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: info@ptipep.com

Web: www.ptipep.com

Figure 7: Example of an Overlapping Peptide Library.

PharManufacturing: The International Peptide Review24