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though acetamides are more common, a trifluoroacetamide functionality offers some specific advantages in synthesis be- cause incorporation of the trifluoroacetyl group deactivates the amino acida-position towards radical reactions.[26, 27]It is

apparent that there are limitations to the removal of amino protecting groups by the S30 extract, however, as illustrated by the lack of reaction of carbamate 17 a, sulfonamide17 b, andN-decanoylleucine17 c.

Most of the compounds examined had either thea-amino

ora-carboxyl group protected, but the reactions of glutamyl hydrazide8, serine acetate13 a, and aspartate and glutamate benzyl esters 13 b,c, respectively, demonstrate that side- chain carboxylic acid derivatives are also cleaved. As for doubly protected amino acids, both protecting groups are re- moved from the N-acetylated leucine methyl ester 14, but the corresponding hydrazide18is inert.

It does not affect the utility of the results, but it is none- theless interesting to consider the enzymes likely to be in the S30 extract and responsible for the reactions observed. Leucine aminopeptidases (LAP, E.C. 3.4.11.1) are known to hydrolyse hydrazide 1 a[28] and the presence of this enzyme

ester 3 b,[28] but in separate studies with this enzyme we

found it does not show activity for other protected amino acids, such as valine hydrazide 1 c and ester 3 c. Therefore other enzymes, such as aminopeptidase N (PepN, E.C. 3.4.11.2), which is known to have broad substrate specifici- ty,[29–31] are also likely to be involved, as well as those en-

zymes with amino acid selectivity, such as methionine ami- nopeptidase Type 1 (MAP 1, E.C. 3.4.11.18);[32]aminopepti-

dase B (PepB, E.C. 3.4.11.23), which is selective for acidic amino acids;[33] and oligopeptidase B (Protease II, E.C.

3.4.21.83), which shows selectivity for basic amino acids.[34, 35]

Hydrazide cleavage appears to be dependent on enzymes that require their substrates to have a free amino group, such as LAP,[28] because 1 a and 2 react, whereas the des-

amino hydrazide 16 andN-acetylated leucine hydrazide 18

do not react. Ester cleavage is not limited in this way, how- ever, as shown by the reaction of the acetylated leucine methyl ester 14.b-Aspartyl peptidase (E.C. 3.4.19.5),[36, 37]g-

glutamyl transpeptidase (E.C. 2.3.2.2),[38, 39]and glutaminase

A (E.C. 3.5.1.2)[40]are likely to be involved with the removal

of side-chain protecting groups, whereas carboxypeptidases probably account for the amino acid deacylation reactions. In any event, given the large number of enzymes expected to be in the S30 extract, there is likely to be redundancy.

Having determined the scope of the deprotection of amino acid derivatives by the S30 extract, examples of those shown in Figures 1 and 2 were tested for deprotection and incorporation into peptidyl-Pro cis–trans isomerase B (PpiB) in situ through cell-free protein synthesis.[8, 10, 21, 22]

Figure 4 shows a representative SDS-PAGE analysis of the purified proteins: lane B shows the PpiB produced when each of the twenty normal amino acids (1 mm) was added, lane C shows a decreased amount of that protein when (S)- leucine was not added and only the background concentra- tion of this amino acid was present (shown through separate HPLC analyses to increase from negligible to around 0.01 mmduring the course of the experiment), and lanes D and E show the PpiB formed when hydrazide 1 a (2 mm)

Figure 2. Amino acid derivatives partially deprotected (>20–<80 %) byE. coliS30 extract under standard conditions for cell-free protein synthesis.

Figure 3. Amino acid derivatives unaffected (<10 % reaction) by theE. coliS30 extract under standard conditions for cell-free protein synthesis. Boc=tert-butyloxycarbonyl.

illustrate that PpiB is efficiently produced by using either (S)-leucine, hydrazide1 a, or ester3 aand that conversion of hydrazide 1 a and ester 3 a into the free amino acid occurs sufficiently easily that it does not limit cell-free protein syn- thesis under the standard conditions. In identical fashion, it was demonstrated that ester3 d, amide 4, acetamide5 a, tri- fluoroacetamide6 a, and the doubly protectedN-acetyl-(S)- leucine methyl ester 14resulted in unrestricted synthesis of PpiB. Less protein formed with hydrazides1 band9 a, ester

10 a, and amide 10 b, but even in those cases unrestricted PpiB production was achieved by adding more of the S30 extract, by preincubating the mixture before finally adding the PpiB DNA, or by increasing the assay time to 24 hours. In this context it is relevant to note that cell-free protein synthesis occurs continuously throughout the experiment (6 h) performed under standard conditions (as evident from the SDS-PAGE analysis provided in the Supporting Infor- mation), whereas 50 and 75 % of methyl ester3 ahad hydro- lysed after 0.5 and 1 h, respectively, whereas only 50 % of tert-butyl ester 10 a was converted into free (S)-Leu after 6 h. Irrespective of this, the reactions carried out in situ were shown to be compatible with the cell-free protein syn- thesis for a variety of carboxylic acid derivatives including hydrazides, methyl esters, benzyl esters,tert-butyl esters and amides, and amino protecting acetamides and trifluoroaceta- mides.

The utility of these observations is demonstrated by the incorporation of the unnatural amino acids (S)-4-fluoroleu- cine (26), (S)-4,5-dehydroleucine (27), and (2S,3R)-4-chloro- valine (29) into PpiB through the direct use of their respec- tive protected forms 23, 25, and 28in the cell-free system. The latter compounds were prepared through side-chain elaboration of (S)-leucine and (S)-valine[20, 41–43] (Schemes 1

and 2, respectively). All attempts to prepare the free amino acids 26 and27 by deprotection of the corresponding leu- cine derivatives 21 and22 were frustrated by formation of lactone24. Eventually, removal of the phthaloyl group from

fluoride 21 was achieved by treatment with hydrazine to give hydrazide23, which underwent oxidative cleavage with NBS to give amino acid 26in addition to lactone24. Even by using this approach, the protected alkene22afforded hy- drazide25, but treatment of it with NBS gave only lactone

24. Nevertheless, as shown by the SDS-PAGE analysis illus- trated in Figure 5, PpiB was efficiently produced through cell-free synthesis by using hydrazides 23 and 25 (lanes G and H, respectively), as substitutes for (S)-leucine (lane E). The most abundant peak at 19 340 Da in the mass spectrum

Figure 4. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of synthesized His6-PpiB with A) no DNA, B)

DNA, C) no (S)-leucine, D) no (S)-leucine but with hydrazide1 a, and E) no (S)-leucine but with methyl ester3 a.

Scheme 1. Side-chain elaboration of (S)-leucine. Leu=leucine, NBS=N- bromosuccinimide.

Scheme 2. Side-chain elaboration of (S)-valine.

Chem. Eur. J.2013,19, 6824 – 6830 !2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 6827

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