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Microwave-Enhanced Solid Phase Peptide
Synthesis
Jonathan M. Collins
Bioscience Division
CEM Corporation
Microwave SPPS Reaction Vessel
Standard Microwave Fmoc SPPS Parameters
Deprotection
Time
Temperature
Bubbling
Enhanced
Deprotection,
and
30 seconds (5)*
40ºC (25ºC)* Coupling,
N
3 minutes (15)*
80ºCReactions
(25ºC)*
N
Cleavage
2
2
Coupling
Time
Temperature
Bubbling
5 minutes (60)*
80ºC (25ºC)*
N2
*Typical Conventional Parameters
Galanin Synthesis
GWTLNSAGYLLGPQQFFGLM-NH2
Conditions
Scale:
0.10mmol w/ MBHA Rink Amide Resin, 0.59meq/g. (Novabiochem)
Enhanced
Deprotection, Coupling, and
A= Conventional
B= Microwave Cleavage Reactions
Reaction
Reagents
Time
Max Temp.
(ºC)
Deprotection
20% Piperidine / DMF
3 min
21=A, 75=B
Coupling
HBTU/HOBt/DIEA 0.9/1/1 ,
x10 excess
4 min
21=A, 75=B
Cleavage
Reagent K
18 min
38=A and B
GWTLNSAGYLLGPQQFFGLM-NH2
™
Conventional
(Purity = < 10 %)
™
Microwave
(Purity = 84.8%)
108-123Angiogenin
ENGLPVHLDQSIFRRP
Conditions
Enhanced
Deprotection, Coupling, and
Scale:
0.25mmol w/ Pro-Chlorotrityl Resin, 0.61 meq/g. (Anaspec)
Cleavage Reactions
Reaction
Reagents
Time
Max Temp.
(ºC)
Deprotection
20% Piperidine / DMF
3 min
75
Coupling
HBTU/DIEA 0.9/1 , x4 excess
4 min
75
Cleavage
TFA/TIS/H2O
18 min
38
ENGLPVHLDQSIFRRP
™ Crude purity =
86.7%
™ Total synthesis
time = 5 hours
Proinsulin C-peptide
EAEDLQVGQVELGGGPGAGSLQPLALEGSLG
Conditions
Scale:
0.25mmol w/ MBHA Rink Amide Resin, 0.60meq/g. (Novabiochem)
Enhanced Deprotection, Coupling, and
Cleavage Reactions
Reaction
Reagents
Time
Max Temp.
(ºC)
Deprotection
20% Piperidine / DMF
3 min
75
Coupling
PyBOP/DIEA 0.9/2 , x6 excess
4 min
75
Cleavage
TFA/TIS/H2O
18 min
38
EAEDLQVGQVELGGGPGAGSLQPLALEGSLG
™ Crude purity =
75.0%
™ Total synthesis
time = 10 hours
Microwave Peptide Synthesis
I.
Microwave Application to SPPS
II.
Microwave SPPS
A. Racemization
Enhanced
Deprotection, Coupling, and
B. Aspartimide
formation Reactions
Cleavage
C.
(δ-lactam formation) - Arginine
D.
Difficult peptides (microwave vs. conventional)
E.
Phosphopeptide synthesis
Aggregation
™ The peptide backbone, coupling reagents, and
solvent (DMF) are all polar and will attempt to
align with the alternating electric field of the
microwave. At 2450MHz, 1 alteration ~ 10-9s.
™ Random motion of the chain breaks up aggregation
A. Racemization during SPPS
™ Conventional synthesis has shown racemization
can be an issue with His and Cys residues
Enhanced
Deprotection,
Coupling,
and
™ Aspartimide Formation of Asp-Gly segments can
CleavageofReactions
lead to racemization
Asp and other side
products
™ Microwave coupling is performed with a 5-min.
reaction, at 80°C maximum temperature
Racemization of Cysteine
O
H
N
OH
H
SH
N
R3
R1
1Han
R2
™ α-carbon proton is
susceptible to direct
proton abstraction by
tertiary amines during
coupling1
™ Preactivation can
significantly elevate
racemization levels for
Cys
et al. (1997) J. Org. Chem. 62, 4307-4312.
Racemization of Histidine
H
N
O
OH
H
Nπ
Nτ
H
1Harding,
™ π-nitrogen is closer to the
α-carbon and sufficiently
basic to abstract a proton
™ τ -nitrogen more
accessible and easier to
protect (Trt)1
™ Racemization at higher
temperatures is an issue
during activation state
S.J.; Jones, J.H.; Sabirov, A.N.; Samukov, V.V.; J. Pept. Sci.; 5, 368 1999.
