resin 2 100%

X + + Asp X = tbu, All, Bzl α-peptide β-peptide Giralt et al. Tetrahedron Lett. 30, 497, 1989. Yan, Mayer et al. Bioorg. Med. Chem Lett. 15, 1065, 2005. ide reactions: Aspartimide Formation

Difficult sequences in PP Experimental evidence (often from end product analysis) shows the occasional appearence of sequencedependent drops in coupling or deprotection efficiency at one or several consecutive residues. Problem is related with a tendency of the growing peptide chain to aggregate (β-sheet propensity, etc.) Attempts to predict these regions/situations are only partially successful. emedies include: a) Environmental changes: solvents (fluorous, DM), chaotropes, temperature b) Temporary backbone modification by the mb amide protection Fmoc Fmoc Pfp 2 Fmoc Fmoc piperidine Fmoc X Me Me Me Fmoc Fmoc Fmoc -to- acyl shift PP Me Me Me c) Temporary backbone modification by pseudoprolines Fmoc ' After PP & deprotection ' For optimal disruption of β- sheet (aggregation), prolines or Ψ-prolines must be incorporated every 6-7 residues

ynthesis of large peptides and proteins Convergent peptide synthesis The stepwise character of standard PP becomes a limitation when peptides of certain large size (i.e., >50 residues) are attempted. The inevitable buildup of impurities (deletions, truncations, etc.) decreases the yield of expected product and complicates its purification. witching from a stepwise to a convergent scheme (condensation of partially protected fragments, as in classical solution chemistry) is one of the possibilities. Purified protected segments are coupled either in solution or on solid phase to give the target molecule. egments can be prepared by a variety of ways (solid phase or solution). They are usually - and side chain-protected (fully or minimally), with a free C. There are a number of approaches to protected segments: Protected peptide acids from very acid-labile resins Fmoc Prot Prot Prot C Purif Mild acidolysis Fmoc PP Me iniker handle Fmoc Fmoc Prot Prot Prot Prot Prot Prot 2 Prot Act Prot Dissection of target sequence ideally at Pro or Gly to minimize epimerization on C activation Prot Prot Prot Prot Me Cl Cl Trityl handle AL handle etc. Me

Cyclic peptides head-to-tail cyclization 6 1 ide-chain to side chain cyclization 2 5 4 3 2 3 7 6 8 C disulfide bonds (native and non-native)

Allyl-based chemistry for PP - rthogonality Amine protection by the Alloc group ( α or ε ) PdL 2 L Pd L L Pd L Alloc -C 2 ; + u u 2 Carboxyl protection (Asp, Glu or ) PdL 2 (as above) u A peptide synthesis protection scheme is termed orthogonal when the three types of protecting groups (-, C-anchoring, side chain) can be removed selectively in any order and under completely different chemical conditions

pz as Alternative to Alloc, Fmoc and Boc ynthesis of Multicyclic Peptides equires rthogonal and Mild Conditions emovable Protecting Groups 2 C 2 Mechanism pz 1 2 C 2 1 eduction C 2 2 C 2 C 2 2 C 2 C 2 + - C 2 emoval conditions 6 M ncl 2,1.6 mm Cl/dioxane in DMF, r.t. 2 x 30 min

Examples of orthogonal protection Two fully orthogonal schemes Fmoc piperidine TFA Pd 0 If the -terminal Fmoc protection is desired in the final product, -methylaniline must replace morpholine (which deprotects Fmoc) in the allyl deprotection cocktail. Another possibility is to use an hydrostannolytic cleavage procedure: (Ph 3 P) 2 PdCl 2 + Bu 3 n Fmoc piperidine Pd 0 Pd 0 TFA

ynthetic approaches to cyclic peptides A. Conventional method for head-to-tail cyclization Prot linear precursor-resin Prot 2 linear precursor C-activation high dilution head-to-tail cycle B. n resin head-to-tail cyclization by side-chain anchoring -Terminal Amine 2 n C-Terminal Carboxyl ide-chain Functionality ide-chain Carboxyl

ide chain anchoring schemes Anchoring through Asx/Glx side chains X W W X X = peptide acids X = peptide amides PP Prot X ide chain anchoring of Lys/rn via urethane linker Prot u u u PP 2 Prot Prot Main application: head-to-tail cyclizations

ynthetic approaches to cyclic peptides C. Backbone anchoring through the BAL linker Backbone Amide -Terminal Amine 2 n C-Terminal Carboxyl ide-chain Functionality ide-chain Carboxyl

