Bromelain Catalyzed Synthesis of Peptides in Organic Solvent
J. Chin. Chem. Soc., Vol. 50, No. 1, 2003 181
As shown in Table 1, Sephadex G-50 is the best adsorp-
tion material. In addition, the more the amount of adsorbent,
the better the yield. Polysaccharide or even monosaccharide
play a very important role in the stabilization of enzyme. We
also used XAD-7 and XAD-8 as adsorption material. How-
ever, these materials yielded poor results. Without adsorption
of enzyme onto an adsorbent, the yield of the dipeptide prod-
uct was generally below 5% in organic solvent.
decreased the yield of the dipeptide. The major reason was
due to secondary hydrolysis.
The reuse of enzyme
Bromelain and the adsorption material recovered after
the reaction were able to be reused. However, due to the deac-
tivation or loss of the enzyme during the reaction process, the
amounts of active enzyme were reduced. Upon the recovery
of the enzyme, ethyl acetate probably took some water out
from the system. Therefore, addition of proper amounts of
buffer is necessary to maintain enzyme activity, otherwise a
lot of enzyme activities was lost. This is realized when the en-
zyme was reactivated with the addition of excess amounts of
buffer, followed by incubation. The amount of water is the
most important factor. As the amount of water is not enough,
so is the recovery of the activities of the enzyme. It is worth
noting that addition of the same amount of water resulted in
the same activity as addition of the buffer. This result was in
accordance with Klibanov15 “it’s a good idea to saturate ethyl
acetate with water, which quenches their thirst for the en-
zyme’s essential water”. Addition of reducing reagent 2-mer-
captoethanol does not increase enzyme activity.
The effect of organic solvent
Several organic solvents were tested for promoting the
coupling, including dioxane, acetonitrile, CH2Cl2, ethyl ace-
tate, dioxane/ethyl acetate or acetonitrile/ethyl acetate. To
consider the solubility of the substrate, hydrophilic solvent
was preferred. The water content in the reaction system was
also varied. Ethyl acetate was chosen not only for its polarity
to dissolve the substrate but also its hydrophobicity to form a
biphasic system.
The effects of buffer content and pH
Bromelain is able to function under a wide range of pH
(3.5 ~ 7.5) conditions. For the synthesis of Cbz-Gly-L-Leu-
OMe, the best yield was obtained at pH 5. The reaction yield
was affected by different types of buffer. This effect was
tested by using water, 3 M, pH = 5, KCl-potassium phosphate
buffer, citrate-potassium phosphate buffer, citrate-sodium
buffer, or citrate-potassium buffer to undergo the reaction.
For the same type of buffer, as the concentration of buffer is
increased, the yield of the reaction improved. Among the cat-
ions we tested, the presence of Na+ ion activated bromelain
better than K+. And citrate is the best anion thus found.
The best result was obtained when the amount of buffer
was around 10% (v/v) to the solvent. The denaturation of
bromelain was minimized using a hydrophobic solvent-ethyl
acetate. The yield was lower when the amount of water was
too low to activate (dissolve) enough enzymes in active con-
formation. Water is also a product of the reaction and is ther-
modynamically unfavorable in reversing hydrolysis. Water
also participates in other side reactions. Therefore, the yield
will also be lower when an excess amount of water is added.
Lyophilizing the enzyme with buffer was attempted; al-
though the water content was decreased, no improvement
was observed for the yield.
The effect of triethylamine
We added triethylamine to convert the hydrochloride
salt of NH2CRHCOR’ to NH2CRHCOR’. The amount of nu-
cleophile in free a-amine form was generated in situ. How-
ever, using LeuOMe (0.2 mmole) to replace HCl·LeuOMe
(0.2 mmole) decreased the synthesis of Cbz-Gly-L-Leu-OMe
to 41%. Obviously, Et3N·HCl was also an important factor in
this reaction. As shown in Table 2, the yields were affected by
changing the amount of triethylamine salt and/or triethyl-
amine. The presence of Et3N·HCl (0.2 mmole) increased the
yield to 75%. Et3N·HCl is miscable in water and in organic
solvent. We suggest that Et3N·HCl promoted the transfer of
the substrate to the enzyme by minimizing the barrier be-
tween organic phase and aqueous phase.
Table 2. The Effect of the Triethylamine and Its Hydrochloride
Salt
LeuOMe·HCl
(mmole)
LeuOMe Et3N·HCl
Et3N
(mmole)
Yield
(%)
(mmole)
(mmole)
0.20
0.15
0.10
0.05
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0.20
0.15
0.10
0.05
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77
76
70
40
41
75
0.05
0.10
0.15
0.20
0.20
The rate of reaction
We found the kinetics of enzymatic reaction obeyed the
Michaelis-Menton equation. According to the reaction con-
dition to synthesize Cbz-Gly-L-LeuOMe, the maximum yield
(85%) was reached in 7 hours. Extending the reaction time
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0.20
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