5. Chemical Synthesis of Peptides
Peptides, proteins, pseudopeptides, peptidomimetics ---- chemistry, biology, biophysics
peptide synthesis ( > 3,000 – 10,000 Da) [protecting group, racemization-free condensation]
Synthetic methodology [solution-phase and solid-phase peptide synthesis]
[1] General Considerations
Development of rapid, highly stereospecific, high yield synthesis
Two major impetuses for peptide synthesis
to improve potency, selectivity, stability, diminution of toxic side effects of the native
ligands such as peptides and pseudopeptides
to predict “the second code” for the three-dimensional structure of a peptide/protein
[2] Solution Phase Synthesis
Choice of Na-protecting groups
peptide synthesis [C --- N synthesis to minimize racemization]
temporary a–amino protecting groups
[coupling strategy, side chain protection, final deprotection]
(a) tert-butoxycarbonyl (t-Boc) [deprotection by mild acidic condition]
(b) 9-fluorenylmethoxycarbonyl (Fmoc) [basic condition for deprotection]
<
no need for acidic condition
hydrophobic seq. 합성

7. (2) Side chain protection
orthogonal: stable during the deprotection of the N-protecting group
readily removable at the final deprotection step
benzyl group --- orthogonal to the tBoc
tert-butyl group --- orthogonal to the Fmoc
(3) Coupling methods
dicyclohexylcarbodiimide (DCC)
1H-hydroxybenzotriazole (HOBt)
BOP (benzotriazole-1-yl-oxy-tris-(dimethylamino) phosphonium hexafluorophosphate)
HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)
(4) Deprotection strategies
1) Boc-benzyl protecting groups ---- HF or TFMSA (trifluoromethane sulfonic acid)
2) Fmoc-t-butyl protecting groups ---- TFA
cf) choice of scavengers to avoid side reactions
(5) Protection of the C-terminal carboxyl group
C-terminus with carboxyl or amide group --- protection via ester or amide formation
(6) Stepwise and Fragment Condensation
1) stepwise addition and Na-deprotection
2) ligation of fragments separately synthesized
※ carboxylic group activation prior to aminolysis ---- leading to condensation
1) reactive acylating agent
2) stable acylating agent

8. Coupling Methods
Carbodiimide [DCC, diisopropylcarbodiimide, water soluble carbodiimide]
Drawbacks: racemization, dehydration (Asn & Gln), by-products (N-acylurea)
O-acylisourea causes the side reactions.
Trapping agents
a) p-nitrophenol
b) pentachloro and pentafluorophenols
c) N-hydroxysuccinimide (HOSu)
d) N-hydroxybenzotriazole (HOBt)
DCC/HOBt: no major drawbacks (good pair)

10. (2) Mixed carbonic anhydride method
racemization [5(4H)oxazolone]
unstable mixed anhydride
in situ reaction with the amine
exothermic reaction (-15ºC)
Side reactions
urethane formation
racemization
appropriate solvents and short reaction
DCC method preferred !!!

11. (3) Active ester method (part of DCC method)
mixture of Na-protected A.a. and DCC --- trapping agent --- active ester ---- amine
---- acetylating the amine group
(4) Azide method
i) hydrazide formation
ii) azide formation
iii) aminolysis with the amine component
(5) BOP reagent (Castro’s reagent)
i) stable, nonhygroscopic, soluble
ii) high yield peptide synthesis
with low racemization
iii) difficult coupling
iv) on-resin cyclization
via lactam formation
v) BOP > DCC/HOBt

12. vi) carcinogenic hexamethylphosphoric triamide formation (BOP reaction)
PyBOP / BrBOP / PyBroP / BroP
(6) HBTU reagent
[2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]
side chain to side chain cyclic lactam formation
(7) Amino acid halides
Fmoc protected amino acid chloride
rapid synthesis of short peptides
coupling in immiscible solvents
high efficiency
fast
ease of synthesis of the activated amino acid at a low cost
Solid phase peptide synthesis
Fmoc A.a. chlorides + DIEA (basic coreactant) ---- oxazolone formation
HOBt addition (1:1) --- acylation (peptide formation)
Protected amino acid fluoride with Fmoc, Boc, Z
stable and effective for coupling

13. (8) Urethane protected amino acid N-carboxyanhydrides (UNCAs)
the best coupling reagent (better than BOP or HBTU)
difficult coupling steps (highly sterically hindered amino acid)
stable ---- stability, reactivity, solubility
Boc, Fmoc, Z derivatives ---- commercially available
highly reactive toward Nu like amines ---- good yield & high purity w/o racemization
(9) Difficult couplings
UNCA, A.a. fluorides, HBTU, PyBroP > BOP or Carbodiimide
[hindered peptides or amino acids bearing N-terminal a,a’-dialkylation]

