Conventional and microwave-assisted SPPS approach: A comparative synthesis of PTHrP(1-34)NH2

Article abstract of DOI:10.1002/psc.1395

Attracted by the possibility to optimize time and yield of the synthesis of difficult peptide sequences by MW irradiation, we compared Fmoc/tBu MW-assisted SPPS of 1-34 N-terminal fragment of parathyroid hormone-related peptide (PTHrP) with its conventional SPPS carried out at RT. MWs were applied in both coupling and deprotection steps of SPPS protocol. During the stepwise elongation of the resin-bound peptide, monitoring was conducted by performing MW-assisted mini-cleavages and analyzing them by UPLC-ESI-MS. Identification of some deletion sequences was helpful to recognize critical couplings and as such helped to guide the introduction of MW irradiations to these stages.

Full text of DOI:10.1002/psc.1395

Protocol
Received: 23 March 2011
Revised: 17 June 2011
Accepted: 20 June 2011
Published online in Wiley Online Library: 1 August 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1395
Conventional and microwave-assisted
SPPS approach: a comparative synthesis
of PTHrP(1–34)NH2
Fabio Rizzolo,a Chiara Testa,a,b Duccio Lambardi,c Michael Chorev,d,e
Mario Chelli,a Paolo Roveroc and Anna Maria Papinia,b∗
Attracted by the possibility to optimize time and yield of the synthesis of difficult peptide sequences by MW irradiation, we
compared Fmoc/tBu MW-assisted SPPS of 1–34 N-terminal fragment of parathyroid hormone-related peptide (PTHrP) with its
conventional SPPS carried out at RT. MWs were applied in both coupling and deprotection steps of SPPS protocol. During the
stepwise elongation of the resin-bound peptide, monitoring was conducted by performing MW-assisted mini-cleavages and
analyzing them by UPLC-ESI-MS. Identification of some deletion sequences was helpful to recognize critical couplings and as
c
such helped to guide the introduction of MW irradiations to these stages. Copyright ꢀ 2011 European Peptide Society and
John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: microwave irradiation; solid-phase peptide synthesis; microwave-assisted mini-cleavages; difficult peptide sequences;
parathyroid hormone-related peptide; UPLC-ESI-MS analysis
REACTION SCHEME
GENERAL OPTIMIZED PROCEDURE
RT and MW-assisted SPPS of parathyroid hormone-related peptide (PTHrP)(1-34)NH2 were performed using Liberty™ MW peptide synthesizer (CEM) on Rink-amide NovaSyn® TGR resin
(0.2 mmol/g, 500 mg). After deprotection of the N-terminal Fmoc group with a 20% solution of piperidine in DMF, Fmoc-protected amino acids (5 equiv),TBTU (5 equiv) and DIEA (10 equiv) in
DMF were used in the coupling steps.The RT-SPPS protocol consists of two consecutive deprotection steps of 5 and 10 min employing deprotecting reagent, and 20 min for the coupling steps. In
MW-assisted SPPS protocol, the deprotection steps were performed at 75 °C using 35 W for 0.5 min for the first one and 60 W for 3 min for the second one, while the coupling steps were performed
at 75 °C using 30 W for 5 min for all amino acids except Arg and His for which the conditions reported in Table 1 were used.The ongoing of synthesis was monitored by UPLC-ESI-MS of
MW-assisted mini-cleavages of intermediate fragments. A small sample of Fmoc-protected peptide-resin beads (10 mg) was removed from the reactor vessel, weighted into a fritted polypropylene
tube and treated with a 20% solution of piperidine in DMF (2 × 1 ml) for 5 min.The sample was then washed with DMF (2 × 1 ml) and DCM (3 × 1 ml), and dried under vacuum. Mini-cleavages
were performed in a 10 ml glass tube containing 2 ml of cleavage mixture consisting of TFA/TIS/water solution (95 : 2.5 : 2.5 v/v/v).Tubes were inserted into the MW cavity of Discover™ S-Class
(CEM) and the cleavage was carried out at 45 °C, using 15 W for 15 min assisted by external cooling of the reactor vessel.The crude fragments were analyzed by UPLC-ESI-MS with a Symmetry
300 C18 column (100 mm × 2.1 mm ID, 3.5 µm,Waters) using the following conditions: flow rate 0.15 ml/min with a linear gradient from 0 to 30% of B (solvent A: 2% ACN, 0.1% formic acid in
H2O; solvent B: 2% H2O, 0.1% formic acid in ACN) in 7 min, 30-60% B in the next 3 min, then to 95% B for 2 min, and returned to initial conditions for 3 min for re-equilibration.
