Protein structure and function are highly dependent upon the fidelity of protein biosynthesis and the susceptibility of proteins to chemical modifications. Alterations to the defined primary sequence of proteins or to amino acid residue side chains through chemical reactions have been implicated in the progression of numerous diseases.[1-5] Protein
oxidation in particular has been strongly associated with the development of several degenerative conditions.[6-10] This oxidation has been previously postulated to occur post-
synthesis rather than pre-synthesis, resulting from reactions with free radicals commonly produced through normal cellular processes.[11-13] However, despite the highly
discriminatory process through which amino acids are selected for protein synthesis, oxidised amino acids have been incorporated into proteins, suggesting that this oxidation could also occur pre-translationally. For example, (S)-m-hydroxytyrosine produced under normal cellular conditions is an effective surrogate of (S)-tyrosine and is incorporated by the native translational machinery to produce oxidised proteins.
The aliphatic amino acids (S)-valine, (S)-leucine and (2S,3S)-isoleucine are highly susceptible to side chain modification and in particular oxidation.[15-19] Of interest in this
present work was the possibility that unsaturated analogues of the aliphatic amino acids would be misincorporated during protein biosynthesis resulting in oxidised protein. The seven compounds assessed represent all possible alkene analogues of (S)-valine, (S)-leucine and (2S,3S)-isoleucine, other than the hydrolytically labile α,β-unsaturated amino acids
(Figure 3.1). Their misincorporation was assessed in vitro using cell-free protein synthesis
(CFPS) with translational machinery sourced from native E.coli cellular lysate.
Figure 3.1 Dehydro amino acids under investigation in this chapter.
Synthesis of Dehydro Amino Acids
A literature search revealed that of the seven dehydro amino acids 1-7 of interest, the (S)-4,5- dehydroleucine (2) and (2S,3S)-4,5-dehydroisoleucine (4) had been previously reported as replacements for (S)-leucine and (2S,3S)-isoleucinerespectively, during protein synthesis in bacterial auxotrophs.[21,22] None of the other dehydro amino acids 1, 3, 5-7 had been studied
in this context, although the dehydro amino acids 5-6 are naturally occurring.[23,24]
Only the (S)-4,5-dehydroleucine (2) was commercially available and was purchased,
H3N O O H3N O O H3N O O H3N O O H3N O O H3N O O H3N O O 1 2 3 4 5 6 7
the amino acids 1, 3-7 were selected based on the number of steps and common methodologies. The chosen synthetic approaches reflect what was considered to be the most time efficient method for preparation. The dehydro amino acids 1, 3-7 were prepared as racemates. The use of racemic material is common for this type of incorporation study as there is negligible misreading of (2R)-amino acids.[22,25,26]
3.2.1Synthesis of 3,4-Dehydrovaline
It was anticipated that deprotection of N-phthaloyl-3,4-dehydrovaline methyl ester would
provide the desired dehydrovaline 1 (Scheme 3.1). N-Phthaloyl-3,4-dehydrovaline methyl ester was prepared as described previously.
Scheme 3.1 Synthesis of 3,4-dehydrovaline 1.
The two protecting groups of N-phthaloyl-3,4-dehydrovaline methyl ester were successfully removed by acid-hydrolysis to give 3,4-dehydrovaline 1. The amino acid was purified by precipitation upon the addition of propylene oxide to a solution of the amino acid 1 in hydrochloric acid and ethanol. The spectral characteristics of the 3,4-dehydrovaline
1 are consistent with those previously reported for this amino acid.
3.2.2Synthesis of 3,4-Dehydroleucine
3,4-Dehydroleucine 3 was synthesised by a reported approach (Scheme 3.2). (E)-4-
Methylpent-2-enoic acid was brominated by treatment with N-bromosuccinimide to give (E)-4-bromo-4-methylpent-2-enoic acid. Analysis of the product by 1H NMR spectroscopy
confirmed the synthesis of (E)-4-bromo-4-methylpent-2-enoic acid by the doublet signals at both 7.29 and 5.90 ppm integrating for one proton each and possessing matching coupling constants, which correspond to the olefinic protons. This reaction proceeds via the allyl
N O O O O H3N O O HCl, AcOH, H2O 73%
stabilised radical, due to the stability of the tertiary radical (the major resonance contributor) bromination was only observed at the 4- position.
