685844

research-article2017
JLAXXX10.1177/2472630316685844SLAS TechnologyGali

Technology Brief
SLAS Technology

An Open-Source Automated Peptide

2017, Vol. 22(5) 493­–499
© 2017 Society for Laboratory
Automation and Screening
Synthesizer Based on Arduino and Python DOI: 10.1177/2472630316685844
https://doi.org/10.1177/2472630316685844
journals.sagepub.com/home/jala

Hariprasad Gali1

Abstract
The development of the first open-source automated peptide synthesizer, PepSy, using Arduino UNO and readily available
components is reported. PepSy was primarily designed to synthesize small peptides in a relatively small scale (<100 µmol). Scripts
to operate PepSy in a fully automatic or manual mode were written in Python. Fully automatic script includes functions to carry
out resin swelling, resin washing, single coupling, double coupling, Fmoc deprotection, ivDde deprotection, on-resin oxidation,
end capping, and amino acid/reagent line cleaning. Several small peptides and peptide conjugates were successfully synthesized on
PepSy with reasonably good yields and purity depending on the complexity of the peptide.

Keywords
peptide synthesizer, Arduino, Python, do-it-yourself, laboratory automation, SPPS

Introduction (Geel, Belgium). Fmoc-bestatin was synthesized as previously

Significant advancement has been happening since the past
decade in the development of very low-cost open-source
microcontrollers such as Arduino and Raspberry Pi.1,2 The use
of these microcontrollers in combination with the powerful
general-purpose and easy to learn programming languages
such as Python allows scientists to develop certain automated
instruments relatively easily at a small fraction of the cost of
their commercially (if) available counterparts.3 In addition,
these instruments can be fully customized for their laboratory
needs.4–7 Here, the development of the first open-source auto-
mated peptide synthesizer, PepSy, developed using Arduino
UNO and readily available components, is reported. The
scripts to operate PepSy were written in Python.
PepSy uses the solid-phase peptide synthesis (SPPS) method
employing the traditional Fmoc chemistry.8 The SPPS method
and its application in the automated synthesis of peptides was
first reported by Merrifield in the mid-1960s.9,10 Later after the
introduction of the Fmoc-based SPPS and the availability of
improved hardware as well as software simplified the design of
automated peptide synthesizers.11,12 Since then, several auto-
mated peptide synthesizers with a variety of options were
developed and became commercially available.13,14 However,
even today they remain relatively expensive (>$30,000) and
require recurring service contracts, which limits their use in the
laboratories with limited funding. In this regard, PepSy will
serve as a low-cost (<$4000) do-it-yourself alternative to the
commercially available automated peptide synthesizers.
reported.15 Rink amide MBHA resin (100–200 mesh, 0.3–0.8
mmol/g) and Fmoc-Thr(tBu)-Wang resin (100–200 mesh)
were obtained from EMD Chemicals (Gibbstown, NJ). Fmoc-
8-amino-3,6-dioxaoctanoic acid (PEG2) was obtained from
Peptides International (Louisville, KY). Fmoc-DAP(ivDde)-OH
was obtained from Bachem Americas (Torrance, CA). Other
Fmoc-protected amino acids and coupling agents were obtained
from Advanced ChemTech (Louisville, KY) or Chem-Impex
International (Wood Dale, IL).
General SPPS Procedure
Swelling of the resin. The resin (50 µmol) was initially
swelled for 15 min in 2 mL of 1:1 methylene chloride
(DCM)/N,N-dimethylformamide (DMF) and drained. The
resin was then washed once with 2 mL DMF.
Coupling of the amino acid (primary amine).  A Fmoc-protected
amino acid was coupled to the deprotected N-terminal amine
on the resin (1 eq.) using Fmoc-protected amino acid (3.3 eq.)
in 0.5 mL DMF, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HBTU) (3.3 eq.) in 0.5 mL
1
Department of Pharmaceutical Sciences, College of Pharmacy, The
University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
Received September 27, 2016.
Supplementary material is available online with this article.