The total run time per sample was 15 min
∗
Scope and Comments
Correspondence to: Anna Maria Papini, Department of Chemistry ‘‘Ugo Schiff’’, Laboratory
of Peptide & Protein Chemistry & Biology, Via della Lastruccia 3/13, Polo Scientifico e
Tecnologico, University of Florence, I-50019 Sesto Fiorentino, Italy.
E-mail: annamaria.papini@unifi.it
The introduction in 1992 of MW irradiation in peptide chemistry
stimulated a host of interest followed by numerous efforts to use
it to overcome synthetic difficulties resulting in sluggish reactions,
low yields and complex reaction crude products that were hard
to purify [1]. During the last decade the growing interest in MW
field has been marked by the progress in developing scientific MW
equipment to embrace specific research needs [2–5]. In general,
introduction of MW technology led to the shortening of reaction
time that resulted in higher purity of crudes and increased yields
of the pure final product.
a
Department of Chemistry ‘‘UgoSchiff’’, Laboratory of Peptide& Protein Chemistry &Biology,
ViadellaLastruccia3/13,PoloScientificoeTecnologico,UniversityofFlorence,I-50019Sesto
Fiorentino, Italy
´
b
c
Laboratoire PeptLab-SOSCO – EA4505 Universite de Cergy-Pontoise, 5 mail Gay-Lussac,
Neuville-sur-Oise, Cergy-Pontoise 95031, France
Department of Pharmaceutical Sciences, Laboratory of Peptide & Protein Chemistry &
Biology, ViaUgoSchiff 6, PoloScientificoeTecnologico, University of Florence, I-50019Sesto
Fiorentino, Italy
d
e
LaboratoryforTranslationalResearch, HarvardMedicalSchool, Cambridge, MA02139, USA
Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
Abbreviations used: ACN, acetonitrile; MW, microwave; NMP, N-methyl-2-pyrrolidone;
RT, room temperature; TIS, triisopropylsilane.
c
J. Pept. Sci. 2011; 17: 708–714
Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.

CONVENTIONAL AND MICROWAVE-ASSISTED SPPS APPROACH
The application of MW irradiation in peptide chemistry has
been reported in several publications, most of which describe
case studies of successful syntheses of difficult peptide sequences
[6–8]. However, a definitive comparison of conventional RT and
MW-assisted-SPPS protocols must be performed on identical in-
struments. In fact, comparing conventional RT and MW-assisted
SPPS carried out on different instruments that apply protocols
that differ in the equivalent excess of reagents and their molar
ratios, stirring techniques and the nature and number of washing
steps, may be misleading. Moreover, the automated synthesizers
are generally developed to produce peptides without monitoring
of the progress of the synthesis. Therefore by-products caused by
side reactionssuchasaspartimide anddiketopiperazine formation,
incomplete couplings and deprotections may be detected only
after the final cleavage of the deprotected peptide from the resin.
In this study we compared the Fmoc/tBu RT-SPPS with the MW-
assisted synthesis of the 1–34 N-terminal fragment of PTHrP using
the same instrument (Liberty, CEM, Matthews, NC, USA), and
monitoring the progress of the synthesis by UPLC-ESI-MS of MW-
assisted mini-cleaved fragments of the growing peptide chain.
PTHrP is an autocrine, paracrine and intracrine regulator of
processes such as endochondrial bone formation and epithe-
lial–mesenchymal interactions during the development of mam-
mary glands. In analogy to PTH, most of the known biological
functions are exerted by the N-terminal PTHrP(1–34) fragment
that has 60% sequence similarity to PTH(1–34). The sequence of
PTHrP(1–34)NH2 is shown in Figure 1. In the past, this sequence
was the subject of numerous structure–activity–conformation
relationship studies [9,10]. Importantly, the presence of clusters
of arginine, of sterically hindered and hydrophobic amino acid
residues in the sequence represents a synthetic challenge that
was the subject of our comparative study reported herein.
