Solid-Phase Peptide Synthesis (SPPS)
• Chain assembly
• Cleavage from resin and removal of side-chain
protecting groups
• Purification
• Additional chemical modification
• Characterization
~ first introduced by Bruce Merrifield in 1963
Comparison of Boc and Fmoc SPPS
Boc FmocRequires special equipment
Yes No
Cost of reagents Lower Higher
Solubility of peptides Higher Lower
Purity of hydrophobic peptides
High May be lower
Problems with aggregation
Less frequently More frequently
Synthesis time ~20 min/amino acid~20-60 min/amino acid
Final deprotection HF TFA
SafetyPotentially dangerous
Relatively safe
Solid Support - Resin
• Resin for SPPS: polystyrene bead with 1% divinyl-benzene, a
cross-linking agent.
• Dry resin beads: 40-100 microns, 100-200 or 200-400 mesh
• When in contact with solvents, the beads swell to approximately
10 times their dry volume.
• Macroscopically, the resin appears as an insoluble solid
support. However, on the molecular level the resin is “in
solution” or fully solvated.
• This solvation enhances coupling of the peptide resin with the
protected amino acids.
Fmoc Resins
• HMP resin (4-hydroxymethyl-phenoxymethyl-copolystyrene-1% divinylbenzene resin), also known as Wang resin produces a carboxylic acid terminal peptide
• Amide resin – produces an amide terminal peptide• MAPS resin (multiple antigenic peptides resin)
Protected Fmoc Amino Acid Derivatives
• Asp(OtBu) ; Glu(OtBu) ; Asn(Trt) ; Gln(Trt)• Arg(Pmc) ; His(Trt) ; Lys(Boc)• Ser(tBu) ; Thr(tBu) ; Tyr(tBu) ; Cys(Trt)
General Protocols
- Fmoc chemistry
Loading
Deprotection
wash
Activation
Coupling
wash
Repeat ~
Cleavage from
resin
Loading and Capping
DCC (N,N’-dicyclohexylcarbodiimide)
DMAP (4-Dimethylaminopyridine)
Acetic (benzoic) anhydride
Coupling Efficiency Vs. Peptide Length
PeptideLength
Coupling Efficiency
Coupling Efficiency
Coupling Efficiency
Coupling Efficiency
Coupling Efficiency
0 0.995 0.99 0.98 0.97 0.965 0.98 0.95 0.92 0.89 0.8510 0.96 0.91 0.83 0.76 0.6915 0.93 0.87 0.75 0.65 0.5620 0.91 0.83 0.68 0.56 0.4625 0.89 0.79 0.62 0.48 0.3830 0.86 0.75 0.56 0.41 0.3135 0.84 0.71 0.50 0.36 0.2540 0.82 0.67 0.45 0.30 0.2045 0.80 0.63 0.41 0.26 0.1750 0.78 0.60 0.37 0.22 0.1455 0.76 0.58 0.34 0.19 0.1160 0.74 0.55 0.30 0.17 0.0965 0.73 0.53 0.27 0.14 0.0770 0.71 0.50 0.25 0.12 0.06
Activation – HBTU/HOBt
HBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluroniumHOBt: 1-hydroxybenzotriazole
HBTU activation ~ FastMoc chemistry !
