Lara Malins Baran Group Meeting 04/23/16 Highlights 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 H N N H O H N O O HN O HN O N H O H N S S O NH 2 NH NH 2 OH HO OH O N H O OH H N O O N H O N H N O NH 2 HN NH NH OH O NH O HN O NH O HN NH N H N O N H O NH 2 Goselerin (AstraZeneca) Topics not covered : 1882 1901 1932 1953 1963 present 1970 1992 1994 dipeptide Gly-Gly Cbz oxytocin SPPS Fmoc NCL ligation Timeline: Theodor Curtius H 2 N OAg O + Ph Cl O Ph N H O H N O OH O Ph OH O + + 2AgCl 2 2 Ph N H O N 3 O H 2 N H N O OEt O N H H N O OEt O O H N O Ph N H H N O N 3 O O H N O Ph 1) H 2 NNH 2 2) HNO 2 A A N H H N O N H O O H N O Ph Acyl azide coupling H N O OEt 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 O conc. HCl " H 3 N H N O OH O Cl Ber. Dtsch. Chem. Ges. 1901, 34, 2868 Ber. Dtsch. Chem. Ges. 1905, 38, 605 N H O Cl O Br H 2 N O OEt N H O OEt O H N O Br N H O OH O H N O H 2 N base/ NH 3 Emil Fischer Acid chlorides in peptide synthesis and the #-bromoacyl method glycylglycine leucylglycylglycine "masked" Leu amine
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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."
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."
"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
• 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
*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
• Construction of native amide bonds in aqueous media• Completely chemoselective (alcohols, amines, acids, internal thiols, etc. are all tolerated) - allows use of unprotected peptides
• 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)