The synthesis was initially carried out following the con-
ventional RT protocol using the Liberty automated peptide
synthesizer excluding MW irradiations. As modern automated
SPPS protocols allow the assembly of larger and increasingly
complex peptides, a precise control of the coupling reactions
is a crucial prerequisite in peptide synthesis. In fact monitoring
the progress of synthesis allows the detection of undesirable
products caused by side reactions, incomplete couplings or
deprotections. Although different methods have been developed
for monitoring of SPPS, we observed that the use of colorimetric
monitoring or continuous-flow UV absorbance of the reaction
column effluent was not informative enough to identify difficult
steps in the synthesis. Therefore, we decided to monitor the
progress of PTHrP(1–34)NH2 synthesis by UPLC-ESI-MS analyses
of small aliquots of cleaved peptide fragments obtained by
MW-assisted mini-cleavages. The application of MW-assisted
mini-cleavages of resin-bound peptides has been proposed as a
fast, reliable method to monitor SPPS [11]. After specific coupling
cycles, suspected to be difficult, we stopped the synthesizer and
withdrew a small aliquot for analysis by UPLC-ESI-MS. In particular,
we focused our attention on the PTHrP fragments related to the
19–28 sequence, characterized by clusters of Arg residues and
highly hydrophobic residues (see Reaction Scheme and Figure 1).
By UPLC-ESI-MS analyses of intermediate fragments of the
PTHrP(1–34)NH2 included in the 19–34 sequence we noticed the
presence of the desired peptide as well as of some by-products
(Table 1 and Figure 2). The fragmentation patterns of these by-
products in ESI-MS/MS allowed us to confirm the formation of dele-
tion sequences as reported in Supporting Information (Figure S1).
As the length of the resin-bound peptide increases, the related
UPLC-ESI-MS analyses become much more complex. We report
as an example the characterization of the 12–34 fragment
of PTHrP(1–34)NH2. The deconvoluted spectrum obtained for
the cleaved mixture of this sample resulted in several deletion
sequences (Figure 3). The fragments desLys13/Gln16-f(12–34) and
desLys13/Gln16,Leu27-f(12–34) were identified as two isobaric
peptide sequences lacking either Lys13 or Gln16 residues.
The UPLC-ESI-MS/MS analyses of the intermediate resin-bound
fragments obtained from the RT-SPPS of PTHrP(1–34)NH2 confirm
that it is a difficult sequence for SPPS. The desired peptide was
usually present as the major component in the cleavage mixture,
but it was accompanied by some deletion peptides mainly lacking
Arg, Leu and His residues. It is well known that Arg-containing
peptides are difficult to synthesize due to the sterically hindered
Pbf group as side-chain protection and the tendency to form
γ -lactam leading to low yield couplings [12].
With the above information in hand we sought to improve
the synthesis of this difficult sequence of PTHrP(1–34)NH2 by
employing MW-assisted SPPS using Liberty automated peptide
synthesizer with MW irradiations. To address the difficulties
observed during the incorporation of Arg and His residues
we applied the protocols reported in Table 2. Specifically, we
have decreased the power of MW irradiation and lowered the
temperature in order to avoid side reactions such as γ -lactam
formation for Arg and racemization for His [13].
Indeed, after semi-preparative purification the MW-assisted
SPPS of PTHrP(1–34)NH2 yielded 27 mg of >95% pure peptide,
whereas RT-SPPS gave only 18 mg of >95% of pure peptide. This
improvement is attributed to the higher purity of the crude cleaved
peptide mixture (Table 3 and Figure 4).
On the basis of the results of the analytical RP-HPLC of the crude
PTHrP(1–34)NH2, we conclude that the use of MW irradiations
in SPPS has enhanced the efficiency of crucial coupling cycles
improving the final yield and purity of crude peptide and speeding
up the remaining coupling cycles. This improvement can be
attributed to the prevention of peptide backbone aggregation
and acceleration of deprotection and coupling steps.
