Solid-Phase Peptide Synthesis

(SPPS) and Applications of

Synthetic Peptides

Rajendra A Sonawane

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

Strategies for SPPS Chain Assembly

Boc (t-butyloxycarbonyl) Fmoc (9-fluorenylmethoxycarbonyl)

Protecting Group Strategies in SPPS

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

General Protocols- Fmoc chemistry

Loading and Capping

DCC (N,N’-dicyclohexylcarbodiimide)

DMAP (4-Dimethylaminopyridine)

Acetic (benzoic) anhydride

Deprotection - Piperidine

Conductivity monitoring

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)

Cleavage from Resin and Removal of Side-Chain Protecting groups

TFA Cleavage – 95% TFA + Scavengers

Scavengers

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

HPLC- Purity Analysis

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 Analysis-Standard

Amino Acid Composition Analysis-Sample

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

ABI 433A Peptide Synthesizer

ABI 433A – Front View

ABI 433A – Rear View

ABI 433A – Flow Schematics

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

Peptide Vaccine

Peptide Vaccine

Peptide Vaccine

Antimicrobial Peptides (AMPs)~ Host-Defense Peptides (HDPs)

Antimicrobial Peptides (AMPs)~ Host-Defense Peptides (HDPs)

Peptide Array (Peptide Chips)

Peptide Array (Peptide Chips)

Peptide Array (Peptide Chips)

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