of the protein formed from hydrazide23(Figure 6 b) corre- sponds to substitution of fluoride 26 for all five of the (S)- leucine residues found in native PpiB (Figure 6 a), while the next most abundant peak at 19 321 Da in Figure 6 b corre- sponds to substitution of four of the five (S)-leucine resi- dues. (Each substitution increases the mass by 18 Da. How- ever, due to the natural 13C isotope abundance, PpiB gives
four dominant peaks 1 Da apart, which differ in intensity by less than 15 %. These peaks were not resolved and any one may be labelled by the spectrometer, so the mass difference indicated for one substitution is 18!4 Da.) Based on the
mass-spectrometric data, the average degree of incorpora- tion of fluoride26is approximately 90 %. In the case of hy- drazide 25, the mass spectrum (Figure 6 c) shows that four or five of the (S)-leucine residues are replaced with alkene
27 (in this case, each substitution decreases the mass by 2 Da). This extent of incorporation was confirmed through amino acid analysis of the PpiB. There has been an earlier report of the incorporation of alkene27into leucine zipper peptides,[44]although no details of that study have been pub-
lished.
Previously, we had found that chloride29is not incorpo- rated into PpiB in place of (2S,3S)-isoleucine, despite sever- al other chlorides proving to be suitable substitutes for ali- phatic amino acids.[11]These studies had been based on the
use of a crude mixture of chloride29, its 2S,3Sdiastereomer, (S)-3-chlorovaline, and recovered starting material, obtained by chlorination of (S)-valine,[43] because it had not been
practical to separate and test the pure material. Close analy- sis of the mixture showed that chloride 29is unstable and converts into lactone 30 under the conditions of cell-free protein expression (i.e., pH 7.5, 378C), with a half-life of ap- proximately 15 minutes. By comparison, when the valine methyl ester3 cwas chlorinated, the chloride28that formed was sufficiently stable for it to be separated through HPLC without lactonization. With the cell-free system, this materi- al underwent deprotection in situ and product29was incor- porated into PpiB (Figure 5, lane D) as a substitute for (2S,3S)-isoleucine (Figure 5, lane C). With chloride 28, the most abundant peak in the mass spectrum of the PpiB at
Figure 5. SDS-PAGE analysis of synthesized His6-PpiB with A) no DNA,
B) DNA, C) no (2S,3S)-isoleucine, D) no (2S,3S)-isoleucine but with chloride28, E) DNA, F) no (S)-leucine, G) no (S)-leucine but with hy- drazide23, and H) no (S)-leucine but with hydrazide25.
19 455 Da (Figure 6 d) corresponds to substitution of the chlorinated free amino acid29 for all ten of the (2S,3S)-iso- leucine residues found in native PpiB (Figure 6 a), while the next most abundant peak at 19 431 Da results from nine re- placements (each substitution that corresponds to replacing a methyl group with chlorine increases the mass by an aver- age of 20.5 Da, although the natural carbon and chlorine isotope abundance in each of these chlorinated PpiBs results in five dominant ions 1 Da apart, differing in intensity by 15 % or less, so the difference indicated by the spectrometer is (20!5) Da). With chloride 29, the mass spectrum shows that the average degree of incorporation is above 90 %. For hydrazides23and25, the S30 extract simply provides a way to avoid having to carry out problematic deprotection as a separate step; however, for chloride28the S30 extract also continuously replenishes the free amino acid29as it is con- sumed through lactonization. As a result, incorporation is observed by using ester28, but not with the same initial con- centration of the free amino acid29.