Backbone anchoring through the BAL linker Me Me ATU/DIEA DMF Me Me BAL + 2 2 Allyl acb 3 Me Me PP Me PG Me Allyl Allyl Main applications: head-to-tail cyclization C-terminal elaboration (alcohol, ester, 2ry amide, thioester, aldehyde)

Disulfide bridges in synthetic peptides ative bridges Folding processes and intermediates Contribute to structural stabilization Artificial bridges: Conformational restriction Main synthetic approaches A. Deprotection and oxidation of a polythiol precursor X X -2X [] - 2

Disulfide bridges in synthetic peptides ative bridges Folding processes and intermediates Contribute to structural stabilization Artificial bridges: Conformational restriction Main synthetic approaches B. imultaneous deprotection and oxidation of suitable Cys derivatives X X I 2-2 XI

A. Deprotection and oxidation of a polythiol precursor X X -2X [] - 2 Most usual Cys protecting groups in this approach Meb Mob Tmob Trt Mmt Boc-compatible Fmoc-compatible

A. Deprotection and oxidation of a polythiol precursor xidation conditions 1. Atmospheric (peptide (~10 µm), p~8, air or 2 ) Main indication Peptides 1 2. xidizing agents K 3 Fe(C) 6 (p 7-8) Cl-Pt IV complexes (p 3-6) C C Cl Pt Cl C C 2 2 Cl Pt Cl 2 2 Peptides 1 DM (p 4-7) Peptides 2 3. edox systems Peptide (~10 µm, p ~8, glutathione, [G]/[GG]=10, denaturing agents, Ar atmosphere) Cysteine/cystine (similar to G/GG) Peptides 2

B. imultaneous deprotection-activation of Cys derivatives X X I 2-2 XI Also Tl III and (only for Fm) piperidine or DBU Most usual Cys protecting groups in this approach Trt Tmob Acm Fm Fmoc-compatible Boc/Fmoccompatible Boc-compatible

Cleavage from resin and global deprotection The F reaction Acidolysis of the peptide resin with anhydrous F in the presence of carbocation scavengers (e.g., F-anisole 9:1) at 0ºC for 1h remains the method of choice in Boc PP to cleave the peptide from the resin and simultaneously deprotect the side chains. TFA acidolysis in Fmoc PP A number of TFA-containing cocktails have been developed, depending on the presence of certain residues and protecting groups. Peptide contains Arg(Mtr) or free Trp o Peptide contains Cys(Trt) or Met Yes 94% TFA 2.5% EDT, 2.5% 2, 1% TI Yes o o Peptide contains > 2 Arg(Mtr) residues Yes 95% TFA, 2.5% 2, 1% TI 1M TMBr/thioanisole in TFA with m/cresol/edt 81.5% TFA, 5% thioanisole, 5% phenol, 5% 2, 2.5% EDT, 1% TI

Workup, purification and characterization What can one expect to find in a crude peptide preparation? the target peptide! 2000 1000 + non-peptide materials! Gly-Ile-Gly-Ala-Leu-Phe- Leu-Gly-Phe-Leu-Gly-Ala- Ala-Gly-er-Lys-Lys-Ahx- Lys-Asn-Glu-Gln-Glu-Leu- Leu-Glu-Leu-Asp-Lys-Trp- Ala-er-Leu-Trp-Asn- 2 0 0 2 4 6 8 10 12 14 deletion sequences incompletely deprotected sequences

Workup, purification and characterization Preparative P-PLC is the standard method for synthetic peptide purification 600 400 200 0 0 2 4 6 8 10 12 14 Mass spectrometry (PLC-EI and MALDI-TF) are the usual methods for peptide characterization

Peptide ynthesis Examples Polyalanine. Thymosin α1. β-amyloid (1-42). ATE. xathiocoraline