Materials and Methods
All chemicals obtained commercially were used without fur-
ther purification. Bestatin was obtained from Acros Organics
Corresponding Author:
Hariprasad Gali, Department of Pharmaceutical Sciences, College of
Pharmacy, The University of Oklahoma Health Sciences Center, 1110 N.
Stonewall Avenue, Room 305, Oklahoma City, OK 73117, USA.
Email: hgali@ouhsc.edu
494 SLAS Technology 22(5)
DMF, 1-hydroxybenzotriazole (HOBt) (3.3 eq.) in 0.25 mL
DMF and N,N-diisopropylethylamine (DIPEA) (6.6 eq.) in
0.25 mL DMF for 1 h at room temperature. The resin was then
washed five times with 2 mL DMF.
Coupling of the amino acid (secondary amine).  An Fmoc-pro-
tected amino acid was coupled to the deprotected N-termi-
nal amine of the resin (1 eq.) using Fmoc-protected amino
acid (3.3 eq.) in 0.5 mL DMF, HBTU (3.3 eq.) in 0.5 mL
DMF and DIPEA (6.6 eq.) in 0.25 mL DMF for 1 h at room
temperature. The solvent was then drained and the same
procedure was repeated once more. The resin was then
washed five times with 2 mL DMF.
Deprotection of the Fmoc group. Fmoc deprotection after
each amino acid coupling was accomplished using 1 mL of
20% piperidine in DMF for 1 × 10 min and 1 × 20 min at
room temperature. The resin was then washed five times
with 2 mL DMF.
Deprotection of the ivDde group. ivDde deprotection was
accomplished using 1 mL of 2% hydrazine hydrate in DMF
for 1 × 10 min and 1 × 20 min at room temperature. The
resin was then washed five times with 2 mL DMF.16
On-resin disulfide bond formation.  On-resin oxidation of cys-
teine thiols was carried out by treating the acm-protected
cysteines containing peptide-resin with thallium (III) tri-
fluoroacetate (2 eq.) in 2 mL DMF for 1 × 60 min and 1 ×
60 min at room temperature. The resin was then washed
five times with 2 mL DMF.17
End capping. End capping of the N-terminal amine was
accomplished using 50 eq. of acetic anhydride and pyridine
Figure 1.  A fluidic flow diagram of
PepSy. The reagents and solvents
are delivered in precise volumes to
the reactor (RC) by the solenoid
micro pump (SP) via the VICI stream
selector valve (PS). Mixing of the
reaction mixture is achieved by the
nitrogen bubbling. The nitrogen is
also used to push the reagents and
solvents from the reactor.
in 1 mL DMF for 30 min at room temperature. The resin
was then washed five times with 2 mL DMF.
Cleavage of the peptide from the resin.  Before cleavage, resin
was washed five times with 2 mL DCM and dried for 30
min by nitrogen purging. Cleavage was performed by treat-
ing the peptide-resin with 2 mL of cocktail containing tri-
fluoroacetic acid (TFA), ethanedithiol or triisopropylsilane,
water, and thioanisol in a ratio of 90:5:2.5:2.5 for 4 h at
room temperature. The peptide-containing cocktail was
separated and collected in a test tube. The resin was then
washed with 0.2 mL TFA, and the washings were added to
the cocktail. The crude peptide was precipitated out by add-
ing 0.5 mL of the cocktail to 50 mL of ice-cold methyl tert-
butyl ether (MTBE) in a 50-mL falcon centrifuge tube. The
tube was centrifuged at 2000 rpm for 10 min, and MTBE
was carefully decanted each time. Finally, the crude peptide
was washed twice with 50 mL MTBE and separated by cen-
trifugation. The crude peptide was then dried by purging
nitrogen for 2 h at room temperature and purified by high-
performance liquid chromatography (HPLC).
HPLC conditions
HPLC solvents consisted of water containing 0.1% v/v
TFA (solvent A) and acetonitrile containing 0.1% v/v TFA
(solvent B). A Sonoma C18 column (ES Industries, West
Berlin, NJ; 5 µm, 100 Å, 4.6 mm × 250 mm) was used
with a flow rate of 1.5 mL/min. The HPLC gradient sys-
tem began with an initial solvent composition of 95% A
and 5% B for 2 min followed by a linear gradient to 50%
A and 50% B in 15 min, after which the column was
reequilibrated.
Gali 495

Figure 2.  An electrical wiring diagram of PepSy. The solenoid isolation valves and the micro pump are connected to the digital I/O
pins of the Arduino board via the power switching board. Both the Arduino board and the VICI stream selector valve are connected
to the USB ports of a PC running Windows OS. The Arduino board is powered by the USB port itself, whereas the power switching
board/solenoid valves/pump and the VICI stream selector valve are powered by an external 24-V DC power supply.

