Peptide and peptide nucleic acid syntheses using a DNA/RNA synthesizer

Article abstract of DOI:10.1002/bip.22574

The use of an ABI 394 DNA/RNA synthesizer for peptide and peptide nucleic acid (PNA) syntheses is described. No additional physical part or software is needed for the application. A commercially available large DNA synthesis column was used, and only about half of its volume was filled with resin when the resin was fully swollen. With additional space in the top portion of the column, agitation of reaction mixture was achieved by bubbling argon from the bottom without losing solution. Removing solutions from column was achieved by flushing argon from top to bottom. Two peptide and two PNA sequences were synthesized. Good yields were obtained in all the cases. The method is easy to follow by researchers who are familiar with DNA/RNA synthesizer.

Full text of DOI:10.1002/bip.22574

Peptide and Peptide Nucleic Acid Syntheses Using a DNA/RNA
Synthesizer
Durga Pokharel, Suntara Fueangfung, Mingcui Zhang, Shiyue Fang
Department of Chemistry, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931
Received 7 July 2014; revised 18 September 2014; accepted 27 September 2014
Published online 9 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22574
INTRODUCTION
olid phase peptide and oligonucleotide (ON) synthe-
ABSTRACT:
ses are traditionally performed on different synthe-
sizers. Such different designs are a result of several
considerations. For example, the commonly used
solvent for peptide synthesis is DMF, while that for
The use of an ABI 394 DNA/RNA synthesizer for peptide and
peptide nucleic acid (PNA) syntheses is described. No addi-
tional physical part or software is needed for the application.
A commercially available large DNA synthesis column was
used, and only about half of its volume was filled with resin
when the resin was fully swollen. With additional space in the
top portion of the column, agitation of reaction mixture was
achieved by bubbling argon from the bottom without losing
solution. Removing solutions from column was achieved by
flushing argon from top to bottom. Two peptide and two PNA
sequences were synthesized. Good yields were obtained in all
S
ON is acetonitrile. The former has a higher viscosity than the
latter, which causes different mechanical designs to drive liq-
uid transfer. In addition, the number of monomer positions
on the synthesizers is different. For peptide synthesizer, more
than 20 are preferred, while for DNA/RNA synthesizer, 4–8
are enough. Furthermore, peptide synthesis is mostly per-
formed at 1–100 mmol scales while ON synthesis is normally
performed below 10 lmol. However, one of the most signifi-
cant differences that cause different synthesizer designs is the
kinetics of the reactions. The reactions for peptide synthesis
are slow, while those for ON synthesis are fast. Because of
this, peptide synthesis is usually performed batch-wise, and
the reaction chamber on peptide synthesizers is a flask that
can be shaken or a synthesis column through which the reac-
tion solutions can be circulated. These designs are also bene-
ficial to the usually larger scale peptide synthesis, where
better agitation of reaction mixture and more efficient use of
regents are desirable. In contrast, ON synthesis can be per-
formed using a continuous-flow system, and the reaction
chamber on a DNA/RNA synthesizer is usually a synthesis
column without a circulation function. This design reduces
instrument cost, and may be even more suitable for small
scale synthesis compared to using a reaction flask, where
small volumes of solutions may be used less efficiently. The
ABI 394 DNA/RNA synthesizer adopted such a synthesis col-
umn design. Mainly due to this continuous-flow feature,
DNA/RNA synthesizers are considered not suitable for pep-
tide synthesis. However, there are good reasons to enable this
function. Peptide synthesizers are expensive and take signifi-
cant lab space. The use of DNA/RNA synthesizer for peptide
synthesis will enable one instrument for two applications,
which will save money on instrument and increase lab space
the cases. The method is easy to follow by researchers who are
C
V
familiar with DNA/RNA synthesizer. 2014 Wiley
Periodicals, Inc. Biopolymers (Pept Sci) 102: 487–493, 2014.
Keywords: solid phase synthesis; synthesizer; DNA;
RNA; peptide; peptide nucleic acid
This article was originally published online as an accepted
preprint. The “Published Online” date corresponds to the pre-
print version. You can request a copy of any preprints from the
past two calendar years by emailing the Biopolymers editorial
office at biopolymers@wiley.com.
Additional Supporting Information may be found in the online version of this
article.
