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Lara Malins Baran Group Meeting04/23/16Highlights in Peptide and Protein Synthesis
"My entire yearning is directed toward the synthesis of the first enzyme. If its preparation falls into my lap with the synthesis of a natural protein material, I consider my mission fulfilled."
Emil Fischer
Peptides: The Greatest Hits
Peptides: Greatest Hits Collection(1882-present)
Scope of this meeting: (aka Lara's peptide mixtape):
www.thepeptides.com
The PepTides - "Love Question Mark" (2013)
...Rugby league player suspended after admitting to the "use and trafficking of peptides"
(August 2013)
• Brief historical perspective on peptide synthesis, outlining challenges and discoveries in the early years (1882-1960s)• Development of solid-phase peptide synthesis (SPPS)• Advances in coupling reagents and protecting groups• Chemoselective ligation chemistry for the synthesis of large peptides and proteins• Perspectives and unmet challenges• A collection of some personal favorites!
Peptides in the news:
• Non-amide bond forming ligation methods (e.g. bioconjugation, see Sella GM, May 2014 - ADCs)• Biological expression or semi-synthetic approaches (e.g. expressed protein ligation, unnatural amino acid incorporation - cf. P. Schultz)• Unnatural amino acid/peptide derivatives (!-peptides, peptoids, peptidomimetics)• Post-translational modifications (glycoslyation, sulfation, phosphorylation) and the synthesis of post- translationally-modified peptides or proteins
The Golden Oldies:Peptides as Therapeutic Leads:• ~100 therapeutic peptides currently on market• Octreotide, Goselerin, Leuprolide - top sellers• Approval rate for peptides (20%) vs. for small molecules (10%)• Anticipated global market of US$23.7 billion by 2020
Octreotide(Novartis)
"peptides can assist you during your journey to health and well-being" (https://peptidesdirect.com.au)
-EuPA Open Proteomics 2014, 4, 58 HN N
HO
HN
O
O
HN O
HN
ONH
OHN
S
SO
NH2
NH
NH2
OHHO
OH
O
NH O
OH
HN
O
O
NH O
NHN
O
NH2HN
NH
NH
OH
O
NH
OHN O
NH
O
HNNH
N
HNO
NH
O
NH2
Goselerin(AstraZeneca)
Topics not covered:
1882
1901
1932
1953
1963 present
1970
1992
1994dipeptideGly-Gly
Cbz
oxytocin
SPPS
Fmoc NCL
ligationTimeline:
Theodor Curtius
H2N OAg
O+ Ph Cl
O
Ph NH
O HN
OOH
O
Ph OH
O
+
+ 2AgCl
22
Ph NH
ON3
OH2N
HN
OOEt
O
NH
HN
OOEt
OOHN
O
Ph
NH
HN
ON3
OOHN
O
Ph
1) H2NNH22) HNO2
A
ANH
HN
ONH
OOHN
O
Ph
Acyl azide coupling
HN
OOEt
O
pentaglycine peptide
J. Prakt. Chemie 1882, 26, 145
J. Prakt. Chemie 1904, 70, 57
Theodor Curtius
- Emil Fischer, 1905(letter to Adolph von Baeyer)
HN NH
O
Oconc. HCl
"H3N
HN
OOH
O
Cl
Ber. Dtsch. Chem. Ges. 1901, 34, 2868
Ber. Dtsch. Chem. Ges. 1905, 38, 605
NH O
ClO
Br
H2NO
OEt
NH O
OEtOH
N
OBr N
H O
OHOH
N
OH2N
base/NH3
Emil Fischer
Acid chlorides in peptide synthesis and the #-bromoacyl method
glycylglycine
leucylglycylglycine"masked" Leu amine
H2NHN
O
NHO
N
O
NHO
HN
O
NH2
O O
HN
NH2
O
O
HN
ONH
O
OH
H2N
SS
Total Synthesis of Oxytocin
Vincent du Vigneaud• Head of Biochemistry - Cornell University Medical College• Studied the biochemical importance of sulfur-containing compounds (insulin, penicillin, oxytocin, vasopressin)• First total chemical synthesis of a bioactive peptide hormone• Nobel Prize in Chemistry (1955)
J. Am. Chem. Soc. 1953, 75, 4879 (Vigneaud)• Prepared using a 3-fragment condensation approach• Coupling methods: acid chlorides, anhydrides, pyrophosphite method• Purified by counter-current distribution (CCD) • No HPLC, mass spec, high resolution NMR!• Overall yield <<1%
CCD apparatus(invented by Lyman Craig)
Practical Limitations: • lack of readily cleavable N-terminal protecting groups • limited access to enantiopure amino acids
Octadecapeptide - Fischer
Leu-(Gly)3-Leu-(Gly)3-Leu-(Gly)8-Gly
Ber. Dtsch. Chem. Ges. 1907, 40, 1754
NH O
HN
OHN
OBr
O
Cl
Lara Malins Baran Group Meeting04/23/16Highlights in Peptide and Protein Synthesis
NH
R
O
PGHN
R'
OOR* N
R
O
OR*
OPGHN
R'
N
O OPGHN
R'
R
N
O OHPGHN
R'R
H N
O OPGHN
R'
R
Racemization through oxazolone formation (major)
O
O
NH
R O
O
NH
RO
O
NH
R
Cbz (Z) BocFmoc
CarpinoJACS 1957, 79, 4427McKay and AlbertsonJACS 1957, 79, 4686
Bergmann and ZervasBer. Dtsch. Chem. Ges.