(Ala) 10 Val -Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Val- Ala residues have the highest propensity to form α-helixes -econdary structure highly dependent on environment: - α-helixes in non-polar organic solvents - unwinding to form β-sheet or random coils occurs in polar aqueous media Levy, Y.; Jortner J.; Becker,. M;. PA 2001, 98, 2188 2193

ynthesis of -(Ala) 10 -Val- by ChemMatrix and P resin P - 5Ala - 4Ala -(Ala) 10 -Val- - 3Ala - 2Ala - 1Ala ChemMatrix -(Ala) 10 -Val- 100 90 4700 eflector pec #1[BP = 849.4, 2791] 849.3565 2790.8 80 70 ABI 433A CTU-DIEA % Intensity 60 50 40 30 20 10 438.9618 444.9713 460.9444 482.1754 494.2012 506.1258 524.0522 546.7827 565.2255 582.3868 595.9114 615.2542 636.2585 655.9890 671.9622-2Ala - 1Ala 689.9221 707.3022 727.1047 744.0919 761.0737 778.3237 0 417.0 551.4 685.8 820.2 954.6 1089.0 Mass (m/z) 796.2936 809.0622 844.9844 860.9822 865.3311 887.3066 905.5911 921.3929 936.1615 957.4049 977.4352 991.5001 1004.5093 1021.3715 1045.5337 1063.4335

Thymosin α 1 1 5 10 Ac - er Asp Ala Ala Val Asp Thr er er Glu Ile Thr Thr Lys Asp 15 Leu Asn Glu Ala Glu Glu Val Val Glu Lys Lys Glu Lys 28 25 20-28-residue acetylated peptide hormone used to treat chronic hepatitis B - Earlier syntheses suffered from low yields

Thymosin α 1 (P vs PEG) CTC resin Zadaxin - Automatic linear stepwise synthesis on an ABI 433-0.1 mmol scale - Coupling conditions: CTU/DIEA ink-cm resin

β-amyloid (1-42) Formation of amyloid plaques is thought to contribute to the degradation of the neurons in the brain and the subsequent symptoms of Alzheimer's disease. Lührs, T.; itter, C.; Adrian, M.; iek-loher, D.; Bohrmann, B.; Dobell,.; chubert, D.; iek,. PA 2005, 102, 17342-17347.

β-amyloid (1-42) Previous synthetic attempts: - ynthesis in solution - olid-phase synthesis using different resins: polystyrene, Tentagel, PEG-P and Pepsyn K - everal strategies have been studied to overcome the difficulties of this synthesis: introduction of an oxidized Met-35, the use of DM as a coupling co-solvent, the use of DBU, the introduction of mb backbone amide protection. - ecently, the use of an intramolecular acyl migration reaction of the corresponding -acyl isopeptide has been reported..

β-amyloid (1-42) - Automatically - CM resin (0.6 mmol/g) - AB linker - BTU/Bt/DIEA Major peak was the desired peptide: 91% purity García-Martín, F. et al. J. Comb. Chem. 2006, 8, 213 220.

ATE - Chemokine peptide of 68 residues - Important in clinical therapy in the quest to treat diseases such as asthma, rheumatoid arthritis and AID - β-sheet flanked by 2 α-helixes Chung, C.; Cooke,.M.; Proudfoot, A. E. I.; Wells, T.. C. Biochemistry 1995, 34, 9307 9314.

ATE - Manually - Wang-P resin - ATU/At/DIEA The final peptide was not achieved. The fragment comprising Arg 44 -er 68 was observed

Pseudoprolines (ψpro) - Pseudoproline dipeptides consist of a dipeptide in which the er or Thr residue has been reversibly protected as proline-like TFA-labile oxazolidine. - The insertion of a pseudoproline dipeptide into a sequence disrupts the formation of the secondary structures thought responsible for problems during peptide assembly http://www.merckbiosciences.co.uk/html/bc/pseudoproline.htm

Pseudoprolines (ψpro) http://www.merckbiosciences.co.uk/html/bc/pseudoproline.htm

ψpro s in ATE

ATE with P resin - Automatically - P resin o ψpro - ATU/DIEA amples at Arg 44 With ψpro

ATE with P resin - ynthesis with no ψpro was stopped after 38 cycles since sample at er 31 did not show the target peptide - ynthesis with ψpro reached 45-aa but not the final peptide P resin With ψpro ample at Ile 24

ψpro + PEG +

ATE with CM resin and ψpro - Automatically - CM resin & 4 ψpro - ATU/DIEA Final peptide 31 % purity

ATE with CM resin and ψpro Purified by reversedphase PLC MALDI-TF García-Martín, F.; White, P.; teinauer,.; Côté,.; Tulla-Puche, J.; Albericio, F. Biopolymers (Peptide cience) 2006, 84, 566 575.