PepSy Design and Assembly
The fluidic flow diagram of PepSy is shown in Figure 1.
Figure 2 shows the electrical wiring schematic used for
connecting all the electrical/electronic components of
PepSy. All the components used for assembling PepSy are
shown in Table 1. Scripts to operate PepSy in a fully auto-
matic mode or manual mode (Fig. 3) were written in Python.
Results and Discussion
PepSy was primarily designed to synthesize small peptides
on a relatively small scale (<100 µmol). The current reaction
conditions were optimized for a 50-µmol scale in our labora-
tory and tested for synthesizing peptides with up to 30 amino
acid residues.18 As shown in Figure 1, all the reagents and
solvents are delivered in precise volumes to the reactor (RC,
a 10-mL disposable polypropylene syringe barrel fitted with
a porous polyethylene frit) by a solenoid micro pump (Bio-
Chem Fluidics, Boonton, NJ), labeled SP, via a VICI stream
selector valve (Valco Instruments, Houston, TX), labeled PS.
Mixing of the reaction mixture is achieved by bubbling nitro-
gen through the reaction mixture. Two nitrogen lines were
used to achieve a homogeneous mixing during the reaction
and washing steps. The nitrogen is also used for removing
reactants/solvents from the reactor after each reaction and
washing step by pushing them to the waste.
The SPPS method used by PepSy is mainly composed of
four unit operations: (1) priming, (2) reagent/solvent addi-
tion, (3) mixing, and (4) removing the reactants/solvents
from the RC and drying. For priming (filling the lines
between the reagent/solvent bottle/tube and the SP), the
actuator on the PS is moved to the corresponding position,
and the solenoid isolation valve (Bio-Chem Fluidics),
labeled Prime (SV5), is opened to enable flow of the
reagent/solvent to the waste; other valves remain in their
normal state. For the reagent/solvent addition, the actuator
on the PS is moved to the corresponding position, and the
solenoid isolation valve, labeled Reagent (SV3), is opened
to enable the flow of the reagent/solvent to the RC; other
valves remain in their normal state. For mixing, the sole-
noid isolation valve, labeled Nitrogen (SV1), is opened to
enable flow of the nitrogen to the RC and escape through
the vent while sparing the reaction mixture, and other valves
remain in their normal state. For removing the reactants/
solvents from the RC and drying, the SV1 is opened; the
solenoid isolation valve, labeled Vent (SV2), is closed; the
solenoid isolation valve, labeled Waste (SV4), is opened;
and other valves remains in their normal state. This arrange-
ment of solenoid valve states allows nitrogen to pass
through the RC into the waste. A nitrogen pressure of ~2 psi
was found to be optimal for mixing, removing reactants/
solvents, and resin drying for 50-µmol scale synthesis. The
PTFE tubing internal diameter (1/16 in.) and their lengths
496 SLAS Technology 22(5)

Table 1.  Parts List of PepSy.