Correspondence to: Shiyue Fang; e-mail: shifang@mtu.edu
D.P. and S.F. contributed equally to this work
Contract grant sponsor: US National Science Foundation
Contract grant numbers: CHE-0647129 and CHE-1111192
Contract grant sponsor: NSF Equipment Grant
Contract grant numbers: CHE-9512445 and CHE-1048655
Contract grant sponsors: MTU Biotech Research Center, Michigan Universities
Commercialization Initiative, MTU Research Excellence Fund-Technology Com-
mercialization, and The Royal Thai Government Scholarship (S.F.)
C
V
2014 Wiley Periodicals, Inc.
PeptideScience Volume 102 / Number 6
487

488
Table I Bottle Assignments and Amount of Reagents for Peptide Synthesisa
Solution for One Cycle
Equivalentb
ml
Pokharel et al.
Bottles
Reagents/Solvents
mmol
Concentration (M)
Total (ml)
0.4
—
1–8
9
Amino acids in DMF
Blocked
3
—
0.4
—
0.168
—
0.168
0.420
—
c
10
11
12
14
15
18
19
DIEA/2,6-lutidine in DMF
Ac2O in DMF for capping
Pyridine in DMF for capping
Piperidine in DMF for de-blocking
HATU in DMF for activating
DMF
3/3
0.4
0.6
0.6
1.2
0.4
—
0.420
0.935
3.733
ꢀ20%
0.392
—
c
c
c
c
c
c
10 3 3
40 3 3
50 3 3
2.8
0.560 3 3
2.240 3 3
2.800 3 3
0.156
—
—
—
—
DCM
—
—
a The synthesis was performed using 100 mg 2-chloro-trityl resin (0.56 mmol/g loading, 56 lmol).
b The synthesis column could retain 0.6 ml solution while bubble argon from bottom, but 1.2 ml (0.4 ml amino acid solution 1 0.4 ml DIEA/lutidine
solution 1 0.4 ml HATU solution) solution of activated amino acid solution was delivered to improve volume accuracy. The actual equivalents of amino
acids and activator used were therefore 1.5. The actual equivalents of capping and de-blocking reagents were also half of the values shown in table.
c Excess prepared.
usage. Furthermore, the conjugates of DNA/RNA with pep- synthesizer, and the DMF remained in the column was
tides are receiving increasing attention for biological stud- pushed out into a beaker by reverse flush. The volume was
ies.1–3 The synthesis of such conjugates on a single determined to be about 0.6 ml.
synthesizer is expected to be more convenient. In this Report,
we show that peptides and peptide nucleic acids (PNAs) can
Determine Times Required for Reverse Flush and
Reagent Delivery
Reagents and solvents were placed on the synthesizer according
be synthesized on an ABI 394 DNA/RNA synthesizer. No
additional physical part or software is needed. The proce-
dures are easy to follow by anyone who is familiar with DNA
to Table I (see protocol 3 in Supporting Information). The col-
synthesis, and good yields can be obtained in both cases.
umn containing swollen resin and filled with DMF was flushed
from the top to record the time for emptying the column by
reverse flush. The time was recorded in Table II (entry 1). The
EXPERIMENTAL
time required for filling the column with DMF was also
recorded (entry 2). For measuring the times needed to add 0.4
ml base solution (from bottle 10, entry 3) and 0.4 ml activator
Determine Column Volume
The 2-chloro-trityl polystyrene resin (100 mg, 0.56 mmol/g solution (from bottle 15, entry 4) to amino acid bottles 1–8,
loading, 56 lmol, 1% DVB, 100–200 mesh) was loaded in a the solutions were first pressurized and the lines from the bot-
10–15 lmol DNA/RNA synthesis column (Glen Research, tles (10 and 15) to block were filled. The block and the lines to
Sterling, VA), placed on an ABI 394 DNA/RNA synthesizer, the amino acid bottles were cleaned and emptied. The amino
and swollen in DCM for 30 min (see protocol 3 in Support- acid bottle is removed. A graduated cylinder (or graduated 1.5
ing Information). DCM was removed by reverse flushing, ml centrifuge tube) is placed under the liquid line. Then, the
and the column was filled with DMF. To remove residue solution from either bottle 10 or 15 was delivered to the cylin-
DCM, the DMF was removed by reverse flush with argon der for an arbitrarily chosen time using an appropriate func-
from the top of the column and the column was refilled tion (e.g., 10–5, 15–5) followed by cleaning up the block (18 to
with DMF. The column was then flushed with argon from waste, block flush) and flushing the liquid in the line between
bottom using the flush to waste function for 5 s. After wait- the block and amino acid bottle to the cylinder (e.g., flush to
ing for 30 s, the flush was repeated. Because there was signif- 5). These need to be tested several times until the correct
icant empty space within the top portion of the column time for delivering 0.4 ml solution is determined. The times
after a portion of DMF was removed during the first flush for delivering activated amino acid (entry 5) and capping
and the column had a much bigger internal diameter than (entry 6) and de-blocking solutions (entry 7) to column were
the argon port, not all DMF could be removed with the measured by first empty the column followed by appropriate
flushes. The bottom of the column was detached from the delivery functions shown in Table II.
