Introduction
UV-monitoring and robotic peptide library platforms
are major innovations in automated peptide synthesizer
technology which were first developed in the 1990’s,
however not much has changed since then. Recent
product releases from Protein Technologies, Inc.
represent the first major advances in these two areas
in almost two decades.
UV-Monitoring
The IntelliSynth UV-Monitoring and Feedback
Control System available on the Tribute Peptide
Synthesizer from Protein Technologies, Inc. is an
invaluable tool for peptide synthesis. Unlike other
UV-monitoring systems available on the market
today, the IntelliSynth system is the only on-line
system that can monitor the deprotection reaction
in real-time.
Other UV-monitoring systems use the old technology
developed in the 1990’s: an external monitor that
can only take one measurement after a 10 minute
deprotection step. They require the nitrogen and
NMP flow rates to be calibrated prior to every
synthesis, and require extra methanol rinses
between UV-measurements to remove bubbles
and clear the lines. They cannot control the length
of the deprotection reactions.
The IntelliSynth UV-system uses an on-line UV-
monitor and detector. It takes UV-measurements
every 10 seconds during the deprotection reaction,
not just at the end. It is the only system that can
modify the deprotection times as well as repetitions
and coupling times based on the feedback data.
Unlike other systems, no special rinsing is required
for the measurement area because the flow cell is
the tube itself, so it gets rinsed when the resin is
rinsed. It does not interfere with the reaction, and
no solvent flow tests or nitrogen adjustments are
required. It can graph individual deprotection
reactions or the overall data for a synthesis, and
when used with PTI Peptide Predictor software, it
can identify difficult couplings before, during and
after a synthesis.
To i l lustrate th is, a 9-mer analog, 9Pbw0
(RLWLAIGRG-NH2), of the 43-mer antimicrobial
peptide Protaetiamycine [1], was synthesized on
a Tribute Peptide Synthesizer using the IntelliSynth
Improved Technologies for Peptide Synthesis: New Methods for
UV-Monitoring and Robotic Peptide Library SynthesisProtein Technologies, Inc.
IDENTIFY DIFFICULT SEQUENCES:BEFORE:
Figure 1: PTI peptide predictor analysis of 9Pbw0
sequence.
DURING:
Figure 2: UV-monitoring data taken during the synthesis
of 9Pbw0. Synthesis summary data is shown on the
bottom, where each peak represents an individual
deprotection repeat for each cycle in the synthesis. The
individual deprotection data for one of the repeat peaks is
shown on top, where each peak represents an individual
UV measurement. Both types of data may be viewed at
any time during a synthesis.
Figure 3: Synthesis summary data
for the entire synthesis of 9Pbw0.
AFTER:
Synthesis Summary:
Each peak represents
an individual deprotection
repeat for each cycle in
the synthesis
Individual Deprotection:
Each peak represents
an individual UV
measurement
PharManufacturing: The International Peptide Review22
UV-Monitoring and Feedback Control System to
control the deprotection reactions. Fast couplings
were performed for 2 x 1 minute using the activator
HCTU as has been reported previously [2].
PTI Peptide Predictor software was used to identify
potentially difficult cycles prior to the synthesis
(Figure 1). UV data could be viewed as individual
measurements during a deprotection reaction, or
as individual repeats after each deprotection
reaction during (Figure 2) and after the synthesis
(Figure 3). HPLC analysis showed a high purity
crude peptide product, and the identity of the
peptide was confirmed by mass spectrometry
(Figure 4).
The UV-monitoring system was able to show the
extent of the deprotection reactions during and
after the synthesis.
UV-monitoring is also a useful tool for comparing
deprotection reagents when synthesizing difficult
peptides. Poly-alanine tracts are extremely difficult
to synthesize by conventional Fmoc solid phase
peptide synthesis due to their high propensity to
aggregate after the fifth residue [3]. Using DBU
(Diaza(1,3) bicycle[5.4.0] undecane) in the
deprotection solution has been found to improve
the synthesis of poly-alanine peptides [4].
(A)10K-OH was synthesized using 20% piperidine
in DMF alone, or with 2% DBU added to the solution.
The results show that 20% piperidine alone
(Figure 5) was unable to completely remove the
Fmoc group during cycle 7, resulting in a significant
impurity peak (7-mer = Fmoc-AAAAAAK-OH). The
UV-monitoring data showed cycle 7 had the most
number of repeats, and was therefore the most
difficult cycle for deprotection. Adding 2% DBU to
the 20% piperidine deprotection solution (Figure 6)
was found to be more efficient than piperidine
a)
b)
Figure 4: a) HPLC and b) mass spectrometry data of crude 9Pbw0
(MW = 1040.27, [M+H] = 1040.73, [M+2H] = 521.16).
a) a)
b) b)
Figure 5: a) HPLC and b) UV-monitoring data of crude A10K-OH
synthesized with 20% piperidine as the deprotection solution.