1932, 65, 1192Carpino
JACS 1970 , 92, 5748
H2/PdNa/NH3 (l)HBr/AcOH
TFAHCl
base (amines)NH3 (l)
DeprotectionConditions:
Essential protecting groups (urethanes)
Ph NH
OCl
O
A convenient finding...
R
Ph NH
ONHR'
O
RH2NR'
racemizationbenzoyl
O NH
OCl
O
R
configurationallystable
PhCbz
O NH
ONHR'
O
R
Ph
H2NR'
NH
R'
O
OHO
R''O
Boc, Fmoc, Cbz (racemization-suppressing
urethane group)
NH
R
O
HN
HN
R'
O
O
R''O
CN
3
2 1
chain growth
OMe
OH2N
R
O
HN
1 OMe
O
23
NH
R
O
HN
R'
O
O
R''O2
3 OH
N-acyl groupracemization-prone
H2N1
OMe
O
NH
R
O
HN
HN
R'
O
O
R''O2 1
OMe
O3
CNchain growth
X
C-to-N:
N-to-C:
epimerization at C-2
Unidirectional chain growth:
Oxytocin
J. Am. Chem. Soc. 1959, 81, 5688 (Vigneaud)
Ber. Dtsch. Chem. Ges. 1932, 65, 1192
Improved oxytocin synthesis published in 1959: stepwise, p-nitrophenyl ester couplings, C-to-N elongation, 38% overall yield
I
II
III
du Vigneaud
Racemization through enolization (minor)
NH
R
O
PGHN
R'
OOR*
NH
R
O
PGHN
R'
OOR*
2
Lara Malins Baran Group Meeting04/23/16Highlights in Peptide and Protein Synthesis
Bruce Merrifield and Solid-Phase Peptide Synthesis (SPPS)
• Biochemist by training, moved to Rockefeller Institute for Medical Research (1949)• Work on the structure of peptide growth factors required the synthesis of a pentapeptide:
29 May 1959 - Merrifield records concept of SPPS in his lab book
"my overall yield of pentapeptide was 7%, and it took me 11 months. Certainly, an experienced peptide chemist would have done better, but not without considerable effort."
O
OLinker N
HO
O
LinkerNH
OH
OLoad resin
DeprotectN-terminus
H2NLinker
NH
OH
O
NH
O
O
LinkerOH
NLinkerPeptide 1Repeat: deprotect
and couple
i) Deprotect (global)ii) Cleave from resin
Peptide 1H2NO
OH
= solid support (resin)
= N-terminal amino protecting group
= side-chain protecting groups
Synthetic peptide product
coupleamino acid
Solid-phase peptide synthesis (SPPS)
iii) HPLC purify
Common linkers:
RCl
Trityl linker (R = H)2-Cl trityl linker (R = Cl)
ClMerrifield
(chloromethyl)
Linker =O
NH2OMe
OMe
Rink amide linker
HN
PAM linker(4-hydroxymethyl-phenylacetamidomethyl)
OOH
Common Resins:
Cle.g.
Merrifield linker
Cl
DVBcross-link
Merrifield resin
• Resin loading: 0.2 - 1.5 mmol/gram (number of functionalizable sites)• "Swellability": determines appropriate solvents
Properties to consider:
CbzHN O
ONHEt3
1)
O
O
NHEt3
2) O
OCbzHN
1) Cbz removal: 30% HBr in AcOH
(2-4 h)Cl
H2NO
NH O
HN
OOHN
H O
J. Am. Chem. Soc. 1963, 85, 2149 (Merrifield)
1984 - Awarded the Nobel Prize for SPPS
A reviewer's thoughts: the approach is "a travesty...not chemistry at all, a concept which should be surpressed by the community."