Biopolymers (Peptide Science)

Peptide and Peptide Nucleic Acid Syntheses
489
Table II Times for Reverse Flush and Reagent Deliveries for
Peptide Synthesisa
Cleavage, Deprotection, and Analysis of Peptide 1
A portion of the resin (30 mg) was taken from the column and
swollen with DCM for 30 min. The DCM was removed and
the resin was treated with a TFA cocktail containing 81.5%
TFA, 1.0% triisopropylsilane (TIPS), 5.0% water, 2.5% ethane
dithiol (EDT), 5.0% thioanisole, and 5.0% phenol (300 ll) at
rt for 100 min. The supernatant was removed, and the resin
was washed with TFA cocktail (100 ll 3 2). The peptide in the
combined solutions was precipitated by cold ether (4.5 ml) at
278ꢁC. The supernatant was removed. The crude peptide was
dissolved in 1.2 ml 50% acetonitrile, and 20 ll was injected to
RP HPLC to generate trace A (Figure 1). The peak at 23 min
was collected and one-third was re-analyzed with HPLC to
generate trace B. The total pure peptide from the 30 mg resin
was determined to be 10.7 mg, which is a white pellet. The
yield was calculated to be 80% based on the resin loading value
provided by the manufacturer. The peptide was analyzed with
ESI-MS, calcd for C39H57N10O8 [M1H]1 793.4, found 793.4
(see Supporting Information).
Entry
Function Name/Function Number
Reverse flush/2
18 (DMF) to column/42
Time (s)
1
2
3
4
5
6
7
30
35
10 (bases) to amino acid/for example, 216
15 (activator) to amino acid/for example, 200
1–8 (amino acid) to column/33
4.2
10.5
38
11112 (capping solutions) to column/39
14 (de-blocking solution) to column/40
35
35
a Argon pressure for regulators 1–3 on the synthesizer were 6.3, 6.3, and
8.6 psi, respectively.
Synthesis of Peptide 1
The six-amino acid peptide 1 (LWTRFA) was synthesized
on a 56 lmol scale using 100 mg resin (2-chloro-trityl poly-
styrene with Ala attached, loading 0.56 mmol/g, 1% DVB,
100–200 mesh; see protocol 1 in Supporting Information).
The resin was placed in a commercially available 10/15
lmol ON synthesis column. The Fmoc protected amino
acids Phe, Arg(Pbf), Thr(tBu), Trp(Boc), and Leu were dis-
solved in DMF and placed in bottles 1–5 on the synthesizer,
respectively (see Table I for reagent amounts). HATU was
used as activator and placed in bottle 15. The acid gener-
ated during activation was neutralized with diisopropyle-
thylamine (DIEA) and 2,6-lutidine, which were placed in
bottle 14. Capping failure sequences were achieved with
acetic anhydride and pyridine from bottles 11–12. Fmoc
removal was achieved with piperidine from bottle 14. The
reagents and times for reverse flush and reagent delivery are
summarized in Tables I and II. Using these data, the begin
(Supporting Information Table S3), synthesis (Supporting
Information Table S1), and end cycles (Supporting Infor-
mation Table S4) were composed (see Supporting Informa-
tion), and the synthesis was set up accordingly (see
protocol 1 in Supporting Information). For attaching
amino acid bottles, ensure that the lines from block to the
bottles are empty after bottle change (a suggested cycle to
achieve this is in Supporting Information Table S2). For
attaching other bottles, ensure that the lines from block to
them are filled after bottle change. This is especially impor-
tant for bottles 10 and 15. The sequence was edited as
5TCGA for Leu, Trp(Boc), Thr(tBu), Arg(Pbf), and Phe,
respectively (note, the synthesis starts from A to 5). For set-
ting up synthesis, select appropriate begin cycle (Support-
ing Information Table S3), synthesis cycle (Supporting
Information Table S1), column, sequence, and end cycle
(Supporting Information Table S4). The cycles are in
Supporting Information.
