A convenientmicrowave-enhanced solid-phase synthesis of short chain N-methyl-rich peptides

Article abstract of DOI:10.1002/psc.1209

Structural modification of the peptide backbone via N-methylation is a powerful tool to modulate the pharmacokinetic profile and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)-assisted Fmoc/tBu solid-phase methodto prepare short chain N-methyl-rich peptides, using Rink amide p-methylbenzhydrylamine (MBHA) resin as solid-phase support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc-N-methyl amino acid coupling. Copyright

Full text of DOI:10.1002/psc.1209

Research Article
Received: 28 September 2009
Revised: 25 November 2009
Accepted: 30 November 2009
Published online in Wiley Interscience: 2 February 2010
(www.interscience.com) DOI 10.1002/psc.1209
A convenient microwave-enhanced
solid-phase synthesis of short chain
N-methyl-rich peptides
Hortensia Rodr´ıguez,a,b∗ Margarita Suarezb and Fernando Albericioa,c,d∗
Structural modification of the peptide backbone via N-methylation is a powerful tool to modulate the pharmacokinetic profile
and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)-assisted Fmoc/tBu solid-phase
methodtoprepareshortchainN-methyl-richpeptides, usingRinkamidep-methylbenzhydrylamine(MBHA)resinassolid-phase
support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc-N-methyl amino acid
c
coupling. Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: solid-phase peptide synthesis; microwave; Fmoc-N-methyl amino acids
Introduction
Experimental Procedures
General
The replacement of natural amino acids for N-methyl amino
acids in biologically active peptides has resulted in analogs
with improved pharmacological properties, such as enzymatic
stability [1], receptor selectivity [2], enhanced potency [3], and
bioavailability [4,5]. The N-methylation of backbone confers
high affinity toward the targets, proteolytic stability, membrane
permeability, and conformational rigidity to the peptides. Thus,
in peptide chemistry N-methylation is considered as one of the
most attractive and subtle modifications of a peptide structure
[6].
Resin preparation
FmocRinkamideMBHAresin(0.015 mmol)wasplacedinapeptide
synthesis vessel, swollen in DMF, and deprotected with 5 ml of
20% piperidine/DMF for 4 min.
Washings between the first deprotection, coupling, and
subsequent deprotection steps were carried out with DMF
(5 × 0.5 min) and DCM (5 × 0.5 min) using 10 ml of solvent/g
of resin each time.
In SPPS, protocols to generate a very wide range of pep-
tides are well established, although the coupling of N-methyl
amino acids generally occurs in low yield and in many cases
requires expensive coupling reagents and double coupling. The
most promising reagents used to couple N-methylated amino
acids are bis(2-oxo-3-ox-azolidinyl)phosphonic chloride (BOP-
Cl) [7], HATU/HOAt [8] bis-(trichlororomethyl)carbonate (BTC)
[9], and (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium
hexafluorophosphate (PyAOP) or PyBOP/HOAt [10].
Furthermore, since 1986, microwave (MW) irradiation has
been applied to promote difficult organic reactions [11] and a
considerable number of articles have been published in the field
of MW-assisted organic chemistry [12]. However, the use of MW
irradiation in combination with SPPS has extended only in recent
years [13] as a result of the commercial availability of specialized
equipment for chemistry-dedicated MW applications with built-in
direct temperature control, magnetic stirring, and software for
temperature and pressure control.
MW-SPPS
Protected amino acid (3 eq.) and HOBt (3 eq.) in DCM (1–3 ml/g
resin), followed by DIPCDI (3 eq.) were sequentially added to the
resin and the mixture was irradiated in a CEM Discover MW for
20 min at 250 W with a maximum temperature of 35 ◦C.
∗
´
Correspondence to: Hortensia Rodr´ıguez, Laboratorio de S´ıntesis Organica,
FacultaddeQuimica,UniversidaddeLaHabana,10400-CiudadHabana, Cuba.
E-mail: horten@fq.uh.cu
Fernando Albericio, Institute for Research in Biomedicine, Barcelona Science
Park, Baldiri Reixac 10, 08028-Barcelona, Spain. E-mail: albericio@pcb.ub.es
a
b
c
Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10,
08028-Barcelona, Spain
´
Laboratorio de S´ıntesis Organica, Facultad de Quimica, Universidad de La
Habana, 10400-Ciudad Habana, Cuba
On the basis of a previous report on the synthesis of difficult
peptidesequencesusingtheMWirradiation[14],andtheimproved
results in terms of yields and purities with the use of standard
HOBt/N,N-diisopropylcarbodiimide (DIPCDI) couplings [15], here
we describe MW-assisted SPPS (MW-SPPS) with Fmoc/tert-butyl
(tBu) strategy of short chain N-methyl-rich peptides.
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona,
Spain
´
d
DepartmentofOrganicChemistry,UniversityofBarcelona,Mart´ıiFranque1-10,
08028-Barcelona, Spain
c
J. Pept. Sci. 2010; 16: 136–140
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