O
OH2N
2) Et3N/DMF
CbzHN OH
ODIC, DMF, rt, 18 h
1)
2) Ac2O/Et3Nrt, 2 h
O
OHN
OCbzHN
SPPSthen
NaOH/EtOH
Seminal Paper
Biopolymers 2008, 90, 175 (Mitchell)Leu-Ala-Gly-Val (8% yield of purified compound)
Merrifield
H2N O O O NH2
H2N O OO
H2N O OO
NH2
NH2
n
n
n
- polystyrene +1-2% divinylbenzene (DVB) cross-link:
- ChemMatrix polyethylene glycol (PEG):
more polar resindecreases hydrophobic aggregation
(PCT Int Appl 2005, WO 2005012277 A1)
J. Am. Chem. Soc. 1966, 88, 5319
For development of solid-phase oligonucleotide synthesis, see:
3
NN
Lara Malins Baran Group Meeting04/23/16
NH
O
O
O Oside-chain
benzyl PGs
N-terminal Boc
O
O
Boc SPPS (Boc/benzyl) Fmoc SPPS (Fmoc/tBu)
NH
O
O
O Oside-chain tBu
or Trt
N-terminal Fmoc
O
O
piperidine labile
TFA labile
TFA labile
HF labile
Pros: • Optimizable to 50-60 residues (less aggregation prone)• Stable thioester linkages
Cons:• HF safety hazard• Limited FG compatibility
Pros: • mild and safe (no HF required) • spectrophotometric quantification of couplings
Cons:• More aggregation prone• Incompatible with base sensitive substrates (e.g. thioesters)
"Assembly of the 124 amino acid residues into the protected, resin-bound straight-chain precursor of RNase required 369 chemical reactions and 11,931 steps of the automated peptide synthesis machine."
J. Am. Chem. Soc. 1969, 91, 501 (Merrifield)
Merrifield and his peptidesynthesis machine
Automation
NH
O
OR H2N
RNH
HN
-CO2
Fmoc Spectrophotometric Quantification
piperidine-fulvene adduct!= 7800 cm-1 (" = 301 nm)
fast
ResinHF labile
ResinTFA labile
"Automated Synthesis of Peptides"Science 1965, 150 , 178
Synthesis of an Enzyme with Ribonuclease A (RNase) Activity
• N-terminal Boc protecting groups• DCC as the coupling reagent - JACS 1955, 77, 1067 (Sheehan)
NN
N
OH
NHHOAt
HOBt
J. Am. Chem. Soc. 1993, 115 , 4397 (Carpino) - 1367 citations
NO
O
HO
HOXt/EDC
HOAt: complete at 22 hHOBt: trace product
OMeH2NO
OHNH O
O+
HNN
H O
O
OMe
Oconditions
DCC, 24 hHOBt, DCC, 24 hHOAt, DCC, 24 hHOAt, DCC, DCM, 24 h
DL-isomer (%)coupling conditions61.5%41.9%14.4%< 1-2%
HOAt as a peptide coupling additive
• superior leaving group• neighboring group effect
O
ONNN
X
via:
NH
R R
N NOH
N NN
N
OO
R
Improvements in Coupling Reagents
HOBt in peptide synthesisChem. Ber. 1970, 103 , 788 (König) - 965 citations
NN
N
OHHOBt
Racemizationsuppressor
Reviews: Chem. Rev. 2011, 111 , 6557 (Albericio); Tetrahedron 2004, 60, 2447 (Han)
Tetrahedron 1991, 47, 259 (Coste)
HOAt:
Oxyma: A Safer Alternative
COOEtNC
NOH
• Slightly more reactive than HOAt• Non-explosive (unlike HOBt, HOAt)
Chem. Eur. J. 2009, 15 , 9394 (Albericio)
COOEtNC
NO K
K-Oxyma: for highly acid-labile resinsEur. J. Org. Chem. 2013, 6372 (Albericio)
• non-acidic alternative to Oxyma
**Final product had enzymatic activity (though less than native RNase)
NN
N
O PNMe2
Me2NNMe2
PF6
BOP
NN
N
O PN
NN
PyBOP
PF6
Br PN
NN
PyBrOP
PF6
Tet. Lett. 1975, 16 , 1219(Castro)
Highlights in Peptide and Protein Synthesis
4
NH NH
N
NH2
OHO
NHNH
NH2Cl
H3N
H3N
NHNH
NH2
ClH3N
N
NH
HN NH2
O
HN
EDC/HOBt
HATU, HBTU, TCTU, DPPA, T3P, EDC, DIC, DCC, BOP, BOP-Cl, PyAOP, PyBOP, PyBrOP, cyanuric chloride
Other attempts:
"Alphabet Soup": Why are there so many coupling reagents?