Item Vendor Catalog Number Quantity

Cheminert low-pressure stream selector: 6 to 40 fittings for 1/16- VICI C25G-24524EUTB 1
in. tubing, 24 port
Bio-Chem Fluidics solenoid operated micro pump Western Analytical 130SP2420-1TP 1
Bio-Chem Fluidics solenoid operated isolation valve, normally open Western Analytical 075T2NO24-32 1
Bio-Chem Fluidics solenoid operated isolation valve, normally Western Analytical 075T2NC24-32 4
closed
LabJack PS12DC power switching board LabJack PS12DC 1
Arduino UNO R3 Amazon B008GRTSV6 1
Jumper wire pack, male to female Amazon B00AC4NQYG 1
Hookup wire kit, 22 gauge Amazon B00B4ZRPEY 1
AC 100- to 240-V to DC 24-V 5A power supply Amazon B00LUIKY4S 1
Female 5.5 mm × 2.1 mm DC power cable connector plug Amazon B005CMP434 1
Bellofram subminiature regulator, 0 to 5 PSIG; 1/16-in. NPT(F) Cole-Parmer EW-68826-36 1
Cole-Parmer digital pressure gauge, 0 to 5 psi, 1/4-in. NPT(M) Cole-Parmer EW-68950-50 1
NPT male pipe adapter, Kynar, 1/16-in. NPT × 1/16-in. ID barbed Cole-Parmer EW-30704-00 2
NPT female pipe adapter, Clear PP, 1/4-in. NPT × 1/8-in. ID Cole Parmer EW-31501-41 1
barbed
Female luer tee, PP Cole-Parmer EW-45500-56 1
Male luer with lock ring × 1/16-in. hose barb, PP Cole-Parmer EW-45503-00 3
Silicone (platinum-cured) tubing, 1/16-in. ID × 1/8-in. OD, 25 ft Cole-Parmer EW-95802-02 1
PTFE tubing, 0.03-in. ID × 1/16-in. OD, 5 m VWR 97056-980 3
Flangeless male nut, Delrin, 1/4 to 28 flat-bottom, for 1/16-in. OD VWR P-201 11
tubing
Flangeless ferrule, Tefzel (ETFE), 1/4 to 28 flat-bottom, for 1/16-in. VWR P-200NX 13
OD tubing
Flangeless headless nut, PEEK, short, 1/4 to 28 flat-bottom, for VWR P-283 2
1/16-in. OD tubing
Tee assay, PEEK, 1/4 to 28 FB (F), for 1/16-in. OD tubing VWR 21511-206 2
Adapter, PEEK, luer (F) to 1/4 to 28 FB (F) VWR 14221-486 1
VWR storage bottles, 500 mL VWR 89000-238 2
VWR test tube rack, for 13-mm OD tubes, half-size VWR 89215-716 1
VWR test tube rack, for 30-mm OD tubes VWR 89215-744 1
VWR Talon regular clamp holder VWR 21572-501 11
VWR Talon rod, stainless steel, 12 in. VWR 60079-533 1
VWR Talon rod, stainless steel, 18 in. VWR 60079-534 1
VWR Talon rod, stainless steel, 24 in. VWR 60079-535 1
VWR Talon support stand, cast iron, rectangular base, 5 × 8 in. VWR 12985-070 1
VWR Talon three-prong extension clamp, dual adjustment, small VWR 21570-200 9
Suba-Seal septum stopper, 14/20 to 14/35 outer joint VWR 80062-444 1 PK
Bulk syringes, nonsterile, all plastic, 10 mL, luer-lock VWR BD301029 1 PK
Fritware porous polyethylene sheet, medium porosity (45–90 VWR 70680-110 1 CS
micron)
VWR centrifuge tubes, PP, 50 mL VWR 21008-177 1 CS
BD Falcon round-bottom tubes, PP, 12 × 75 mm VWR 60819-794 1 CS
VWR Safe-T-Flex caps, for 12-mm culture tubes VWR 60828-720 1 CS
PTFE thread tape, 1/2 in. VWR 300008-843 1
VWR laboratory labeling tape, 1 in. VWR 73390-060 1

