# Effect of Microwave Radiation on Enzymatic and Chemical Peptide Bond Synthesis on Solid Phase

Authors: Alessandra Basso; Loris Sinigoi; Lucia Gardossi; Sabine Flitsch
Journal: International Journal of Peptides
 (2009)

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## Abstract

Peptide bond synthesis was performed on PEGA beads under microwave radiations. Classical chemical coupling as well as thermolysin catalyzed synthesis was studied, and the effect of microwave radiations on reaction kinetics, beads' integrity, and enzyme activity was assessed. Results demonstrate that microwave radiations can be profitably exploited to improve reaction kinetics in solid phase peptide synthesis when both chemical and biocatalytic strategies are used.


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## Body

## 1. Introduction

The use of microwave (MW) heating has found successful
applications in solid phase organic synthesis (SPOS) [1–7].As
far back as 1992, Wang described the use of a single-mode microwave as a heating
source to accelerate the chemical coupling
[8] of many amino acids. Reactions on SP often suffer from unsatisfactory
reaction kinetics due to slow diffusion. Since microwave energy activates any
molecule with dipole moment, a rapid heating at a molecular level is achieved,
along with an improved diffusion of solutes.As an alternative to
chemical coupling strategies, peptide bond can be synthesized also via protease-catalyzed
reactions [9–11]. Proteases catalyze the thermodynamically controlled
synthesis in aqueous buffer while avoiding racemization and the needs for side chain protection of amino acids [12].Thanks to their small
molecular weight, proteases, such as thermolysin (35 kDa), diffuse efficiently in a range of different
swelling and rigid polymers, such that high conversions can be achieved
especially when hydrophobic amino acids are used. Moreover, it was
observed that the formation of the peptide bond is thermodynamically favored
when performed on SP—as compared to the process carried out in solution—even
though the heterogeneous reaction takes place in a bulk aqueous environment [12].In
this paper, we report the effect of microwaves in the formation of peptide bond
on SP, which also represents the first example of MW application in solid phase
biocatalysis.The swelling PEGA polymer
(cross-linked acrylamide and polyethylene glycol) was chosen as solid support
since it was previously found to be completely
permeable to proteins with molecular masses up to 35 kDa [9–15].As depicted in Scheme1, the dipeptide Fmoc-Phe-Phe
was synthesized using an MW assisted procedure starting from PEGA-Phe, which
was chemically synthesized.Scheme 1
Synthesis of Fmoc-Phe-Phe-PEGA1900 under
microwave radiation via
enzymatic and chemical coupling. (a) (i) DIC/DMAP in DMF; (ii) Pyp 20%. (b)
Chemical coupling (DIC/HOBt in DMF) or enzymatic coupling (Thermolysin in
aqueous buffer) (see Supplementary Material available online at doi:10.1155/2009/362482).The biocatalyzed reaction was carried out using a
thermostable protease, thermolysin (Thermolysin fromBacillus thermoproteolyticus rokko) which had been previously
employed for peptide synthesis on PEGA beads [9].As a comparison, MW assisted chemical coupling was
carried out using the traditional HOBt/DIC approach, which has been
previously employed in the MW assisted
peptide synthesis in solution [16].The MW assisted enzymatic synthesis of the dipeptide
Fmoc-Phe-Phe-PEGA1900,
Scheme 1,
was monitored for a range of temperatures from 50°C to 100°C at a power of 50 W
(see Supplementary Material). The reactions were followed by determining the
amount of Fmoc on the resin (Figure 1) [15]. Product was also analyzed by HPLC after
cleavage from the resin and verifying its chemical purity and enantiopurity.Figure 1
Reaction profiles of the enzymatic synthesis of
Fmoc-Phe-Phe-PEGA1900, under microwave radiation at 50°C (bold
circles), 60°C (empty circles), 70°C (bold triangles), 80°C (empty triangles),
90°C (bold squares), and 100°C (empty squares).Figure2 reports the initial rates [15] of the MW assisted enzymatic synthesis as
function of the temperature.Figure 2
Initial rates of enzymatic reactions
performed under microwave radiation (black squares) and without microwave