14. Protecting Groups
Amino acid i) N-terminal amino group
ii) C-terminal carboxyl group
iii) reactive group in the side chain
Considerations i) peptide synthesis from C to N-terminus
ii) racemization through 5(4H)oxazolone formation
iii) C-terminal protection (solid phase synthesis)
tert-Butyloxycarbonyl (t-Boc) protection
i) acid cleavage (but stable to base, sodium in NH3, catalytic hydrogenation)
ii) scavengers against reactive tert-butyl carbocations
anisole, ethanedithiol
iii) side chain protection [HF with appropriate scavengers ---- deprotection & cleavage]
Arg tosyl group
His tosyl group HOBt deprotection
dinitrophenyl thiophenol in DMF before HF deprotection
BOM group HF & TFMSA-TFA
Cys p-methylbenzyl group
Asp/Glu cyclohexyl esters
Lys Z or substituted z group (benzyloxycarbonyl)
Met unprotected
Met(O) reduction by low-high HF procedure

15. Trp formyl group piperidine in DMF or low-high HF procedure
Ser/Thr/Tyr benzyl ethers Tyr --- benzylation by strong acid
Tyr 2’,6’-dichlorobenzyl or Br-Z group protection
(2) 9-Fluorenylmethyloxycarbonyl (Fmoc) Group
deprotection under mild basic conditions
[dil. liq. ammonia, ethanolamine, morpholine,piperidine]
Na-Fmoc amino acid preparation [Fmoc-succinimide (Fmoc-ONSu)]
Deprotection: 20% piperidine in dimethylformamide
Cleavage of peptide from the resin: TFA
[side group protecting groups --- basic stable and TFA removable]
Arg PMC group TFA labile
Asn & Gln unprotected
trityl or benzhydryl groups (dehydrated)
Asp & Glu tert-butyl esters
Tyr tert-butyl ether
Cys trityl group TFA
Acm (acetamidomethyl) TFA resistant (S-S bond formation)
His trityl group
Lys Z or substituted z group
Met no protection
Trp no protection or Boc-indole derivative

16. (3) Na-Allyloxycarbonyl type protection
many undesired side reactions during the deprotection-cleavage steps
[carbocations and other active species generated by HF or TFA]
Na-allyoxycarbonyl group for amine and alcohol protection
allyl ester for carboxylic group protection
ethers for aryl alcohol protection
[reaction with allyloxycarbonyl chloride]
easy deprotection via mild hydrogenation
[complexation of allyl groups with Pd(0) and tributyl tin as H2 donor]
all allyl and alloc side chain protected amino acids
& Na-alloc protected amino acids available
allylic anchor groups (resin linkers)
peptide cleavage from the resin – neutral and extremely mild conditions

17. [3] Solid phase peptide synthesis (SPPS)
Scheme 2-13
unreacted A.a. and coupling reagents
-- filtration and washing
2x – 4x excess of reagents
-- complete coupling
automated SPPS
Considerations
i) protecting groups
ii) coupling methods
iii) choice of the solid support (resin)
Resins
i) linkage of the 1st A.a.
ii) functionalization of C-terminal group
iii) swelling property
iv) pressure
v) chemicals

19. Kaiser test
Abs at 570 nm

20. Solid support
polymer of styrene crosslinked with m-divinylbenzene (1%)
X-linking
i) rigidity and physical stability of resins
ii) swelling property in the solvents
iii) accessibility of the reacting groups
1% X-linked polystyrene resin with resin handles (spacers)
i) swelling property
ii) decreasing the interaction between the growing peptides and the resin
iii) different anchoring and cleavage techniques
Solid supports and resins for SPPS
handles
i) high yield for peptide synthesized
ii) increased optical purity
iii) better control of cleavage conditions
※ Resins with different handles
a) Wang resin
p-alkoxybenzyl ester linkage [Merrifield resin + 4-hydroxybenzyl alcohol]
deprotection and cleavage of the peptide with 50% TFA in dichloromethane

21. b) PAL (peptide amide linker) handle
PAL-COOH + p-methylbenzhydrylamine (MBHA) resin
cleavage: 70-90% TFA in dichloromethane
peptide with a C-terminal primary amide
c) Rink resin [Fmoc-t-butyl strategy]
Fully protected (A.a.) peptide 합성
cleavage: mild acidic conditions
(10% acetic acid or 0.2% TFA in dichloromethane)
d) Sasrin resin
disubstituted phenol with the Merrifield resin
protected peptide 합성
cleavage: 0.5% TFA in dichloromethane
e) p-methylbenzhydrylamine resin (Boc-benzyl protection strategy)
peptide amide with Boc-protected A.a.
cleavage: HF or TFMSA in TFA
f) PAM resin (phenylacetamidomethyl) or Oxymethyl-PAM resin
peptide acids
high resistance to acidolysis

22. g) Oxime resin
fully protected peptide
cyclization of protected peptides on oxime resins
cleavage: aminolysis or hydrazinolysis
h) Nitrobenzyl resin
cleavage: photolysis at 350 nm
i) Allylic resins [Hycram resin]
alloc group for the a–amino group protection
fully protected peptide 합성
cleavage: neutral and mild conditions
(3) Deprotection methods
removing the side chain protecting groups and cleavage of the peptide from the resin
a) deprotection and cleavage at different time
fragment condensation (cleavage without deprotection)
orthogonal synthetic – deprotection strategy
b) deprotection and cleavage at the same time
Boc-benzyl protected A.a. (HF, TFMSA in TFA, HBr in HOAc)
Fmoc-t-butyl protection scheme
(TFA in dichloromethane at the last step, Scavengers to reduce carbocation)
cf) HF strong acid in the cleavage and deprotection step --- milder methods preferred