Thiocoraline - Potent antitumoral - Produced by marine actynomycetes - Bicyclic octathiodepsipeptide - C 2 symmetry - ighly rich in Cys - Presence of consecutive Me-Cys and D-Cys - Bisintercalates to the DA through its heterocyclic units omero, F.; Espliego, F.; Pérez Baz, J; García de Quesada, T.; Gravalos, D.; De la Calle, F.; Fernández-Puentes J. L. J. Antibiot. 1997, 50, 734 737.

Limitations in the synthesis of xathiocoraline - Due to the presence of the ester functionality: - Diketopiperazine formation - at the resin level - along the chain - Lability in front of certain protecting group removal conditions - piperidine (Fmoc) - ncl 2 (pz) depending on the position of the group - Due to the presence of Me-Cys(Me): - β-elimination didehydroalanine formation - xidation during cleavage - Due to the presence of two consecutive Me-amino acids: - Diketopiperazine formation is favored - trong coupling conditions needed - Cleavage between Me-AA s can occur under high acidic conditions

Protection scheme PG 5 PG 1 -D-er- PG 1 -Gly- C PG 2 PG 3 -Me-Cys(Me)- PG 4 -Me-Cys(Acm)- - ptimization: Wang resin PG 3 Alloc or pz PG 1 Fmoc PG 4 Boc PG 2 Trt PG 5 pz

ynthesis of the tetrapeptide (i) piperidine-dmf (1:4) (ii) Fmoc-D-er(Trt)-, ATU/At/DIEA, DMF Fmoc-Gly-, DIPCDI, (iii) piperidine-dmf (1:4) Fmoc DMAP, C 2 Cl 2 -DMF (9:1) (iv) pz-cl, DIEA, DMF Wang resin pz Trt (i) TFA TI C 2 Cl 2 (2:2.5:95.5) (ii) Alloc-Me-Cys(Me)-, DIPCDI, DMAP, C 2 Cl 2 -DMF Alloc (i) Pd(PPh 3 ) 4, Phi 3, C 2 Cl 2 (ii) Boc-Me-Cys(Acm)-, ATU/At/DIEA, DMF Boc pz Acm A combination of protecting groups (Fmoc, Trt, pz, Alloc and Boc) gives the tetrapeptide with good purity.

Diketopiperazine minimization tepwise: ctadepsipeptide Fragment coupling: Tetradepsipeptide 2 Tetradepsipeptide 1 - In situ neutralization protocols, introduction of dipeptides, tandem alloc removal-coupling.. failed in overcoming DKP formation Dimerization: Tetradepsipeptide Acm Tetradepsipeptide Acm I 2 (5 equiv) in DMF Tetradepsipeptide Tetradepsipeptide

estricted conformation-based protecting group PG 1 PG 2 removal PG 2 Acm -er-peptide 1) I 2, DMF 2) PG 2 removal PG = protecting group Acm PG 1 -er-peptide PG 1 -er-peptide peptide-er-pg 1 Acm Dimerization restricts the flexibility of the chain preventing DKP formation

ynthesis of protected bicycle Boc pz Boc pz I 2 (5 equiv) in DMF Boc pz Acm TFA 2 C 2 Cl 2 (2:1:7) pz pz PyBP/At/DIEA, DMF pz pz

xathiocoraline pz pz (i) a 2 2 4, AC Et 2 (ii) 3-hydroxyquinaldic acid, EDC Cl, u, C 2 Cl 2 Tulla-Puche, J.; Bayó-Puxan,.; Moreno, J. A.; Francesch, A. M.; Cuevas, C.; Álvarez, M.; Albericio, F. J. Am. Chem. oc. 2007, 129, 5322 5323.

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