were kept as minimum as possible to minimize the reagent
wastage during the priming step.
All solenoid isolation valves and the micro pump were
connected to the digital I/O pins (only six used) of the
Arduino UNO (http://arduino.cc/) via a power switching
board (LabJack Corporation, Lakewood, CO) (Fig. 2). The
power switching board contains 12 digitally controlled
switches (only six used) with LED indicators, resettable
fuse protection, and flyback protection. Arduino board’s
digital pins 2 to 7 were connected to the power switching
Gali 497
Figure 3.  A Python/tkinter-based graphical user interface to
manually control PepSy. The manual mode script was developed
mainly to turn on or off a specific solenoid isolation valve, to
change the VICI stream selector valve position, and to pump
a specific volume of the reagent. It can be used to perform
any one of the four unit operations (priming, reagent/solvent
addition, mixing, or removing the reactants/solvents from the
reactor and drying) by turning on the specific valve(s) and/or the
pump.
board’s P3-14 pin header (pins DI0 to DI5, respectively),
and both boards’ grounding pins were connected together.
The SV1 to SV5 and SP were connected to the power
switching board pins S0 to S5, respectively. Both Arduino
board and the PS were connected to the USB ports of a PC
running Windows OS. The Arduino board was powered by
the USB port itself, whereas the power switching board/
solenoid valves/pump and the PS were powered by an
external 24-V DC power supply.
The open-source software used to operate PepSy in a fully
automatic or manual mode was developed in Python. Python
was chosen for writing scripts mainly because it is a dynamic
high-level programming language that is easy to learn; scien-
tists with no prior software programming knowledge can learn
it fast, and it is freely available.3 In addition, the Python scripts
can be run on the most operating systems. Firmata was used to
interact the Python script with the Arduino board, whereas the
PS was directly controlled by the Python script via serial com-
munication. The Python scripts to operate PepSy in a fully
automatic (PepSy.py) and manual mode (PepSy-manual.py)
are available to download at GitHub.19 Instructions, scripts,
device configuration file, and an example of the sequence con-
figuration file are included in the supplemental material. Fully
automatic mode script generates an output file for each run
with a timestamp for each step. In addition, real-time status
update of the step being carried out is displayed on the com-
puter screen. Examples of an output file and the information
displayed on the screen during a run are included in the supple-
mental material. The manual mode script displays a graphical
user interface, as shown in Figure 3. This script was developed
mainly to turn on or off a specific valve, to change the VICI
stream selector valve position, and to pump a specific volume
of the reagent during the troubleshooting of any mechanical or
electrical issues. However, it can be used to perform any one of
the four unit operations (priming, reagent/solvent addition,
mixing, or removing the reactants/solvents from the reactor
and drying) by turning on the specific valve(s) and/or the
pump. The peptide synthesis is carried out mainly in the auto-
mated mode. However, if the PepSy experiences an unex-
pected error at any stage while running in an automated mode,
then the automated mode script can be closed and the sequence
configuration file can be modified accordingly to begin the
synthesis from where it stopped in the previous run. Fully auto-
matic mode script includes functions to carry out resin swell-
ing, resin washing, single coupling, double coupling, Fmoc
deprotection, ivDde deprotection, on-resin oxidation, end cap-
ping, and amino acid/reagent line cleaning. Additional func-
tions to carry out specific reactions can be added to the script
by the user as needed.
The total cost of the components (Table 1) to assemble
PepSy (Fig. 4a) is <$4,000. PepSy uses disposables com-
monly available in the most chemistry laboratories such as
culture tubes for holding amino acid solutions, and the reac-
tor (Fig. 4b) is made of a disposable 10-mL BD syringe
barrel, a 70-µm polyethylene frit, and a rubber septa.
Operating cost of PepSy is very low due to use of the amino
acids and other reagents purchased in bulk and low-cost dis-
posables. In addition, all the components are readily avail-
able online and can be replaced very easily.
Several small peptides and peptide conjugates were
successfully synthesized on PepSy with reasonably good
yields and purity depending upon the complexity of
the peptide. Three examples are shown in Figure 5.
498 SLAS Technology 22(5)

Figure 4.  A photograph of (a) the

fully assembled PepSy and (b) the
disposable reactor. The assembling of
the PepSy does not require any special
tools.

Figure 5.  Examples of the

peptides synthesized on PepSy.
The crude product purity and yield
of the peptides synthesized by the
PepSy were similar or better than
the peptides synthesized manually.
Gali 499
DOTA-PEG2-Bombesin(7-14) is a 10-residue linear peptide,
whereas DOTATATE is a 9-residue cyclic peptide and used
on-resin oxidation. Similarly, DOTA-PEG2-Probestin used
orthogonal ivDde deprotection. The crude yields and purity
were much better than the method done manually.
This open-source automated peptide synthesizer pro-
vides an encouragement to chemists to develop various
automated synthesizers (e.g., to carry out radiochemistry).
Acknowledgments
The author gratefully acknowledges the assistance of Dr. Gopal
Pathuri, Mr. Benjamin C. Anderson, and Ms. Vy Ho for synthesiz-
ing the peptides on the PepSy.
Declaration of Conflicting Interests
The author declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: This
work was funded by the OU College of Pharmacy startup grant
and an Institutional Development Award (IDeA) from the National
Institute of General Medical Sciences of the National Institutes of
Health under grant number 8P20GM103447.
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