Figure 6: a) HPLC and b) UV-monitoring data of crude A10K-OH
synthesized with 2% DBU/20% piperidine as the deprotection solution.
Product
7-mer
Product
7-mer
PharManufacturing: The International Peptide Review 23
alone at removing the Fmoc group. It resulted in
fewer repeats during the deprotection steps and
the 7-mer impurity peak was significantly reduced.
(The broad pre-peak in both HPLC’s is the DMF
solvent front).
Robotic Peptide Library Synthesizers
The Overture Robotic Peptide Library synthesizer
is an innovative new tool for high-throughput
peptide library applications. Its unique features
make it the most convenient, efficient and
consistent instrument for peptide library synthesis
on the market today.
Other robotic synthesizers use an off-the-shelf
robotic platform that uses a syringe pump to deliver
reagents. This setup wastes solvent because the
syringe pump must be rinsed with 18 mL of
solvent between deliveries, adding time and
expense. In addition, calibration of the robotic arm
is a long and tedious process that must be
performed prior to each synthesis, and the
reaction block septa often wear out during or after
each synthesis and must be replaced. Only one
protocol can be run in all positions, and if cleavage
is desired, the reaction vessels must be transferred
to a completely separate plat form that is
purchased separately, or a secondary plate must
be inserted below the reaction vessel requiring
user intervention prior to the cleavage reaction.
The Overture was designed to accomodate
laboratories interested in library synthesis to
production scales. It features a unique robotic
platform with easy one-point calibration that can
accomodate up to 96 (1.3 or 10 mL) reaction vessels,
or up to 24 (1.3, 10, 40 or 45 mL) reaction vessels.
The Overture uses patented individual amino acid
dispensers which do not require rinsing in
between deliveries. The robot just picks up
whichever primed amino acid dispenser it needs.
The Overture can run 6 different protocols at the
same time, instead of just one protocol for all
positions. This makes the Overture perfect for
method development or running multiple scales
from 5 µmol in a 1.3 mL reaction vessel, to 100
µmol in a 10 mL reaction vessel, to 1 mmol in a
45 mL reaction vessel, all at the same time! Long-
life septa last multiple syntheses without leaking.
Built-in library generation software features easy
sequence importing, RV assignments, and automatic
library generation including overlapping peptide
libraries (Figure 7), alanine scanning libraries,
positional scanning libraries, combinatorial 2- and
3-positional scanning libraries, truncation libraries,
T-cell truncated libraries, and scrambled libraries.
A windows utility version of the software allows
syntheses to be planned on a desktop PC and
uploaded to the Overture using a USB stick. The
Overture also features the popular Safe-Response
and Email Notification features available on the
Tribute and Prelude peptide synthesizers. The
Safe-Response feature automatically drains and
rinses the reaction vessels when a user error
occurs to minimize unwanted side-reactions, while
the Email Notification feature can send an email
at the beginning of each cycle, at the end of the
synthesis, or when an error occurs.
Protein Technologies, Inc. has been a global
leader in automated peptide synthesis instrumentation
for 25 years and strives to continually provide its
customers with the most innovative technologies
for their peptide synthesis needs.
References[1] Shin S, Kim J-K, Lee J-Y, Jung K-W, Hwang J-S, Lee J, Lee DG,
Kim I, Shin SY, Kim Y. J. Pept. Sci. 2009; 15: 559-568.
[2] Hood CA, Fuentes G, Patel H, Page K, Menakuru M, Park JH. J.Peptide Res. 2008; 14: 97-101.
[3] Clausen N., Goldammer C., Jauch K., Bayer E., Peptides: chemistry,structure, and biology 1996, 71.
[4] Kates S.A., Sole N.A., Beyermann M., Barany G., Albericio F., PeptideResearch 1996, 9: 106.
For more information, contact:Protein Technologies, Inc.
4675 S. Coach Dr.
Tucson, AZ 85714, U.S.A.
Toll Free (U.S.A.): 1-800-477-6834
Tel: +1-520-629-9626
Fax: +1-520-629-9806
Email: [email protected]
Web: www.ptipep.com
Figure 7: Example of an Overlapping Peptide Library.
PharManufacturing: The International Peptide Review24