J. Am. Chem. Soc. 2011, 133 , 14710
Lara Malins Baran Group Meeting04/23/16
• Coupling of bulky (and/or non-proteinogenic) amino acids• Considerations for removal of coupling reagent byproducts• Enhancing rate of amide bond formation• Minimizing racemization of activated acid
CbzHN OH
O
R N
NR2
CbzHN O
O
RHN
NR2
CbzHNHN
O
R
NR2
O
CbzHNHN
O
R
NR2
O
Aib couplings with the Aziridine method
Helv. Chim. Acta 1990, 73, 13Helv. Chim. Acta 1987, 70, 102 (Heimgartner)
Et2O, rt, 0.5-6 h
J. Org. Chem. 1992, 57, 5566 (Heathcock)Synthesis of (-)-Mirabazole C - Application of PyBroP
CbzHN OH
O
MeBnS
H3NOMe
O
MeBnS
Cl
HN OMe
O
MeBnS
CbzHNO
MeBnSPyBroP, DIEA
DMAP, DCM, 4 h
90%
1) HBr, HOAc
HN OMe
O
MeBnS
NH O
MeBnS
OCbzHN
MeBnS
A
2) A, PyBroPDIEA, DMAPDCM, 24 h
HN N
H
O
MeBnS
NH O
MeBnS
OHN
MeBnS
O
SBn
S
NNS
S
NMe
Me
S
N
Me S
NNS
S
NMe
Me
S
N
Me
1) Na, NH32) NH4Cl3) TiCl4, DCM NiO2, benzene
reflux, 6 h60%
63%
+ 60%
(-)-Mirabazole C
Case Studies:
Acid chloride, BOP, BOP-Cl, DPPA, DCC were unsuccessful in coupling A
!-aminoisubutyric acid(Aib)
H2NO
OH
HN
O
OHMe
N-Me-valine
Synthesis of Palau'amine
N
N
O
NN
OO N
NBoc H Boc
OH
OH
NBocN
H
NH
OH
MeO2CMe
Boc
NN
Boc H BocF
OH
Synthesis of 5-N-acetylardeemin
DCC/DMAPDCC/HOBt
BOP-Cl
C8
C15b
partial racemization(C15b or C8)
cyanuric fluoridepyridine
DCM, -15 oC
H2N CO2Me
Me+
H-Ala-CO2Me
NaHCO3, H2ODCM
71% (2 steps)
J. Am. Chem. Soc. 1999, 121 , 11953 (Danishefsky)
See also: TFFH as a reagent for acid-fluoride couplings:
JACS 1995, 117 , 5401 (Carpino)
N N
FPF6
TFFH
Choosing a coupling reagent: where to begin?
solid-phase solution-phase
aniline?
acidchloride
base sensitive?
DIC/HOBtDIC/oxyma
bulky AA,difficult
coupling
PyAOPHATU
HOAt/DICPyBrOP
PyBOPHBTU
HOBt/DIC
generalconds bulky AA,
difficultcoupling
HOBt/DIC or DCC
generalconds
HOAt/DICacid fluorideor EDC
(water-soluble)
Highlights in Peptide and Protein Synthesis
Start
5
Lara Malins Baran Group Meeting04/23/16
A Few Notable Protecting GroupsReview: Chem. Rev. 2009, 109, 2455 (Albericio)
SO2
OO
BsmocJACS 1997, 119, 9915JOC 1999, 64, 4324
SO2
OO
!-Nsmoc
SO2
OO
"-Nsmoc
> >
JOC 2007, 72, 1729
H
JOC 2007, 72, 1729
Relative sensitivity to piperidine:
5 g Fmoc-Phe-OH: $16.655 g Bsmoc-Phe-OH: $107.26
Price comparison (Alfa Aesar)
SO2
OO
HN Cl
NH
SO2
CH2
H2N Cl
-CO2
N
SO2
N
J. Am. Chem. Soc. 1997, 119, 9915 (Carpino)
Leu-Leu-Leu O
O
O NH
O
SO2
NH2N NH2
NH2
HN
Bsmoc
Me
Bsmoc removalonly
Fm removalonly10% in DCM
2% in DCM
Alternatives to Fmoc
Michaeladdition
Selective removal of Bsmoc or Fm/Fmoc
JOC 2003, 68, 4894 (Enzon Pharmaceuticals, Inc.)
O
O
O
NHO
O
NHO
O
SO2
N NH
O
O
O
NHO
O
H2N
N
N
O2S
O
OHPEG
EDC, DMAPDCM, rt, 12 h
O
O
O
NHO
O
NH
82% (2 steps)
(1 equiv.)
O
PEG
PEG 2'-paclitaxelglycinate
DCM, rt, 3 h
JOC 2003, 68, 4894 (Enzon Pharmaceuticals, Inc.)
O
O
O
NHO
O
NHO
O
SO2
H3C
O
O
OH
O
CH3 OHO
O
HO
CH3CH3
O
OH
NHO
OO
O
BsmocHN OH
O
EDC, DMAPDCM, 10 oC to rt, 1.5 h
Taxol
Application of Bsmoc protection
!"
#!!"