In summary, although the application of MW-assisted SPPS
to the synthesis of PTHrP(1–34)NH2 led only to a moderate
improvement in final yield (6.3% vs 4.4%), it allowed us to
obtain a crude product of higher quality (77% vs 35%) and in
a shorter time (20 h vs 34 h used for the RT and MW-assisted SPPS
strategies). Moreover, we demonstrated the usefulness of the
combination of an MW-assisted mini-cleavage protocol and the
UPLC-ESI-MS analysis for monitoring the quality of the reaction
step (see Supporting Information). Compared to the ninhydrin
colorimetric monitoring, our strategy is faster and the UPLC-ESI-
MS/MS analysis is more accurate and more informative. We think
that application of the strategy presented in this report will help
to improve many syntheses of difficult sequences.
Experimental Procedures
RT and MW-assisted SPPS of PTHrP(1–34)NH2
All Fmoc-protected amino acids and TBTU were purchased from
Iris Biotech. (Marktdrewitz, Germany). The following amino acid
side-chain-protecting groups were used: OtBu (Asp, Glu), tBu (Ser,
Thr), Pbf (Arg), Trt (Gln, His) and Boc (Lys). Rink-amide NovaSyn
TGR resin was purchased from Novabiochem (Laufelfingen,
Switzerland). ACN, DCM and diethyl ether from Sigma-Aldrich
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J. Pept. Sci. 2011; 17: 708–714 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

RIZZOLO ET AL.
Figure 1. Characteristics of the PTHrP(1–34)NH2 sequence are a cluster of arginine residues in the positions 19–21, sterically hindered and hydrophobic
amino acid sequences.
Figure 2. (A–D) TIC chromatogram of selected crude mixtures of intermediate resin-bound sequences obtained during the synthesis of PTHrP(1–34)NH2
(Fmoc/tBu RT-SPPS) generated by the MW-assisted mini-cleavages. The related MS/MS spectra are shown in Supporting Information (Figure S1).
(St. Louis, MO, USA), DMF and NMP from Scharlau (Barcelona,
Spain), DIEA and TFA from Acros Organics (Geel, Belgium) and
TIS from Fluka/Aldrich (St. Louis, MO, USA). The synthesis of
PTHrP(1–34)NH2 was carried out using Fmoc/tBu SPPS strategy
on a Liberty automated peptide synthesizer with a single-mode
MW reactor (CEM), by RT and MW-assisted strategies. The reactions
were performed in a Teflon vessel and mixed by nitrogen bubbling.
Reaction temperatures were measured by an internal fiber-
optic sensor. The RT-SPPS protocol consisted of two consecutive
deprotection steps of 5 and 10 min, respectively, and a 20 min
coupling step. In MW-SPPS protocol, the two deprotection steps
were performed at 75 ◦C using 35 W for 0.5 min for the first one and
60 Wfor 3 min for the secondone, whereasthe coupling stepswere
performed at 75 ◦C, using 30 W for 5 min for all amino acids except
for Arg and His residues that required specific coupling parameters
performed in two steps (RT followed by MW irradiation, Table 1).
The syntheses were performed on Rink-amide NovaSyn TGR
resin (0.2 mmol/g, 500 mg), which was suspended in a solution
of DMF/DCM (1 : 1 v/v) and swelled for 30 min. During the
general coupling cycle, the N-terminal Fmoc-protecting group
was removed with a solution of 20% piperidine in DMF. Fresh
stock solutions of the Fmoc-protected amino acids (0.2 M) and
TBTU (0.5 M) in DMF, and of DIEA (2 M) in NMP were prepared
in separated bottles and used as reagents during the SPPS. In
particular, the coupling cycles were performed using 2.5 ml of
Fmoc-protected amino acids, 1 ml of TBTU and 0.5 ml of DIEA in
NMP of stock solutions. The fully assembled peptide was cleaved
from the resin by treatment with 7 ml of a TFA/TIS/water solution
(95 : 2.5 : 2.5 v/v/v) for 3 h at RT. The resin was filtered and the
combined filtrates were concentrated under a stream of nitrogen.
The crude peptide was precipitated from the cleavage mixture by
addition of ice-cold diethyl ether and stored for 30 min at −20 ◦C.