Coupling
Objectives: maximize solvation and minimize hydrogen bonding
DMF (dimethylformamide) ; NMP (N-methylpyrrolidone)
DIEA (diisopropylethylamine)
Purification
• Filtration and DCM wash
• Concentration by Rotavapor
• Ether extraction
• Lyophilization
• Purification by HPLC
Additional Chemical Modification
• Disulfide bond formation
• Phosphorylation
• Biotinylation
• Farnesylation
• Glycosylation
• C- and N-terminal modification
• Chromophore and fluorophore labelling
How to Choose Peptide Solvents
• Peptides with a net positive charge:
(1) H2O alone
(2) gently shake / warm up to 30oC
(3) 10% HOAc
• Peptides with a net negative charge:
(1) H2O or HOAc
(2) NH4HCO3
• Peptides with a net zero charge:
(1) H2O, HOAc, warming and shaking
(2) 6M guanidine-HCl, TFA, HCOOH
(3) MeOH, isopropanol, acetonitrile
Characterization
• Purity analysis by HPLC
• Amino acid composition analysis by precolumn PITC
derivatization on a PicoTag HPLC system
• Determination of peptide molecular weight by mass
spectrometry
HPLC- Purity Analysis
(1) Column : JUPITER 5u C18, 250 x 4.60 mm , 300 Å (phenomenex)
(2) Eluent A : 0.1% TFA
(3) Eluent B : 0.08% TFA in 80% CH3CN
(4) Gradient :
Time(min)
Flow rate(ml/min)
Eluent A(%)
Eluent B(%)
Initial 1.00 100 0
30.00 1.00 0 100
40.00 1.00 0 100
40.01 0.00 0 100
(5) Sample preparation : appropriate amount in d.d. H2O
(6) Loading : 1 mL
Amino Acid Composition Analysis
(1) Column : Pico Tag for amino acid composition analysis (Waters)
(2) Eluent A : 0.1 M NH4OAc, 0.03 M NH4(SO4)2, 0.04% AcOH
(3) Eluent B : 0.1 M NH4OAc, 50% CH3CN
(4) Gradient :
Time(min)
Flow rate(ml/min)
Eluent A(%)
Eluent B(%)
Initial 1.00 100 0
10.00 1.00 95 5
35.00 1.00 45 55
42.00 0.00 0 100
42.01 0.00 0 100
(5) Sample preparation : appropriate amount in 2 mM NaOH
(6) Loading : 20 µL
Amino Acid Composition Analysisa.a. standard area pmol area/pmol sample area pmole 理論值 實驗值Asx 146761 250 587.04 116669 199 3 2.9
Glx 131146 250 524.58 105742 202 3 2.9
Ser 141532 250 566.13 29319 52 1 0.7
Gly 156800 250 627.20 142010 226 3 3.3
His 145333 250 581.33 40358 69 1 1.0
Thr 145038 250 580.15 0 0 0 0.0
Ala 159585 250 638.34 0 0 0 0.0
Arg 152570 250 610.28 0 0 0 0.0
Pro 153833 250 615.33 0 0 0 0.0
Tyr 157653 250 630.61 82851 131 2 1.9
Val 160931 250 643.72 43161 67 1 1.0
Met 155710 250 582.84 82829 142 2 2.0
Ile 177989 250 1471.96 0 0 0 0.0
Leu 183749 250 935.00 0 0 0 0.0
Phe 150162 250 560.65 0 0 0 0.0
Lys 229405 250 757.62 68605 91 1 1.3
PS3 Peptide Synthesizer - PTI
PS3- a lot cheaper and easier to use!
- Simple and fast
cycle time under 40 mins/coupling
- Variety of coupling techniques
- Zero-dead-volume fluid valve system
- Self diagnostic program
- Higher productivity
up to 45 couplings automatically
3 different peptides sequentially
Symphony Peptide Synthesizer - PTI
Symphony/Multiplex 12-channel solid-
phase synthesizer
- Fast multiplex operation
operate 12-channel simultaneously
- Patented multiplexing matrix valve
- Lower coupling reagent cost
- Variable scales: 0.005-0.35 mmol
- Automated cleavage
- Easily customized protocols
- Extreme versatility
Microwave Peptide Synthesizer - CEM
Odyssey System on a Discover platform
World’s first microwave peptide
synthesizer wins 2004 R&D 100 Award!
-Significantly increased reaction rates
cycle time less than 10 mins
- Better product purity and yield
- Overcoming chain aggregation
- Automated cleavage within 15 mins
- Lower cost: cheaper reagents
- Useful on multiple programmable scale
- Greater flexibility
PepSy Peptide Synthesizer - Zinsser
Parallel synthesis of peptide libraries in
96-well plate format.
- 9 independent 96-well reactor stations
- 864 peptides in 30 h, 10 mer, ~ 1 mg each
- Dispensing pen for each a.a.
- no washes or flushes needed
- speeds up synthesis
- no cross contamination
- Bar code check for every step
- Software-assisted library design
Applications of Synthetic Peptides
• Peptide Vaccine
• Antimicrobial Peptides (AMPs)
~ Host-Defense Peptides (HDPs)
• Peptide Array (Peptide Chips)
• Stimulus-Responsive Peptides
Applications of Stimulus-Responsive Peptides
Chockalingam, K. et al. Protein Engineering, Design and Selection 2007 20:155-161; doi:10.1093/protein/gzm008