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Cbz-
SPPS
!Protein Functional Domains
Smallest Proteins
Chemical Ligation
!"#$%&'%(
&)*($(
+,$
%-./
"0%
12%&'%/
3"0&%/-",45%
Perspectives on Protein Synthesis
Highlights in Peptide and Protein Synthesis
Annu. Rev. Biochem. 2000 69, 923
• SPPS (and subsequent coupling reagent and PG advances) greatly improved the effiency of peptide preparation but did not substantially expand the size of accessible targets
Limitations of Solid- and Solution-phase couplings:
• On-resin aggregation generally limits routine SPPS to ~50 amino acids• Solution-phase fragment condensations (using protected peptides) are plaqued by the poor solubility of protected peptides
6
Lara Malins Baran Group Meeting04/23/16
OOC
SH
NH3
OH
SHCOO
NH3Chemoselective
ligationOOC
SHHO
NH3
OH
SHCOO
NH3
+
HO
Chemical Ligation
• unprotected peptides• aqueous solution• physiological pH
mutually reactivefunctional groups
Amide bonds through acyl transfer
H3NSPh
OClH3N
O
O
HS+
Na2CO3 H2O 0 oC
H2NO
O
S
OH2N
- PhSH
S to Nacyl shift
NH
O
O
HSO
H2N
R
Template
O S
OPG
Thiolcapture
Template
O SH
O
Template
O S
O
H2N
SH
H2NS
Template activation
O to N acyl
transfer
Template
OH S
NH S
O
O
NH
HSRelease template
Template
Prior thiol capture
A A A
A
A
B
B
B
BHO S
PG
=
Tetrahedron Lett. 1981, 22, 185 (Kemp)Tetrahedron Lett. 1981, 22, 181 (Kemp)
O
O
O
O
OAcMe
OMeS S
CO2EtH2N
O
O
OH
OAcMe
OMeS S
CO2Et
HN
O
Tetrahedron Lett. 1981, 22, 181 (Kemp)
t1/2 = 2.7 h (DMSO)28 h (DMF)
• concentration independent O to N acyl transfer
(intramolecular)N to O acyl
transfer• effective local conc. of
intramolecular amine = 0.6 M
J. Org. Chem. 1986, 51, 1829 (Kemp)
OS S
X
Z
O
X = H, Cl, Br, NO2Z = H, Cl
RO
CO2MeH2N
OS S
X
Z
HO
CO2MeHN
OR
DMSO, 25 oC
ring substituents
9.7 x 10-5
4.8 x 10-4
1.1 x 10-3
7.6 x 10-5 7.8 x 10-4
1.2 x 10-3
2.4 x 10-1
3.5 x 10-3
7.6 x 10-3
R
HBrClHBrClNO2HCl2
HHHHHHH3ClCl2
X Zacyloxy
CH3CH3CH3Z-AlaZ-AlaZ-AlaZ-AlaZ-AlaZ-Ala2
k1(s-1) t1/2k1 /kH
(rel rate)
2 h24 min10 min2.5 h15 min10 min2.8 s 33.3 min1.5 min2
1 (ref)4.9111 (ref)10 153200 345 1002
Effectivemolarity (EM)*
4.6
4.55.1
5.78.6 34.9 2.72
*EM determined from the ratio of k1 to k2 (k2 = rate constant for intermolecular aminolysis of each 1- and/or 3-functionalized 4-(acyloxy)dibenzofuran template with H-Cys(Bn)-OEt in DMSO at 25 oC
(deprotection)
(reduction)
HO S
n = 0, 1
R'
PG
Optimized templates - 4,6-dibenzofurans
acyl shift
MRTGGA
O
OO
SHBocHN
S
O
HN
O
H2N
S
O
S
O
O
+
1) HFIP/H2O (thiol capture)2) DMSO (acyl transfer)3) PEt3, DCM/HFIP (template cleavage)
GGANH
HS
OMRT
O
BocHN
S
O
HN
O
PG
PG
4) Dnp-F, DIEA (Cys protection)5) TFA/thioanisole (remove non-Cys PGs)6) Scm-Cl (convert Acm to Scm)
GGANH
DnpS
OMRT
O
H2N
S
O
S
O
O Repeat 1-6 for2 more ligations
Cys(Acm) Cys(Scm)
BPTI 30-5829 residue peptide
Total Synthesis of BPTI - Basic Pancreatic Trypsin Inhibitor (30-58)
Justus Liebigs Ann. Chem. 1953, 583, 129 (Wieland)
J. Org. Chem. 1989, 54, 2803 (Kemp)
Highlights in Peptide and Protein Synthesis
Annu. Rev. Biochem. 2000 69, 923Chem. Soc. Rev. 2009, 38, 338
7
Lara Malins Baran Group Meeting04/23/16
6 M Gn•HCl/0.1 M Na2HPO4
pH = 4.3O
SHHN
OBr+A B
O
SHN
OA B
Thioester ligation - "Backbone-engineered" proteinsScience 1992, 256, 221 (Kent)
thioester linkage
H2NO
S
OO
NH
NH O
S
OO
NH
HIV-1 PR(1-50)
51
51
NH O
SHHIV-1 PR(1-50) 51
HO
HIV-1 PR(53-99) O
OBr
HIV-1 PR(53-99) OH
OBr
52
52
HIV-1 PR(1-50) NH O
SO
51 HIV-1 PR(53-99) OH52
HFHF
automatedBoc-SPPS