The precipitated product was collected by centrifugation, washed
with diethyl ether (3 × 7 ml) and centrifuged. The remaining solid
was dried under a stream of nitrogen and lyophilized.
MW-assisted Mini-cleavage of PTHrP(1–34)NH2 Fragments
Both RT and MW-assisted SPPSs were monitored by UPLC-ESI-MS
analysis of MW-assisted mini-cleavages of intermediate resin-
boundfragmentsusingDiscover S-Classsingle-modeMWreactor
equipped with Explorer-48 autosampler (CEM). The mixing of the
cleavage reaction was accomplished by magnetic stirring and
the reaction temperature was monitored at the bottom of the
reactor vessels by an IR sensor. A small sample of beads carrying
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CONVENTIONAL AND MICROWAVE-ASSISTED SPPS APPROACH
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RIZZOLO ET AL.
Figure 3. TIC chromatogram and deconvoluted spectrum of the cleaved mixture of the intermediate resin-bound sequence PTHrP(12–34)NH2 (Fmoc/tBu
RT-SPPS) generated by the MW-assisted minicleavages.
Table 2. Fmoc/tBu RT and MW-assisted SPPS deprotection and coupling protocols used for the synthesis of PTHrP(1–34)NH2
First deprotection
Second deprotection
Coupling
Power
Time
(min)
Power
(W)
Temperature
Time
(min)
Power
(W)
Temperature
Time
(min)
Temperature
(◦C)
Protocol
(◦C)
(◦C)
(W)
RT-SPPS
5
–
20
75
10
3
–
20
75
20
5
–
30
–
20
75
20
75
20
50
MW-assisted SPPS
Arga
0.5
35
60
Step 1
Step 2
Step 1
Step 2
–
–
–
–
25
5
25
–
His
2
4
30
a The Arg cycle (step 1 and 2) was performed twice refreshing the coupling solution.
and power, involved in the MW-assisted mini-cleavage reactions
were monitored as reported in Supporting Information (Figure S2).
The reaction mixture was then filtered and the crude peptide was
precipitated from the cleavage mixture by addition of ice-cold
diethyl ether followed by cooling for 5 min at −20 ◦C. The product
was collected by centrifugation and directly subjected to UPLC-
ESI-MS analysis (see Supporting Information).
Table 3. Yield of PTHrP(1–34)NH2 obtained from the RT versus MW-
assisted SPPS
Purity of crude
peptide (%)
Yield of >95%
pure peptide
SPPS strategy
RT
35
77
4.4% (18 mg)
6.3% (27 mg)
MW-assisted
HPLC Analysis of PTHrP(1–34)NH2
Crude PTHrP(1–34)NH2 obtained from both the RT and MW-
assisted SPPSs were analyzed by analytical RP-HPLC (Alliance 2695
HPLC system equipped with a 2996 photodiode array detector,
Waters(Milford, MA, USA))using a Jupiter C18 (5 µm, 250×4.6 mm)
column (Phenomenex, Torrance, CA, USA) at 1 ml/min. The
solvents used were A (0.1% TFA in H2O) and B (0.1% TFA in
ACN).
Fmoc-protected resin-bound peptide (10 mg) was weighted into
a fritted polypropylene tube and treated twice with a 20% solution
of piperidine in DMF (1 ml) each time for 5 min. The beads were
then washed with DMF (2 × 1 ml) and DCM (3 × 1 ml), dried under
vacuum and transferred into a 10 ml glass tube containing the
cleavage mixture that was placed into the MW cavity. The mini-
cleavages were carried out with 2 ml of TFA/TIS/water solution
(95 : 2.5 : 2.5 v/v/v) at 45 ◦C, using 15 W for 15 min with external
cooling of the reactor vessel at the positions shown in the
Reaction Scheme. All the parameters, i.e. pressure, temperature
RP-HPLC Semi-preparative of PTHrP(1–34)NH2
Lyophilized crude peptide was prepurified by solid-phase ex-
traction with an RP-18 LiChroprep silica column from Merck
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CONVENTIONAL AND MICROWAVE-ASSISTED SPPS APPROACH
Figure 4. Analytical RP-HPLC of crude PTHrP(1–34)NH2. Fmoc/tBu RT-SPPS (A) and Fmoc/tBu MW-assisted SPPS (B). HPLC: 10–60% B (0.1% TFA in ACN)
in A (0.1% TFA in H2O) over 20 min.
