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Protease-catalyzed routes to oligopeptides
The vision that motivates this program is to develop protease-catalyzed approaches to synthesize oligopeptides with control of sequence. For this purpose, we developed protease-catalyzed reverse equilibrium synthetic methods that offer some degree of sequence control as well as enantio- and regio-selectivity (Scheme 13). Important progress has been made on influences of pH, ionicity, monomer and enzyme concentration on reaction rates. By carefully controlling reaction parameters and using papain as catalyst, Et2-L-Glu was converted to oligo(γ-Et-L-Glu) in >80% yield, with avg. DP=7, in 20 min (Li et al., 2006) (Figs 14 and 15).
Scheme 13. General mechanism for protease-catalyzed oligomerization of amino acid alkyl ester monomers.
Figure 15. MALDI-TOF spectrum of oligo(γ-ethyl-L-glutamate) synthesized using 0.5 M L-glutamic acid diethyl ester hydrochloride, 8 mg/mL catalyst, at 40 °C for 20 min, pH 7.0, under noncontrolled pH conditions.
Figure 14. Time course of oligo(γ-ethyl-L-glutamate) synthesis. Reactions were conducted with 8 mg/mL papain catalyst, 0.5 M L-glutamic acid diethyl ester hydrochloride, at 40 °C, in phosphate buffer (0.9 M, pH 7) under noncontrolled pH conditions. Note that yields reach >80% in only 20 min. minimum values obtained.
Figure 16. Leucine unit distribution in octamer from MALDI-TOF spectra of oligo(γ-Et-L-Glu-co-L-Leu) synthesized using 0.25 M L-(Et)2-Glu-HCl and 0.25 M L-Et-Leu-HCl, 16 units/mL enzymes, at 40 °C for 4 h, 0.9 M phosphate buffer, pH 8.5. The curve is the theoretical distribution trend for each co-oligopeptide assuming a random distribution of repeat units.
In another published study by our laboratory, four proteases were evaluated for their ability to provide control of oligopeptide sequence for co-oligomerizations of Et2-L-Glu with L-Leu (Li et al, 2008). The cumulative results of LC−MS, monomer relative reactivity ratios and MALDI-TOF lead to the conclusion that papain, bromelain, α-chymotrypsin and protease SG all showed no apparent specificity with respect to a preference for adding either L-Et-Leu to a Et-Glu terminal propagating chain, or L-(Et)2-Glu to a Leu terminated chain (Fig 16). This resulted in the preparation of statistically random oligo(γ-Et-L-Glu-co-L-Leu) with DPavg ∼ 7 units. The compelling question raised by this work is which proteases will provide selectivity that regulates the sequence of this and other amino acid alkyl ester monomers during oligopeptide synthesis? The answer to this question is currently under study. The result of these efforts is expected to provide simple, cost-effective routes to oligopeptides with various degrees of sequence control for a wide variety of applications.
References
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Li G, Raman VK, Xie WC, Gross R.A. “ Protease-catalyzed co-oligomerizations of L-leucine ethyl ester with L-glutamic acid diethyl ester: Sequence and chain length distributions” Macromolecules 41 (19): 7003-7012 (2008).
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Li, Geng; Vaidya, A; Viswanathan, K; Cui, JR; Xie, W.C.; Gao, W; Gross, R.A.; Rapid regioselective oligomerization of L-glutamic acid diethyl ester catalyzed by papain, Macromolecules 39 (23): 7915-7921 (2006).
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