FIGURE 1 RP HPLC profiles: (a) Crude peptide 1. (b) Pure
peptide 1. (c) Crude peptide 2. (d) Pure peptide 2.
Biopolymers (Peptide Science)

490
Pokharel et al.
2 was analyzed with ESI-MS, calcd for C86H132N25O26
[M1H]1 1948.1, found 1947.6 (see Supporting Information).
Synthesis, Cleavage/Deprotection, and Analysis of
PNA 3
The 5-mer PNA 3 (H2N-TAGAC-CONH2) was synthesized on
a 56 lmol scale (90 mg Sieber amide resin, 0.62 mmol/g load-
ing, 1% DVB, 100–200 mesh; see protocol 2 in Supporting
Information) using N-(N-Fmoc-2-aminoethyl)-N-[(N-6-Bhoc-
9-adenyl)acetyl]-glycine, N-(N-Fmoc-2-aminoethyl)-N-[(N-4-
Bhoc-1-cytosyl)acetyl]-glycine, N-(N-Fmoc-2-aminoethyl)-N-
[(N-6-Bhoc-9-guanyl)acetyl]-glycine, and N-(N-Fmoc-2-ami-
noethyl)-N-[(1-thyminylacetyl]-glycine as the monomers.
The reagents and solvents were placed on the synthesizer as in
Table III. The times for reverse flushing column and reagent
delivery were determined as described for peptide, and listed in
Table IV (see protocol 4 in Supporting Information). These
data were used for composing the begin (Supporting Informa-
tion Table S6), synthesis (Supporting Information Table S5),
and end cycles (Supporting Information Table S7). The synthe-
sis was set up in the same way for peptide (see protocol 2 in
Supporting Information). After synthesis, a portion of the resin
(6 mg) was incubated with TFA containing m-cresol (9:1, v/v,
50 ll) at rt for 30 min. The supernatant was removed by filtra-
tion, and the step was repeated for four more times. The cleav-
ing solutions were combined. The PNA was precipitated with
cold ether (1.5 ml) at 220ꢁC. The supernatant was removed by
filtration. The residue was dissolved in 80 ll water; 10 ll was
injected into RP HPLC to generate trace A (Figure 2). The peak
at 21 min was collected, concentrated, re-dissolved in 20 ll
50% acetonitrile, and injected to HPLC to generate trace B. The
total pure PNA from 6.0 mg resin was determined to be 2.1
mg, which is a white pellet. The yield was calculated to be 75%
based on the resin loading value provided by the manufacturer.
The pure PNA was analyzed with ESI-MS, calcd for
C54H70N31O14 [M1H]1 1376.6, found 1376.6 (see Supporting
Information).
FIGURE 2 RP HPLC profiles: (a) Crude PNA 3. (b) Pure PNA
3. (c) Crude PNA 4. (d) Pure PNA 4.