Aspartimide Formation in SPPS
O
O
N
H
NH
N
H
O
N
H
O
O
O
NH
OH
NH
O
N
H
O
O
N
Hydrolysis
N
H
Nu
Nu
X
OH
O
NH
O
NH
N
H
O
Nu
Investigation of Racemization during
Microwave SPPS
Research Peptide
VYWTSPFMKLIHEQCNRADG-NH
2
™ Consists of all 20 amino acids including Cys, His
™ C-terminal “Asp-Gly” Segment for maximum
aspartimide potential and racemization of Asp
VYWTSPFMKLIHEQCNRADG-NH2
™ Unacceptable levels of
racemization of Asp,
Cys, and His residues
™ Addition of 0.1M
HOBt during
deprotection reduced
Asp racemization
™ Addition of HOBt to
the coupling solution
had no benefit
Reducing Aspartimide Formation
Piperazine vs. Piperidine
N
N
N
Piperazine
pKa = 9.8
Piperidine
pK= 11.1
™ Piperazine has a slower Fmoc removal rate, but has shown
decreased levels of aspartimide formation
™ Piperazine is a non-controlled substance and less toxic and
odorous than piperidine
Tregear, G., Macris, M., Mathieu, M.N., Wade, J.D.; Pept. Sci., 7, 107 2000
VYWTSPFMKLIHEQCNRADG-NH2
™ Microwave energy
allows for complete
Fmoc removal in 3minutes w/ piperazine
™ Piperazine
substantially reduced
racemization of Asp
Tertiary Amines - SPPS Coupling
N
O
N
Enhanced Deprotection, Coupling,
N and
Cleavage
NMM ReactionsTMP (Collidine)
DIEA
pKa = 10.1
pKa = 7.41
pKa = 7.43
™ Hindered amines used for reducing racemization
VYWTSPFMKLIHEQCNRADG-NH2
™
NMM elevated
racemization levels
compared to DIEA
™
Possibly due to
slower coupling
rates
™
TMP significantly
reduced Cys
racemization
™
Selection of base
does effect His
racemization
VYWTSPFMKLIHEQCNRADG-NH2
™
Lower coupling
temperatures (55ºC)
significantly reduces
His racemization
™
Cys moderately
effected
™
Racemization limited to
activated ester state
™
Elevated temperatures
up to 80ºC do not
increase racemization
of Cys and His already
incorporated on a
peptide chain
C. δ-lactam formation of Arginine
Fmoc
H
N
O
Act
Fmoc
H
N
O
NH
N
NHR
H
Enhanced Deprotection, Coupling, and
NH
NH Cleavage Reactions
NHR
™ Competitively occurs with peptide bond formation
™ Can be a significant problem for difficult Arg couplings
δ-lactam formation of Arginine
ABRF 1992 Peptide:
GVRGDKGNPGWPGAPY
™ Lactam formation of arginine causes major
deletion during synthesis
GVRGDKGNPGWPGAPY
Synthesis Conditions
1. Non-microwave
2. 20% Piperidine / DMF
3. HBTU/DIEA
4. Tyr(tBu)-Wang Resin
(0.88meq/g)
™
Significant Arg
deletion
GVRGDKGNPGWPGAPY
Synthesis Conditions
1. Microwave
2. 20% Piperidine / DMF
3. HBTU/DIEA
4. Tyr(tBu)-Wang Resin
(0.88meq/g)
™
Significant Arg
deletion
™
Significant aspartimide
formation –18 co-elute
w/ product and
del(Arg)
PEG vs. PS Based Resins
PEG Based
From www.matrix-innovation.com
PS Based
GVRGDKGNPGWPGAPY
Synthesis Conditions
1. Microwave
2. 5% Piperazine w/ 0.1M
HOBt / DMF
3. HBTU/DIEA
4. PAL-ChemMatrix Resin
™
Elimination of Arg
deletion
™
Elimination of
aspartimide
formation
1-42ß-amyloid
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
MW = 4514.12
™ Difficult peptide to synthesize
™ Difficult peptide analyze
Synthesis
PAL-PEG-PS Resin ; 0.2meq/g (Applied Biosystems)
20% Piperidine in DMF
HBTU/DIEA activation
1-42ß-amyloid
DA - EFRHDSGYEV - HHQKLVFFAE - DVGSNKGAII - GLMVGGVVIA
9.6% Purity
37.2% Crude Yield
Conventional
™ Microwave Energy increased purity
™ Total synthesis time of 19 hours
68.8% Purity
40% Crude Yield
Microwave
Chemokine SDF-1a