(Darmstadt, Germany) using H2O/ACN as eluents. The purifica-
tion of the peptide was performed by semi-preparative RP-HPLC
on a Supelco C18 180 Å (250 × 10 mm, 5 µm) column (Sigma
Aldrich, St. Louis, MO, USA); eluents: A 0.1% TFA in H2O; B 0.1%
TFA in ACN: flow 4 ml/min; gradient 30–60% of B in 30 min.
synthesis we applied such type of couplings for the three arginine
residues (R19-R-R21) and for the two histidine residues (H25-H26).
Evidently, carrying out the RT and MW-assisted SPPSs on the same
instrument allowed unbiased side-by-side comparison and the
conclusion that the latter procedure is superior to the RT-SPPS.
Although the modification of the coupling steps for Arg and
His in the MW-assisted SPPS seems to be empirical in nature, it
was guided by strong rational that took into account the distinct
capacity of this methodology to overcome putative hydrophobic
interactions and aggregation. The impact of these phenomena
increases with the progression of the coupling reactions. We
therefore decided to take advantage of the MW radiation only for a
short duration after the bulk of the reaction has been already taken
place. In this manner, the completion of the coupling reaction was
facilitated without causing undesired side reactions. We propose
this MW-based protocol as a general strategy for overcoming
difficult coupling reactions. Admittedly, applying this strategy
to different peptides will require fine tuning that will include
the adjustment of parameters such as duration of coupling and
recoupling steps as well as the level of MW energy employed in
the recoupling step. We are confident that adapting the strategy
outlined in this protocol will be advantageous over the ‘one
generic MW-assisted coupling cycle fits all’.
UPLC-ESI-MS Analysis of PTHrP(1–34)NH2 Fragments
UPLC-ESI-MS system consisted of an ACQUITY UPLC system (Wa-
ters) coupled with a Micromass Q-Tof MICRO mass spectrom-
eter (Waters) equipped with an ESI source. The chromatographic
separation was achieved on a Symmetry 300 C18 column (100 mm
× 2.1 mm, ID 3.5 µm, Waters) with the column temperature set at
30 ◦C. The flow rate was 0.15 ml/min with a linear gradient running
from 0 to 30% of B (solvent A: 2% ACN, 0.1% formic acid in H2O;
solvent B: 2% H2O, 0.1% formic acid in ACN) in 7 min, followed by
30–60% B in the next 3 min, then by 95% B for 2 min, and returned
to initial condition for 3 min for re-equilibration. The total run time
per sample was 15 min. The ESI-MS analysis was carried out in the
positive ESI mode, the optimal MS parameters were as follows:
capillary voltage 3.2 kV, cone voltage 30 kV, source temperature
120 ◦C and desolvation temperature 320 ◦C. Nitrogen was used
as desolvation and cone gas with a flow rate of 450 and 40 l/h,
respectively. For MS/MS analyses, argon was used as collision gas,
and the collision energy was set to 40 eV. Data were acquired and
processed using MassLynx software (Waters).
Acknowledgements
Chaire d’Excellence ANR 2009-2013 and Fondazione Ente Cassa di
Risparmio di Firenze are gratefully acknowledged.
Highlights and Limitations
Supporting information
This protocol describes two advantages of MW radiation in SPPS.
The first one is monitoring of traditional RT-SPPS by MW-assisted
mini-cleavages combined with fast, efficient and sensitive
UPLC-ESI-MS analysis (15 min/analysis vs 30–45 min/analysis for
traditional mini-cleavage). We use this procedure to confirm the
presence of difficult coupling steps that result in truncated and
deletionsequences. The secondadvantage isthe specificallytuned
MW-assisted SPPS that uses modified cycles targeting difficult cou-
plings. In these couplings the first step is carried out at RT (without
applying MW power, 20 ◦C) and for variable duration (25 or 2 min)
and the second step is carried out in the presence of MW power
(75 or 50 ◦C) for shorter time intervals (5 or 4 min). In the reported
Supporting information may be found in the online version of this
article.
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