Synthesis, Cleavage/Deprotection, and Analysis of
Peptide 2
The 16-amino acid peptide 2 (KNRWEDPGKQLYNVEA) was
synthesized under the same conditions for 1 at 56 lmol (100
mg resin) scale using the Fmoc amino acid monomers
Glu(OtBu), Val, Asn(Trt), Tyr(tBu), Leu, Gln(Trt), Lys(Boc),
Gly, Pro, Asp(OtBu), Trp(Boc), and Arg(Pbf). First, an 8-mer
was synthesized, and the resin (the synthesis cycle contains
washing steps that make resin ready for next synthesis) was
then used directly for adding the remaining eight amino acid Synthesis, Cleavage/Deprotection, and Analysis
monomers on the synthesizer. After the synthesis is complete, of PNA 4
a portion of the resin (35 mg) was taken from the column. The 10-mer PNA 4 (H2N-CTGCTTAGAC-CONH2) was syn-
Cleavage and deprotection were achieved under the same con- thesized under the same conditions for 3 at 56 lmol (90 mg
ditions for 1. The crude peptide was dissolved in 1.3 ml 50% resin) scale using the same monomers used for 3. First, a
acetonitrile, and 20 ll was injected to RP HPLC to generate 5-mer was synthesized. A small portion of the resin (ꢀ0.3 mg)
trace C (Figure 1). The peak at 22 min was collected and re- was taken out, and subjected to cleavage and deprotection
analyzed with HPLC to generate trace D. The total pure pep- using conditions for 3. ESI-MS indicated that the synthesis was
tide 2, which is a white pellet, from the 35 mg resin was 11.7 successful. The remaining resin was used directly for adding
mg. The yield was calculated to be 89% based on the resin the remaining five monomers on the synthesizer. After the syn-
loading value provided by the manufacturer. The pure peptide thesis is complete, a portion of the resin (5 mg) was taken
Biopolymers (Peptide Science)

Peptide and Peptide Nucleic Acid Syntheses
491
Table III Bottle Assignments and Amount of Reagents for PNA Synthesisa
Solution for One Cycle
Equivalentb
ml
Bottle
Reagents/Solvents
mmol
Concentration (M)
Total (ml)
0.4
—
1–8
9
Amino acids in NMP
Blocked
2
—
0.4
—
0.112
—
0.112
0.280
—
c
10
11
12
14
15
18
19
DIEA/2,6-lutidine in NMP
Ac2O in NMP for capping
Pyridine in NMP for capping
Piperidine in DMF
HATU in NMP for activating
DMF
2/2
0.4
0.6
0.6
1.2
0.4
—
0.280
0.933
3.733
ꢀ20%
0.280
—
c
c
c
c
10 3 3
40 3 3
50 3 3
2
0.560 3 3
2.240 3 3
2.800 3 3
0.112
—
—
—
—
—
—
DCM
—
—
a The synthesis was performed using 90 mg Sieber amide resin (0.62 mmol/g loading, 56 lmol).
b The synthesis column could retain 0.6 ml solution while bubble argon from bottom, but 1.2 ml (0.4 ml amino acid solution 1 0.4 ml DIEA/lutidine
solution 1 0.4 ml HATU solution) solution of activated amino acid solution was delivered to improve volume accuracy. The actual equivalents of amino
acids and activator used were therefore ꢀ1.0. The actual equivalents of capping and de-blocking reagents were also about half of the values shown.
c Excess prepared.
from the column. Cleavage and deprotection were achieved tion pump and performing the reactions in a flask with a filter-
under the same conditions for 3. The residue was dissolved in ing function. However, these solutions are complicated. We
80 ll water; 10 ll was injected into RP HPLC to generate the envisioned that simply using a larger DNA synthesis column
trace C (Figure 2). The peak at 33 min was collected, evapo- and only filling about half of the column space with resin
rated, and re-dissolved in 20 ll water, and injected to HPLC to when it is swollen would solve the problem. With empty space
generate the trace D. The total pure PNA 4, which is a white over the resin, agitation of the reaction mixture can be carried
pellet, from the 5 mg resin was 1.9 mg. The yield was calcu- out by an argon flow from the bottom of the column (the flush
lated to be 61% based on the resin loading value provided by to waste function) without losing the reaction solution.
the manufacturer. The pure PNA was analyzed with ESI-MS, Removal of reaction solution and washing solvents can be
calcd for C107H138N56O31 [M12H]21 1351.6, found 1351.8 achieved by an argon flow from the top of the column (the
(see Supporting Information).
reverse flush function).
With a solution for agitation in mind, the next issues to
address are the following. (i) Assign bottles for amino acids
and other reagents. We used the option shown in Table I. (ii)
Determine the volume of DMF that can remain in a column
containing swollen resin after bubbling argon from the bot-
tom. This can be measured by filling the column with DMF,
bubbling argon from bottom, detaching the bottom of the col-
umn from synthesizer, and flushing the remaining DMF from
the top by argon into a graduated cylinder (see protocol 3 in
Supporting Information). (iii) Determine the volume and con-
centration of solutions of amino acids, bases, activator, cap-
ping, and de-blocking. This can be calculated using the
column volume, the desired equivalents of reagents, and the
total loading of the first amino acid on resin. (iv) Determine
the times required for filling the column with DMF, removal of
DMF in column by reverse flush, adding base and activator
solutions to amino acid bottles, delivering activated amino
acids to column, and delivering capping and de-blocking solu-
tions to column (see Materials and Methods section). These
are better to be tested using the real solutions instead of DMF
RESULTS AND DISCUSSION
To use the ABI 394 synthesizer for peptide synthesis, a method
to agitate the reaction mixture must be identified. Potential
solutions to solve the problem include installation of a circula-
Table IV Times for Reverse Flush and Reagent Deliveries for
PNA Synthesisa
Entry
Function Name/Number
Reverse flush/2
18 (DMF) to column/42
Time (s)
1
2
3
4
5
6
7
40
40
8
10 (bases) to amino acid/for example, 216
15 (activator) to amino acid/for example, 200
1–8 (amino acid) to column/33
18
80
50
40
11112 (capping solutions) to column/39
14 (de-blocking solution) to column/40
a Argon pressure for regulators 1–3 on the synthesizer were 6.3, 6.3, and
8.6 psi, respectively.