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQV
CIDPKLKWIQEYLEKALNK
MW = 7963.5
0.1mmol synthesis
Lys-NovSyn Resin
20% Piperidine w/ 0.1M HOBt in DMF
HBTU/DIEA activation
™ Crude Purity ~ 50%
™ Total synthesis time of 35 hours
SPS of Polyamides Containing Pyrrole
and Imidazole Amino Acids
™ Polyamides containing (Im) and (Py) amino acids have
affinity for DNA comparable to naturally occuring DNA
binding proteins
FmocHN
FmocHN
N
OH
N
O
Fmoc-Im-OH
(N-methylimidazole)
OH
N
O
Fmoc-Py-OH
(N-methylpyrrole)
OH
Fmoc HN
O
Fmoc-γ-OH
(g-aminobutyric acid)
"hairpin" motif
SPS of Polyamides Containing Pyrrole
and Imidazole Amino Acids
Im-Im-Py-Py-γ-Im-Py-Py-Py-β-Dp
MW = 1280.36
(Dp = dimethylaminopropylamine)
(β = beta-alanine)
Synthesis
0.1mmol synthesis w/ ß-Ala CLEAR Resin; (0.52meq/g)
20% Piperidine in DMF
HBTU/DIEA activation
Pre-activation performed (Fmoc-Im-OH) soluble in DMF w/ HBTU/DIEA
SPS of Polyamides Containing Pyrrole
and Imidazole Amino Acids
Im-Im-Py-Py-γ-Im-Py-Py-Py-β-Dp
MW = 1280.36
™ 90% Crude Purity
™ Total Synthesis
Time of 5 hours
™ Conventionally
180 minute
couplings
required1
1
Org. Lett.(2001), 3, 8, 1201
Phosphopeptide Synthesis
™ Phosphoamino acids derivatives
allow for their direct
incorporation during synthesis
Enhanced Deprotection,
Coupling,
and eliminate
the need and
for
difficult post-synthetic
Cleavage Reactions
phosphorylation
™ Difficult to couple and make
subsequent couplings difficult
Fmoc-Ser(PO(OBzl)OH)-OH
Phosphopeptide Synthesis
Test Peptide: EIVPN(pS)VEQK-OH
Conventional
Deprotection = 5, 15 minutes (20% Piperidine in DMF)
Coupling = 60 minutes (HBTU/DIEA, 5-fold excess)
Microwave (Max T = 80°C for both)
Deprotection = 30 seconds, 3 minutes (20% Piperidine in DMF)
Coupling = 5 minutes (HBTU/DIEA, 5-fold excess)
Conventional Deprotection
Conventional Coupling
Microwave Deprotection
Microwave Coupling
Conventional Deprotection
Microwave Coupling
™ Phosphoserine residues can be coupled faster and more
efficiently with microwave
™ Higher temperatures during deprotection, even at 50ºC (separate
experiment) can cleave phosphate groups Æ [-79 mass]
Summary
™ Microwave energy can be used to successfully enhance
both the deprotection and coupling reactions
™ Complete cycle
times of 25 minutes
are attainableand
even for
Enhanced
Deprotection,
Coupling,
longer sequences
Cleavage
Reactions
™ Piperazine can be used effectively as a replacement for
piperidine
™ Cysteine and Histidine require special conditions for
racemization free couplings
™ Phosphopeptide couplings are substantially accelerated
with microwave
Programming Liberty – Step 1 / 5:
Operation Cycles
Programming Liberty – Step 1 / 5:
Operation Cycles
™ Every operation cycle is fully customizable
Programming Liberty – Step 1 / 5:
Operation Cycles
™ Easy access to the
reaction vessel for
manual additions or
removal at programmable
pauses
Programming Liberty – Step 2 / 5:
Enter a Sequence
Programming Liberty – Step 2 / 5:
Enter a Sequence
™ Ideal for
precious
reagents; ex.
phosphoamino
acids
™ No priming
required
Programming Liberty – Step 3 / 5:
Set Conditions for Sequence
™ Multiple cycles can be applied to an individual sequence
Ease of Use – Running a Peptide
Step 4 / 5: Load method into 1 – 12 position and
check volume usage calculator
Step 5 / 5: Press START!
C. Serviceability – Automated Valve
and Sensor Verification