automatedBoc-SPPS
Synthesis of HIV-1 protease:
6 M Gn•HCl/0.1 M Na2HPO4pH = 4.3, 48 h
[peptides] = 4 mM
assembled HIV-1 PR dimer exhibits enzymatic activity
O
SRHN
OH2N+
HS Transthio-esterification
HN
OH2N
S
OS!N
acyl shift
HN
ONH
HSO
A B
A
A
B
B
thioesterpeptide N-terminal Cys
peptide
Native Chemical Ligation (NCL)
slow fast
O
IL-8 (1-32) S HN
OH2N
+HS
IL-8 (35-72)SH
SHSH
O
NH
HN
O
HS
Folded IL-8
HN 33
34
HN 33
34IL-8 (1-32)
SH
SH
IL-8 (35-72)
SH
HS
6 M Gn•HCl/phosphate buffer
pH = 7.623 oC, 48-72 h
Science 1994, 266, 776 (Kent)
thiol additive*Science 1994, 266, 776 (Kent)
Total Synthesis of Interleukin-8:
*Thiol additive serves as a reductant to prevent disulfide formation and to reverse unproductive thioesterification of internal Cys residues
Rate enhancement with thiol additives
O
STransthio-
esterification+
O
S +HS
Benzyl thioester Thiophenyl thioesterHS
A A
barnase (50-110)*HN
OH2N
HS
49
O
barnase (1-47) SHN 48
+
HN
ONH
HS
49
OHN 48
6 M Gn•HCl/0.1 M Na2HPO4 pH = 7.5
barnase (50-110)*barnase (1-47)
For benzylthiol:
easier to handle(HPLC stable)
more reactivein ligations
HS
complete at t = 7 h< 25% complete at t = 7 h
HS4 vol% or
For PhSH:
J. Am. Chem. Soc. 1997, 119 , 4325 (Dawson, Ghadiri, Kent)
• Construction of native amide bonds in aqueous media• Completely chemoselective (alcohols, amines, acids, internal thiols, etc. are all tolerated) - allows use of unprotected peptides
Comparative synthesis of Barnase (1-110):J. Am. Chem. Soc. 1997, 119 , 4325 (Dawson, Ghadiri, Kent)
Highlights in Peptide and Protein Synthesis
8
Lara Malins Baran Group Meeting04/23/16
J. Am. Chem. Soc. 2006, 128 , 6640 (Kent)
O
SR
HS R'S
RHO SR'
SR'
O
pKa < 4 - inefficient at transthioesterification
H2N
HS
O
pKa ! 9inefficient LG
in ligation
Ideal thiol additive: 6 < pKa < 9
Fine-tuning with thiol additives
HS NH2
HS
HSO
HO
NHSHS
HSO
HO
4-mercaptophenylacetic acid (MPAA)
• stable solid• water-soluble• rapid ligation• no epimerization• minimal stench!
A A A
B
Kinetically-controlled ligations
OSA
O
SBH2N
HS
O
NH2
O
latent alkyl thioester
CH2N
HS
O
ligation #1 ligation #2(after activation)
Angew. Chem. Int. Ed. 2006, 45, 3985 (Kent)
- Exploiting thioester reactivity allows for Iterative ligations
PNAS 2007, 104 , 4846 (Kent)
KVFERCELAR TLKRLGMDGY RGISLANWMCLAKWESGYNT RATNYNAGDR STDYGIFQINSRYWCNDGKT PGAVNACHLS CSALLQDNIADAVACAKRVV RDPQGIRAWV AWRNRCQNRDVRQYVQGCGV
Convergent Synthesis of Human Lysozyme
1-29O
S
130 amino acids - 4 fragments, 3 ligations
O
OH
31-64O
SH2N
HS
O
Acm
R
1-29OAcm
31-64O
SNH
HS
O
R
66-94O
SNH
O
RS
96-130H2N
HS
O
66-94NH
O
S O
NH
HS
O
96-130
AcmAcm
Acm Acm
MeONH2, pH = 4Thzremoval
66-94O
NH
HS
O
96-130
Acm Acm
H2N
HS
O
kinetically-controlledligation (KCL)
ligation
66-94O
NH
HS
O
96-1301-29
O31-64N
H
HS
O
O
NH
HS
O
MPAA
1) ligation: MPAA2) Acm removal: AgOAc, DTT
PNAS 2007, 104 , 4846 (Kent)Convergent Synthesis of Human Lysozyme
66-94O
NH
HS
O
96-1301-29
O31-64N
H
HS
O
O
NH
HS
O
130 amino acids - 4 fragments, 3 ligations
kinetically-controlledligation
native chemicalligation
native chemicalligation
One-pot KCL to homogeneous EPO
1-29O
S
O
OH
Acm
31-78O
SEtH2N
HS
O
80-166H2N
HS
O
Acm
Acm
O
OH
HOHO
NHAc
O O
NHAcHO
OH
O
OH
ONHAc
O
AcHNO
OHOHO
O
HOAcHN
HOHO OH
OHO
AcHN
HOHO
OH
HO2C
CO2H
=
=
1-29
Acm O
NH
HS
O
31-78
Acm O
NH
HS
O
80-166
Acm
KCL, 5 h; then: MPAA,
One-potkinetically-controlled
ligation
Angew. Chem. Int. Ed. 2012, 51, 11576 (Danishefsky)
EPO (1-166) precursor
See also: Science 2013, 342, 1357"At last..."