MICROWAVE FOR SPS OF N-METHYL-RICH PEPTIDES
1) DCM wash (3 x 1)
DMF wash (3 x 1)
2) Fmoc-deprotect
O
O
MW-SPP
HN
N
coupling
N
H
3) DCM wash (3 x 1)
DMF wash (3 x 1)
H2N
N
H
Fmoc
R
II
n = 3 or 4
R
I
RinkAmide-MBHAresin
n times
n=3 or 4
cleavage
Sequence
TFA:DCM (95:5v/v)
O
MeAla-MeGly-MePhe
IIIa
IIIb MeHis-MeAla-MeGly-MePhe
IIIc
IIId
MePhe-MeGly-MeGly
MeVal-MeGly-MeIle
MeAsp-MeIle-MeAla
Val-MeGly-MeAla
HN
NH2
n=3 or 4
R
IIIa-g
IIIe
IIIf
IIIg
MeAla-MeIle-MeGly
Scheme 1. Fmoc/tBu SPPS of short chain N-methyl-rich peptides (IIIa–g).
Figure 1. RP-HPLC of (NMe)Ala-(NMe)Gly-(NMe)Phe (IIIa).
Manual removal of the Fmoc-protecting group
According to standard SPPS protocols, the coupling time per
residue at room temperature usually varies between 2 and
4 h. The coupling time was shortened to approximate 80 min
by raising the temperature to 50 ◦C [17], and it has been
reported that, under conventional coupling conditions, when
only the acid component in the coupling is N-methylated, it
proceeds well under standard coupling. However, when both
components are N-methylated, as in our case, conventional
couplings pose a challenge and can present some problems
[9,10].
The MW-SPPS method provided a satisfactory coupling
efficiency in 20 min and no secondary amines were detected
by qualitative De Clercq [18] and chloroanyl tests [19]. Although,
there have been a few reports stating that the peptide resin
is well established on solid support at 50 ◦C [13e], we kept the
reactiontemperaturebelow40 ◦CunderMWirradiationtoprevent
undesired side reactions.
Two treatments with piperidine : DMF (2 : 8, v/v) for 10 min, and
two extra 5-min treatments with piperidine : DBU : toluene : DMF
(5 : 5 : 20 : 70, v/v) were used.
Cleavage of the peptides
Final unprotected amide peptides were cleaved from the resin
with the following cleavage cocktails : TFA : DCM (95 : 5 v/v) for
90 min (10 ml/g resin). Peptides were precipitated by addition of
cold diethyl ether, the solution was decanted, and the solid was
triturated with cold diethyl ether, which was decanted again. This
process was repeated twice.
Results and Discussion
The short chain peptides were synthesized using a Fmoc/tBu-
based strategy with stepwise coupling, and Rink amide MBHA
was used as solid phase support (Scheme 1). MW irradia-
tion was provided by a CEM Discover LabMate Focused Single
Mode MW Synthesis System, which produced continuous stir-
ring and irradiation with control of pressure and temperature
[16].
The Fmoc removal of peptidyl resins can be accelerated by
MW irradiation [20,14c]. However, to ensure complete Fmoc
removal, we followed standard treatments with 20% piperidine
in DMF solutions and two extra treatments of 5 min with
piperidine : DBU : toluene : DMF (5 : 5 : 20 : 70, v/v) as a previous
report showed that this treatment was optimal for avoiding
c
J. Pept. Sci. 2010; 16: 136–140 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