Conclusion
This study has demonstrated the ability of the S30 extract to remove a range of protecting groups for direct incorporation of the resulting amino acids into a protein. This approach is more efficient because it not only decreases the number of synthetic steps required in the preparation of unnatural amino acids, but also provides a versatile method to circum- vent problems associated with chemical instability of amino acids during both their deprotection and protein synthesis. The method has been demonstrated to be suitable for the incorporation of the fluoro- and dehydroleucines26and27
and chlorovaline29as substitutes for leucine and isoleucine, respectively, at levels of 90 % or above. These high levels are more than adequate for applications such as isotopic la- belling or the fluorination of proteins, for use in spectro- scopic studies for example, in which the unmodified protein is not detectable. These levels are also suitable to investigate general rather than specific effects of amino acid modifica- tions on protein structure and function. Further, the cell- free system with the S30 extract allows for a complete site- specific incorporation of an unnatural amino acid through the addition of a mutant aminoacyl tRNA synthetase and cognate suppressor tRNA, under which conditions the incor- poration levels would be expected to be quantitative.
Experimental Section
Amino acid derivatives: With the exception of hydrazides2and9 aand
ester10 c, all the amino acids illustrated in Figures 1–3 are available from Sigma–Aldrich, Merck Pty. Ltd., Auspep, TCI Chemicals, or Aurora Fine Chemicals LLC, although for the purposes of this investigation many were prepared from the corresponding free amino acids (see the Support- ing Information). Phenylhydrazide2was synthesized from methyl ester 3 aby using phenylhydrazine.[45]Hydrazide9 awas prepared from (S)-
lysine by esterification with thionyl chloride in methanol, followed by
treatment of the corresponding ester with hydrazine hydrate.[46, 47]Ester
10 cwas prepared by the treatment of (S)-leucine with 1-adamantanol, di- methyl sulfite, andpara-toluenesulfonic acid.[48]
(S)-4-Fluoroleucine hydrazide (23) was prepared by the protection of (S)-leucine, bromination of phthalimide19, treatment of bromide20with silver fluoride, and reaction of fluoride21with hydrazine hydrate.[20](S)-
4,5-Dehydroleucine hydrazide (25) was prepared fromN-phthaloyl-4,5- dehydroleucine methyl ester (22)[49] by reaction with hydrazine hy-
drate.[20]M.p. 130–1328C;1H NMR (300 MHz, D
2O): d=5.06 (m, 1 H),
4.93 (m, 1 H), 4.15 (dd,J=9, 6 Hz, 1 H), 2.62 (dd,J=14, 6 Hz, 1 H), 2.54 (dd,J=14, 9 Hz, 1 H), 1.78 ppm (s, 3 H);13C NMR (100 MHz, D
2O):d=
168.9, 138.5, 116.9, 50.8, 39.8, 21.0 ppm; HRMS (ESI,+ve)m/z: calcd for C6H14N3O: 144.1137; found: 144.1140 [M+H]+.
ACHTUNGTRENNUNG
(2S,3R)-4-Chlorovaline methyl ester (28) was isolated through HPLC from the mixture obtained by chlorination of ester 3 c.[43] 1H NMR
(400 MHz, CD3OD):d=4.27 (d,J=4 Hz, 1 H), 3.87 (s, 3 H), 3.74 (dd,J=
12, 8 Hz, 1 H), 3.66 (dd,J=12, 6 Hz, 1 H), 2.35–2.45 (m, 1 H), 1.10 ppm (d, J=8 Hz, 3 H); 13C NMR (100 MHz, CD
3OD): d=170.2, 55.7, 53.8,
47.0, 39.5, 13.5 ppm; HRMS (ESI, +ve) m/z: calcd for C6H13NO2Cl:
166.0635 and 168.0605; found: 166.0633 and 168.0610 [M+H]+; m/z: calcd for C6H12NO2ClNa: 188.0454 and 190.0425; found: 188.0452 and
190.0428 [M+Na]+; further details are provided in the Supporting Infor- mation.