Highlights in Peptide and Protein Synthesis
9
HS R
Lara Malins Baran Group Meeting04/23/16
S S
Thia-zip reaction
Method for the synthesis of cysteine-rich cyclic peptides
cyclopsychotrideJ. Nat. Prod. 1994, 57, 1619
- one of the largest known cyclic peptides (31 AAs)- 6 cysteine residues and 3 disulfide bonds
CSCKSKVCYKNSIPCGESCVFIPCTVTALLG
S SS S
J. Am. Chem. Soc. 1999, 121, 4316 (Tam)
H2N
HS SH SH SH SH SH SR
O H2N
HS SH SH SH SH S O
H2N
HS SH SH SH SH S O
thiolactoneexchange
pH = 7.5(8 M urea)
S
O
NH2
SHSH
acyl shiftsSHSHSH
NH
OHS
SHSH
SHSHSH
S to Nacyl shift irreversible
[O]DMSO
disulfide formation
cyclopsychotride
(20 h - 24 h)
Thia-zip cyclization is 200x faster than unassisted cyclization
blocking terminal thiol prevents
lactam formationblocking internalthiol still leads
to lactam formation
What about Cys-free peptides and proteins?
HS OHN
HS NH
HNHS
HNHS
OMe
HNHS
O2N
HNHSHNHS
MeOOMe
HNHS
MeOOMe
OMe
R R
R
R = H, OMe R = H, OMe
HNHS
NO2
OHN
R
HSA
H2N A
HSN-terminal
Auxiliary-basedCys surrogates
HNHS
JACS 1996, 118 , 5891 Bioorg. Med. Chem. 2001, 9, 2323Tet. Lett. 2001, 42, 1831PNAS 2001, 98, 6554
Tet. Lett. 2003, 44, 6059Bioorg. Med. Chem.
2004, 12 , 2714
Org. Lett. 2000, 2, 23Org. Lett. 2001, 3, 1403
Bioorg. Med. Chem. Lett. 2002, 12 , 1963J. Am. Chem. Soc. 2002, 124 , 4642
=
H2N A
HSSide-chain
Auxiliary-basedCys surrogates
RHN
HS
A
HSO
OHO
NH
OHHO
OHS
OO
ONHAc
OHHO
OHS
ONH
OHSO NH
OHS
O
O
OHO
HN
OH
OHSO
HN
OHO
NH
OHHO
OHSO
HO
JACS 2006, 128 , 5626Chemistry 2007, 13 , 5670
JACS 2006, 128 , 15026JACS 2007, 129 , 7690
Angew. Chem. Int. Ed.2007, 46, 5975 Chem. Commun. 2008, 10 , 1229
=
HSHN
Angew. Chem. Int. Ed.2015, 54, 15055
Relative abundance (Cys) = 1.7%
ReductiveHN
ONH
HSO
A BHN
ONH
O
A B
Cys Ala
Ligation-Desulfurization
Radical
Pd/Al2O3, H2
VA-044, TCEP, tBuSH
Reductive: JACS 2001, 123 , 526 (Dawson) - 373 citationsRadical: ACIE 2007, 46, 9248 (Danishefsky) - 311 citations
NNH
N
NNH
N
VA-044HO2C P CO2H
HO2C
TCEP
BocHNO
OH
RS
O
OHN
TrtS
BocHNO
OH
RSO
OtBu
R
Thiol amino acids:
e.g.Reviews: Curr. Opin. Chem. Biol. 2014, 22, 70Aust. J. Chem. 2015, 68, 521
Highlights in Peptide and Protein Synthesis
10
Science 2000, 287, 2007 (Bertozzi)
OMeO
PPh2
O OH
OMeO
PPh2
O O
OH
Staudinger ligationReview: Angew. Chem. Int. Ed. 2004, 43, 3106
sampleN3
OMeO
PPh2
O O
sampleN
MeO
PPh2
O O
sampleNO
O
PPh2
O O
sampleHNO
aza ylide-N2
"Cell-surface engineering"
• "Bioorthogonal" tagging• aqueous media• on the surface of living cells• inside living animals (see: Nature 2004, 430, 873) fast
Lara Malins Baran Group Meeting04/23/16
O
S +A B"traceless" auxiliary
PPh2 N3
O
SA PPh2N B
O
NA B
PPh2
HS
O
NH
A B
Ph2P SH
O
-N2
H2O
Org. Lett. 2000, 2, 2141 (Bertozzi)Org. Lett. 2000, 2, 1939 (Raines)
Org. Lett. 2001, 3, 9 (Raines)"Traceless" Staudinger Peptide Ligation
HO PPh2
HS PPh2SH
PPh2