Biopolymers (Peptide Science)

492
Pokharel et al.
because they have different viscosity. In our experiments, those acetic anhydride. The profile of purified 1 is also shown (trace
for activator (bottle 15) and base (bottle 10) to amino acid B). The isolated yield of pure peptide was calculated to be
bottles (e.g., bottle 5) were measured using real solutions 80%, and its structure was identified with ESI-MS (see Sup-
because delivering accurate volumes of these solutions is criti- porting Information). To further demonstrate the technique,
cal for the success of synthesis. For others, delivering more the 16-amino acid peptide 2 (KNRWEDPGKQLYNVEA) was
than needed only consumes more reagents but the synthesis synthesized and cleaved under the same conditions. The crude
will not be affected. As a result, we used DMF for the tests peptide was analyzed with RP HPLC. As shown in Figure 1
even though real solutions are suggested in protocol 3. (v) Edit trace C, the synthesis was very good considering the length of
new functions. They are for adding activator and bases to the peptide and the low equivalents of amino acid monomers
amino acids (each eight functions for bottles 1–8), and for agi- we used. The peptide was easily purified (trace D). The isolated
tating the solutions in bottles 1–8 while activating the amino yield was calculated to be 89%, and the structure was identified
acids by an argon flow (see Supporting Information Table S8). with ESI-MS (see Supporting Information).
(vi) Compose begin, synthesis, and end cycles. This can only
To use the method for PNA synthesis, some parameters
be accomplished with the times required for reverse flushing need to be modified (Tables III and IV). Because of the much
column and reagent deliveries, and the new functions. The higher price of PNA monomers than Fmoc amino acids, we
begin cycle includes steps for filling lines to bottles 10–12 and reduced their equivalents to two, which corresponds to
14–15, and for removing the Fmoc group on the resin if there approximately one because about half of the activated mono-
is one. The synthesis cycle mainly includes steps for activating mers could not be retained in the synthesis column during
amino acids, delivering activated amino acids to column, agi- bubbling. For PNA synthesis, the coupling and capping steps
tating the reaction mixture in column, washing column, add- are normally performed in NMP instead of DMF. Because the
ing capping solutions, and de-blocking solution to column. NMP solutions have higher viscosity, the times for delivering
The end cycle includes steps for washing the resin and the lines these solutions and reverse flushing them out of column have
from block to the amino acid bottles. With these issues to be longer. Based on these considerations, we re-tested all the
addressed, a peptide (or PNA) synthesis can be conveniently parameters (see protocol 4 in Supporting Information), and
set up.