Treatment of amino acid derivatives with S30 extract fromE. coliBL21
Star (DE3): S30 extract fromE. coliBL21 Star (DE3) was prepared as
previously reported.[22]Stock solutions of the amino acid derivatives1 a–f,
2,3 a–g,4,5 a–c,6 a,b,7 a,b,8,9 a–c,10 a–c,11 a–d,12,13 a–c,14,15 a,b, 16,17 a–c, and18were prepared in water, ethanol, or dimethyl sulfoxide (DMSO) according to solubility. An aliquot (4mL) of each stock solution was diluted to a final concentration of 2 mmwith 4-(2-hydroxyethyl)-1-pi- perazineethanesulfonic acid (HEPES) buffer (116mL, 50 mm, pH 7.5) and the S30 extract (80mL). The mixtures were incubated at 378C for 6 h and centrifuged at 12 000 rpm for 10 min. Each supernatant was passed through an Amicon Ultra-4 (YM-10) centrifugal filter device, and the fil- trates were analysed with HPLC by using the Waters AccQ.Tag method, with reference to amino acid standard solutions and the background amino acid concentration of the S30 extract. Accordingly, a sample of each filtrate (20mL) was treated with AccQ.Fluor borate buffer (80mL) and reconstituted AccQ.Fluor reagent (20mL). The mixtures were ana- lysed by using an AccQ.Tag column (C18, 4mm, 150"3.9 mm), eluting with a gradient of acetonitrile in AccQ.Tag eluent. Representative HPLC traces are provided in the Supporting Information.
Cell-free protein synthesis: Plasmid DNA encoding for His6-PpiB with
expression under control of the phage T7 promoter (pND1098) was car- ried out according to a previous report.[10]Plasmid DNA was prepared
from E. coli DH5a/pND1098 with the Qiagen Plasmid Maxi kit. T7 RNA polymerase (50 000 U mL"1) was obtained from New England Bi-
oLabs Inc. (MA, USA). Cell-free protein synthesis was carried out by using a reported procedure[8, 10, 21, 22]with the following few modifications.
(S)-Alanine and RNasin were not added to the inner mixture (500mL). T7 RNA polymerase (2mL) was added to each reaction mixture instead of the plasmid encoding for this enzyme. An aliquot of tRNA solution of 5mL was added instead of 10mL. The final concentration of the solvent (ethanol or DMSO) used to dissolve some of the amino acid derivatives 1 a–f,2,3 a–g,4,5 a–c,6 a,b,7 a,b,8,9 a–c,10 a–c,11 a–d,12,13 a–c,14, 15 a,b,16,17 a–c, and18was#2 %, which was established through con- trol experiments to have no effect on protein synthesis. The His6-PpiB se-
quence (with an additional C-terminal asparagine residue;[10] mass=
19 221 Da, N-formyl-His6-PpiB=19 250 Da) is MHHHHHHMVT
FHTNHGDIVI KTFDDKAPET VKNFLDYCRE GFYNNTIFHR VINGFMIQGG GFEPGMKQKA TKEPIKNEAN NGLKNTRGTL AMARTQAPHS ATAQFFINVV DNDFLNFSGE SLQGWGYCVF AEVVDGMDVV DKIKGVATGR SGMHQDVPKE DVIIESVTVS EN. The in vitro cell-free reaction mixture containing expressed His6-
PpiB was centrifuged at 12 000 rpm for 2 min and the supernatant was ap- plied to a Ni-ion affinity column equilibrated with 20 mmsodium phos- phate, 0.5mNaCl, and 20 mmimidazole at pH 7.5 and 48C. Bound pro- teins were eluted by application of 20 mmsodium phosphate, 0.5mNaCl,
Chem. Eur. J.2013,19, 6824 – 6830 !2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 6829
FULL PAPER
In Situ Cell-Free Protein Synthesisand 500 mm imidazole at pH 7.5 and 48C. The eluted protein fractions were concentrated using an Amicon Ultra-4 (YM-10) centrifugal filter device and analysed by 20 % SDS-PAGE and mass spectrometry (see the Supporting Information for further details).
Acknowledgements
We gratefully acknowledge the support provided for this work through the CSIRO Emerging Science Initiative for Synthetic Enzymes and the ARC Centre of Excellence for Free Radical Chemistry and Biotechnolo- gy.
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Received: November 2, 2012 Revised: February 15, 2013 Published online: March 27, 2013