OHPPh2
N
HNPh2P
HS
PAr2 NAr =
J. Am. Chem. Soc. 2007 129 , 11421 (Raines)
NH NH
N
NH2
SO
Ph2P
BH3
NHNH
NH2Cl
N3
N3
NHNH
NH2
ClN3
N
NH
HN NH2
O
HN
DABCO60 oC
20%
Angew. Chem. Int. Ed. 2003, 42, 4373 (Maarseveen)Staudinger Ring-Closure (Medium-Sized Lactams)
J. Am. Chem. Soc. 2011, 133 , 14710
H2O soluble
KAHA (Ketoacid-hydroxylamine) ligationsAngew. Chem. Int. Ed. 2006, 45, 1248 (Bode)
HN
ONH
+R
HN
ONH
ROA B A B
OOH
O
HO-CO2
DMF, 40 oC(50 - 100 mM)
-H2O
BocHN OH
O
R
SNC
Br
BocHN
R
O
S
CN BocHN
R
O
O
OH
HBTU, DIEADCM, rt
Oxone1:1 DMF/H2O
J. Am. Chem. Soc. 2008, 130 , 4253 (Bode)
O O
HN
S
O O
HN
S
Rink resin
CN
PF6
OO
HN
SO
CN
FmocHN
1:2 MeCN/PhMe15 h, 60 oC
PF6 H2C-CN
OO
NH2
SO
CN
HNpeptide
O
O
HNpeptide OH
Fmoc-Phe-OHHATU, HOAt DIEA, DMF
Fmoc-SPPSTFA
OxoneDMF/H2O
cyanosulfur ylideSolid-phase approachOrg. Biomol. Chem. 2009, 7, 2259 (Bode)
modification of cellsurface glycans
Highlights in Peptide and Protein Synthesis
11
OHN
O
A B
OOH
O
0.1 - 5 mM20-25 oC
O
BONH
HN
ONH
O
A B
HO
oxazetidine
KAHA: Increased reactivity through ring-strain
A
OOH
O
oxaproline
1) 10 - 15 mM40-60 oC H
N
ONH
O
A B
OH
Nature Chem. 2015, 7, 668 (Bode)
2) pH = 10.5(O to N acyl
shift)
MeO
OMe
NH
OO
MeO
OMe
NO
OH
BF3•OEt2, DCM-40 oC to 0 oC
NO
OHFmoc
Ooxazetidinebuilding block
Lara Malins Baran Group Meeting04/23/16
Perspectives and Future Directions
Highlights in Peptide and Protein Synthesis
• Routine preparation of small to medium peptides by SPPS is generally rapid and efficient• Small proteins and enzymes are now accessible by chemical ligation methods (NCL, Staudinger ligation, KAHA, etc) employing unprotected peptides from SPPS
• Rapid and efficient on-resin ligation chemistry: e.g. an automated, programmable solid-phase "ligation machine" for protein synthesis
Some Ideals:
• Unnatural amino acids employed in ligation chemistry (oxazetidines, thiol amino acids, ketoacid precursors) should be commercialized and cost-effective (as accessible as coupling reagents)
• Improvements in coupling reagents and protecting groups allows the construction of most amide bonds (particularly between proteinogenic amino acids)
The State-of-the-art:
Unmet Challenges:• "inaccessible" structures: modified peptides (bearing complex post-translational modifications or non-native structural motifs)
HN N
H
O
O
Ph
NH
HN
O
O
H
S
13
22
S to !-Ccross-links
Subtilosin A
• Facile synthesis of peptides bearing non-proteinogenic or heavily modified amino acids
Astexin-1
"Rethinking amide bond synthesis"Review: Nature 2011, 480, 471 (Bode)
O
HR' R NH2
O
NH
R' R
CuI (1 mol%)AgIO3 (1 mol%)
TBHP
Ar
NHBoc
NO2
BrNIS, baseH2O, 0 oCH2N
Me
O
NH O
OMe HN
Me
O
NH O
OMe
O
Ar
BocHN
+
+
J. Am. Chem. Soc. 2010, 132 , 4098
O
SHBocHNHN
O
OBn
Me
TsOH•
O
NBocHN
Me O
OBn+
CN
CHCl3
Nature 2010, 465, 1027
J. Am. Chem. Soc. 2006, 128 , 13064
12