used them to modify the begin (Supporting Information Table
To test feasibility, the six-amino acid peptide (1, LWTRFA) S6), synthesis (Supporting Information Table S5), and end
was synthesized on a 56 lmol scale using 100 mg resin (load- cycles (Supporting Information Table S7). To test feasibility,
ing 0.56 mmol/g; see protocol 1 in Supporting Information). the PNA sequence (H2N-TAGAC-CONH2, 3) was synthesized
The 2-chloro-trityl polystyrene resin with Ala attached was on a 56 lmol scale using the Sieber amide resin (see protocol 2
placed in a normal 10/15 lmol ON synthesis column taking in Supporting Information). At the end of synthesis, a portion
about half of the total volume after swelling. The Fmoc pro- of the resin was taken out from column and treated with a
tected amino acids Phe, Arg(Pbf), Thr(tBu), Trp(Boc), and TFA solution containing m-cresol. The cleaved and fully
Leu were dissolved in DMF and placed in bottles 1–5 on the deprotected PNA was precipitated with cold ether, and ana-
synthesizer, respectively. HATU was used to activate the amino lyzed with RP HPLC (trace A, Figure 2). The results indicated
acids. The acid generated during activation was neutralized that the synthesis was successful. The full-length PNA appeared
with DIEA and 2,6-lutidine. Capping failure sequences were at 21 min. Other peaks were mainly failure sequences. The pro-
achieved with acetic anhydride and pyridine. Piperidine was file for purified 3 is shown in trace B. The isolated yield was
used for Fmoc removal. The reagents and times for reverse calculated to be 75%, and its structure was identified with ESI-
flush and reagent delivery are shown in Tables I and II. Using MS (see Supporting Information). To further demonstrate the
these data, the begin, synthesis, and end cycles were composed technique, the 10-mer PNA 4 (H2N-CTGCTTAGAC-CONH2)
(see Supporting Information), and the synthesis was set up was synthesized and cleaved under the same conditions. The
accordingly. After synthesis, a portion of the resin was taken crude PNA was analyzed with RP HPLC (trace C, Figure 2).
out from the column. Cleavage and deprotection were per- The isolated yield was calculated to be 61%, which could be
formed with a TFA cocktail containing 81.5% TFA, 1.0% trii- easily improved by increasing the equivalents of monomers.
sopropylsilane (TIPS), 5.0% water, 2.5% ethane dithiol (EDT), The product was readily purified with RP HPLC (trace D, Fig-
5.0% thioanisole, and 5.0% phenol. The peptide was precipi- ure 2). Its structure was identified with ESI-MS (see Support-
tated with cold ether, and analyzed with RP HPLC. As shown ing Information).
in Figure 1, trace A contains a major peak at 23 min, which is
Because the 394 synthesizer has eight nucleoside phosphor-
resulted from peptide 1. The smaller peaks around 14 min and amidite bottles, the synthesis of peptides and PNAs shorter
21 min are mainly from failure sequences that are capped with than 9-mer (the first amino acid is usually attached to resin
Biopolymers (Peptide Science)

Peptide and Peptide Nucleic Acid Syntheses
493
before automated synthesis) can be achieved without any inter-
ruption. Synthesis of longer sequences is also not complicated.
It was easily achieved by starting a new synthesis using the
resin of nine-amino acid oligomer. For peptide, sequences lon-
ger than 40-mer are rarely synthesized using automated syn-
thesizer continuously. For PNA, the most commonly used
length is around 12-mer. As a result, the limited number of
monomer positions on the DNA/RNA synthesizer is not a con-
cern for the technique. Normally, continuous synthesis of long
peptides and PNAs should be avoided in most cases and tests
such as ESI-MS, Kaiser, and TNBS should be performed to
monitor the efficiency of coupling.
CONCLUSION
In conclusion, we have developed a method for the synthesis of
peptide and PNA using an ABI 394 DNA synthesizer. It is
remarkable that no modification of the synthesizer is needed,
and the only item needed for the method is a larger DNA syn-
thesis column, which is commercially available and reasonably
priced. No knowledge in areas such as mechanics and electronics
is required. Anyone who is familiar with the DNA synthesizer
can easily follow the procedures, and can set up the synthesizer
in about 1 day. Once set up, peptide and PNA synthesis may be
even more convenient than using some models of peptide syn-
thesizer. The method is expected to be useful in labs that need
peptides, PNA, and their conjugates with ONs.4–6
In the above synthesis examples, only about half of the acti-
vated amino acid solution was retained in the column after
bubbling argon from bottom. This can be considered as a
waste of reagents. We could have reduced the volume of amino
acid, base, and activator solutions to solve the problem, but
mixing smaller volumes in a precise ratio had been predicted
more difficult. However, we were able to partially solve the
problem by inserting a long waiting time between delivering
most of the solution to the column and delivering the remain-
ing solution, the latter was followed by agitating the reaction
mixture by bubbling argon (see the synthesis cycles in Support-
ing Information). A better way to solve the problem is to use a
larger synthesis column or to reduce the synthesis scale.
The assistance from Mr. Dean W. Seppala (electronics) and
Mr. Jerry L. Lutz (NMR) is gratefully acknowledged.
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Biopolymers (Peptide Science)