radiation (empty squares) at different temperatures.It appears that at 80 and 90°C, the highest enzymatic
activity can be achieved. However, from the reaction profiles reported in Figure 2 it is evident that only at 80°C the enzymatic coupling was complete after one hour whereas at 90 and 100°C the
enzyme undergoes a fast denaturation (bold and empty squares) so that complete
conversion was achieved only upon addition of fresh thermolysin. It is interesting to note that similar profiles had
been previously reported for a different enzyme, the lipase B from Candida antarctica, that showed the
highest activity at 60° C [17, 18].The graph reported in Figure1 also shows that at temperature
ranging from 50 to 70°C the reaction achieves completion within 3 to 4 hours.A nonthermal effect due to microwaves is evident when
comparing the initial rates measured at 50 and 80°C in the absence of MW (in a
thermostated bath, Figure 1, bold circles and empty triangles, respectively); at
80°C the initial rate is eight times lower.Similar nonthermal effects have been reported for
protease esterification and transesterification [19], whereas in lipase-catalyzed reactions no microwave effect was
observed [19–23].For comparison, the chemical coupling on SP was also
carried out (Figure3). Complete conversion was obtained at 50°C and 50 W power
after 30 minutes [13]. (All the amino groups, initially present on the resin (0.15 mmol/gdry), were acylated as confirmed by loading determination.) It must be underlined that the same chemical coupling in the absence of
MW radiation at the same temperature requires two reaction cycles, one of 2 hours
and one of 24 hours, respectively [14, 15].Figure 3
Chemical coupling of Fmoc-Phe on PEGA1900 under microwave radiation at 50°C and 50 W. Determination of conversion
(see Supplementary
Material).The swelling capacity of PEGA1900 in water
was not affected by microwave heating so that after 30 minutes incubation in
water at 100°C under microwave radiation the determined swelling capacity
remained 23 mL/g, as in the case of an identical sample incubated at room
temperature without microwave radiation. This observation indicates that the
improvement of reaction kinetics was not due to a variation of the swelling
properties of the polymer upon microwave heating.The physical stability of PEGA1900 polymer
to microwave treatment was verified by analyzing samples of beads after
microwaves irradiation (Figure 4). After 1 hour of
microwave radiation at 100°C
in water no morphological modification of the beads was observed, and after 1 hour
of irradiation in
DMF at 150°C less than 1% of the beads (determined by beads counting) showed
any evident damage in their structure (Figure 4(b)).(a) Physical
appearance of PEGA1900 beads after microwave radiation at 150°C in
DMF; (b) a detail of the damaged beads counted in a Burker chamber (less than 1% of total).
(a)(b)An indirect evaluation of the chemical stability of
PEGA1900 was obtained by comparing the loading capacity of the resin
before and after the irradiation. After 1 hour of treatment at 100°C under microwave irradiation
it was verified that 0.145 mmol/g of functional groups were still accessible to
Fmoc-Phe out of the initial 0.150 mmol/g. This was also confirmed by reacting
the beads–after being
irradiated with MW at 100°C—with dansyl
chloride and analyzing them with two-photon-microscopy. A good uniformity of
the fluorescence was measured (data not shown) demonstrating that MW treatment
does not affect the reactivity.A severe decrease of the loading capacity of the
polymer was observed only after irradiation at 150°C that caused the loss of
90% of the initial accessible amino groups (0.150 mmol/g).In conclusion, we here demonstrated that PEGA1900 is applicable to both chemical and enzymatic
peptide synthesis under microwave irradiation. The benefit of MW radiation in
terms of improvement of reaction kinetics has been demonstrated both in the
chemical and enzymatic coupling. In the case of enzymatic coupling an optimum
temperature of 80°C allows to achieve maximum reaction rates while avoiding the
fast enzyme denaturation observable at higher temperature.



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*Source: 362482-2009-03-25.xml*