´
RODRIGUEZ, SUAREZ AND ALBERICIO
Table 1. Results from the MW-SPPS and theoretical and experimental masses of highly methylated short chain peptides
Product
Sequence
Theoretical MS
Experimentala MS (M+H)
Purityb %
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
MeAla-MeGly-MePhe
MeHis-MeAla-MeGly-MePhe
MePhe-MeGly-MeGly
MeVal-MeGly-MeIle
MeAsp-MeIle-MeAla
MeVal-MeGly-MeAla
MeAla-MeIle-MeGly
334.20
485.28
320.18
328.25
358.22
286.21
300.22
335.20
486.28
321.15
329.25
359.26
287.32
300.38
93.7
68.7
77.3
97.3
81.4
78.2
79.4
IIIg
a Experimental masses obtained by HPLC-MS and exact masses.
b Purity of crude peptides (analytical RP-HPLC peak area, UV absorbance at 220 nm). The chromatograms are reproduced in Figure 1 and the
supporting information.
Figure 2. RP-HPLC of (NMe)Asp(NMe)Ile(NMe)Ala (IIIe) at (a) room temperature and (b) 60 ◦C.
incompleted deprotection for N-methylpeptidyl resins [10]. Final
unprotected amide was cleaved from the resin by treatment with
TFA : DCM (95 : 5 v/v) (Scheme 1, SI-1).
The crude peptides were analyzed by HPLC-MS. Table 1 shows
the purity and the theoretical and experimental masses of the
highly N-methylated peptides obtained. All N-methyl peptides
were obtained in reasonable purity. See supporting information
for full characterization. As an example see Figure 1.
In order to check the true effect of the MW heating mode on the
solid-phase synthesis (SPS), we attempted to obtain at least two N-
methyl-rich peptide sequences (IIIa and IIIg) by the conventional
method, using the same conditions as under MW synthesis
(coupling reagents, time of coupling, temperature, cleavage
conditions, vessels). In both cases, peptides were obtained with
very low purity, even after double and triple coupling, and using
HOAt-based methods.
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 136–140

MICROWAVE FOR SPS OF N-METHYL-RICH PEPTIDES
Teixedo´ et al. [10] have described the SPS and characterization Supporting Information
of N-methyl peptides by chromatography. They observed multiple
Principal results, characterization of short chain N-methyl rich
peptides, details of HPLC-measurements. This material is available
free of charge via the Internet.
peaks in chromatogram profiles for peptides containing clusters of
three or more consecutive N-methylamino acids at the C-terminal
as a result of the slow conversion between individual conformers.
The highly N-methylated tri- and tetra-peptides synthesized
under MW-SPPS conditions generally showed profiles with narrow
symmetrical peaks in RP-HPLC chromatograms (Figure 1 and
supportinginformation).However,peptideIIIe(Sequence:MeAsp-
MeIle-MeAla) presented a broad peak in the chromatogram
profile, which may be due to the presence of high populations
of conformers that differ in their hydrophobic character, and
therefore in retention times (Figure 2(a)). This was confirmed
by the dramatic effect on the peak shape for an increase in
column temperature, since it changed from a broad to uniform
sharp profile as a consequence of accelerated cis/trans isomerism
(Figure 2(a) and (b)). The broad peak in the chromatogram profile
of IIIe is a consequence of the presence of two very close bulky
N-methyl-amino acid residues in this sequence and (NMe)Asp
in the N-terminal position. The free carboxyl group present
in this N-methyl amino acid could generate associations by
hydrogen bonds and promote slower cis/trans interconversion
of this peptide.
Our results indicate that the use of MW irradiation in the
synthesis of small N-methyl-rich peptides dramatically reduces
the reaction time and increases the purity of the product.
One key feature in MW-assisted chemical reactions is the
dipolar polarization mechanism. In this process, the alternating
electric field from MW radiation provides the energy required
for the rotation of the molecules with a dipole moment.
Unlike conventional heating, MW energy activates any molecule
with a dipole moment, thereby resulting in rapid heating at
the molecular level. Since peptide backbones are polar, this
mechanism is useful in preventing the aggregation of a growing
peptide chain during the coupling reactions in SPPS, thereby
improving the coupling efficiency. In addition, the technology
used (CEM Discover) allows the control of temperature at
35 ◦C.
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