Scheme 3.2 Synthesis of 3,4-dehydroleucine 3.
Treatment of (E)-4-bromo-4-methylpent-2-enoic acid with anhydrous ammonia in dry tetrahydrofuran gave a mixture of both 3,4-dehydroleucine 3 and (E)-4-amino-4- methylpent-2-enoic acid. Separation of these α- and γ- amino acids was achieved by anion exchange chromatography. 1H NMR spectroscopy confirmed the identity of 3,4-
dehydroleucine 3 by the olefinic resonance at 5.22 ppm integrating for one proton and by the signal at 4.70 ppm corresponding to the α- proton. 3,4-Dehydroleucine 3 and (E)-4-amino-4- methylpent-2-enoic acid are formed via SN2’ and SN1nucleophilic substitution reactions
3.2.3Synthesis of 4,5-Dehydroisoleucine
4,5-Dehydroisoleucine 4 was prepared by a similar method to that reported previously
(Scheme 3.3). The alkyne product of the Steglich esterification between
N-carbobenzyloxyglycine and but-2-yn-1-ol was reduced stereoselectively to the (Z)-alkene with hydrogen in the presence of Lindlar’s catalyst. 1H NMR spectroscopy confirmed the
identity of the product, (Z)-N-carbobenzyloxyglycine but-2-enyl ester by the olefinic signals at 5.60-5.45 ppm integrating for two protons.
OH O OH O Br 82% NBS, !,!,!-Trifluorotoluene 7% NH3, THF H3N O O
Scheme 3.3 Synthesis of 4,5-Dehydroisoleucine 4.
A [3,3]-sigmatropic rearrangement of the (Z)-alkenyl ester provided (2RS,3RS)-N- carbobenzyloxyglycine-4,5-dehydroisoleucine (de = 78%). This reaction proceeds via the formation of a chelate-bridged metal enolate by treatment with lithium diisopropylamine and zinc chloride, which then undergoes rearrangement with high diastereoselectivity. This high selectivity results from the chair-like transition state of this rearrangement. The identity of (2RS,3RS)-N-carbobenzyloxyglycine-4,5-dehydroisoleucine was confirmed by 1H NMR
spectroscopy, which revealed a distinctive multiplet at 5.70 ppm which corresponds to the olefinic methine proton.
The carbamate protecting group of the monoprotected 4,5-dehydroisoleucine was removed by treatment with iodotrimethylsilane in anhydrous chloroform to give 4,5- dehydroisoleucine 4. This was confirmed by 1H NMR spectroscopy as no signals
corresponding to aromatic proton were observed. The 1H NMR spectra of 4,5-
dehydroisoleucine 4 was consistent with that previously reported.
3.2.4Synthesis of the 3,4-Dehydroisoleucines
It was envisaged the 3,4-dehydroisoleucines 5-7 could be prepared by deprotection of the corresponding N-phthaloyl-3,4-dehydroisoleucine methyl esters (Scheme 3.4 & 3.5), in the
same manner as that described for the synthesis of 3,4-dehydrovaline 1 (Section 3.2.1). The
O N H O OH O HO DCC, DMAP, DCM 91% O NH O O O 97% Lindlar's catalyst, H2, MeOH O N H O O O O N H O OH O LDA, ZnCl2, THF de 78% H3N O O 68% 47% TMSI, CHCl3 de 95% + (2R,3R) + (2R,3R)
N-phthaloyl-3,4-dehydroisoleucine methyl esters were synthesised as described previously. The alkene isomers of N-phthaloyl-3,4-dehydroisoleucine methyl ester were
only partially resolved by reverse-phase HPLC and as such were collected in two fractions. One fraction consisted of (E)-N-phthaloyl-3,4-dehydroisoleucine and N-phthaloyl-3,4’- dehydroisoleucine and the other of (Z)-N-phthaloyl-3,4-dehydroisoleucine.
Scheme 3.4 Synthesis of the 3,4-dehydroisoleucines 5-6.
Scheme 3.5 Synthesis of the 3,4-dehydroisoleucine 7.
Acid-catalysed hydrolysis of the diprotected dehydroisoleucines gave the dehydroisoleucines 5-7, which were purified by reverse-phase HPLC. 1H NMR spectroscopy
confirmed the identity of these dehydroisoleucines 5-7, with spectral characteristics consistent to those reported previously.