23. [4] Some current topics
peptide libraries
peptides and proteins: difficult in practical applications
i) complex structures with multiple functions
ii) little knowledge
iii) complexity in structures
Combinatorial chemistry: good quality control
[complex mixture of peptides: “problem” rather than “opportunity”]
※ Construction of large diverse peptide libraries
(a) Multiple synthetic method
peptide synthesis on the head of polyacrylic and grafted polyethylene rods
(b) Tea bag method
porous polyprolylene containers with a small amount of a solid phase resin
separate coupling and combined deprotection
(c) The one peptide, one bead method
rapid synthesis of large diverse mixtures [proportioning-mixing or split synthesis]
Mixing – Deprotection – Separation – Coupling – Mixing – etc…. (Fig. 2-17)
(d) SPPS with photolithography
chemically diverse peptide mixtures
chemical structures and reactivities
unusual amino acid incorporation

25. (2) Stepwise and Fragment (Segment) condensation strategies
Stepwise strategy [short peptide synthesis]
disadvantage: i) insolubility of the growing peptide
ii) difficult purification
iii) aggregation of the peptide (poor coupling and deprotection)
iv) by-products
Fragment condensation strategy [protected peptides --- coupling]
disadvantage: i) poor coupling
ii) racemization
iii) low solubility
Examples of coupling reagents
DCC/HOBt
CuCl2 in DCC/HOBt
BOP and HOBt
DCC and ethyl-2-(hydroxoimino)-2-cyanoacetate
papain and a-chymotrypsin

26. (3) Cyclization of peptides in solution and on solid supports
[disulfide, lactam (cyclic amide), other functional groups]
intramolecular cyclization
selective cyclization [orthogonal protection of the different reactive groups]
solution cyclization
[under highly diluted condition to prevent intermolecular reaction]
ex) monocyclic disulfide analogues of dynorphin A
[MBHA resin, Boc-benzyl protected A.a., Cleavage with HF (+scavengers)
dilution with water, oxidation, removal with ion-exchanger, lyophilization]

27. cyclization on a solid support
advantage: i) high yield
ii) full automation
iii) pseudo-dilution effect
ex) mixed Boc-Fmoc strategy
for on-resin lactam formation
Na-Boc protected A.a.
Fmoc or Ofm esters
side chains for the lactam bridge
HF for the final deprotection-cleavage step
ex) Oxime or Kaiser’s resin
ex) On-resin disulfide bridge formation
by using N-halosuccimides
(Scheme 2-17)

28. (4) Orthogonal protection and synthesis
Orthogonality
Na-Fmoc; basic condition (piperidine)
tert-butyl or trityl group; TFA
handle cleavage; chemical hydrogenation
Bicyclic (lactam and disulfide bridge) analogue of oxytocin
MBHA resin (HF), Na-Boc A.a. (TFA), benzyl-group for side chain (HF)
side chains for the lactam formation [Glu/Asp and Lys/Ornithine with Fmoc or Ofm]
---- dilute piperidine treatment
peptide cleavage from the resin with HF
deprotection with HF
S-S bond formation with Fe3(CN)6
Parallel dimer of deamino-oxytocin (one cysteine and L-b-mercaptopropionic acid)
cysteine – Acm (thallium trifluoroacetate), b-Mpa – Tmob (7% TFA)
PAL-resin (TFA), Fmoc-A.a. (piperidine)
Mild three-dimensional orthogonal protection scheme
dithiasuccinoyl (Dts) group protected A.a. (thiols)
tert-butyl group protected side chains (TFA)
1st A.a. to the resin via o-nitrobenzyl ester linkage (photolysis at 350 nm)

29. (5) Pseudopeptides: amide bond replacements
backbone modification [aCH group]
i) stability of peptides toward proteases
ii) receptor selectivity
iii) peptide antagonist
Ψ[CH2-NH] : the reduced peptide bond
amino acid aldehyde and Schiff base formation with NaCNBH3
--- biological activity
(6) Modifications at the aCH group
Azapeptide: replacement of aCH with nitrogen atom
(7) Allyl-UNCA strategy
UNCA as powerful acylating agents
allyl-based a-amino and side chain protecting groups and resin handles
ex) allylic handle and A.a. as Fmoc or Boc-protected NCAs
(8) Continuous flow synthesis
flow stable polyethyleneglycol dimethylacrylamide (PEGA) copolymer
[chemical stability, swelling property, spectrophotometric monitoring]
Fmoc-A.a.
Rink linker (4-Fmoc-amino[2,4-dimethoxyphenyl] methylphenoxyacetic acid]
Cleavage with conc. TFA (+ scavengers)

30. ※ No peptide is ever trivial to synthesize !