Access to Any Site Directed Stable Isotope (2H, 13C, 15N, 17O and 18O) in Genetically Encoded Amino Acids

Proteins and peptides play a preeminent role in the processes of living cells. The only way to study structure-function relationships of a protein at the atomic level without any perturbation is by using non-invasive isotope sensitive techniques with site-directed stable isotope incorporation at a predetermined amino acid residue in the protein chain. The method can be extended to study the protein chain tagged with stable isotope enriched amino acid residues at any position or combinations of positions in the system. In order to access these studies synthetic methods to prepare any possible isotopologue and isotopomer of the 22 genetically encoded amino acids have to be available. In this paper the synthetic schemes and the stable isotope enriched building blocks that are available via commercially available stable isotope enriched starting materials are described.


Introduction
Proteins and peptides play a preeminent role in living cells, such as receptor action, enzyme catalysis, transport and storage, hormone action, mechanical support, immune protection, etc.
[1]. The 22 genetically encoded amino acids that are the building blocks of proteins and peptides are depicted in Figure 1 [2]. All amino acids except Gly have the L-configuration at the chiral α-carbon atom. Ile and Thr have two chiral atoms and Pyl has three chiral atoms. Tyr, Val, Met, Leu, Ile, His, Lys, Phe, Arg, and Trp are essential amino acid for mammals. These amino acids must be available in their food,

OPEN ACCESS
as mammals are incapable of synthesizing or of synthesizing them in sufficient amount to meet metabolic needs.  After the primary protein systems are formed via translation of DNA and RNA, post-translational modifications of residues that include acylation, phosphorylation, sulfation, hydroxylation, oxidative methylation, prenylation and cross-linking occur that lead to the active proteins and peptides in the living cell. In order to understand the role of proteins for the structure-function, dynamics and localization of the individual proteins in the complex environment of living cells, techniques such as labeling endogenous proteins have been applied [3]. A drawback of these labeling methods is that modifications are introduced on the protein systems in question. Site-directed stable isotope introduction on the other hand, allows labeling in the protein system without any modification.
Nowadays, 13 C-, 15 N-enriched proteins are detected in prokaryotic cells using NMR techniques [4]. Earlier, methyl groups were used for the NMR detection of proteins in cells [5]. Stable isotope enriched systems have been used to study protein structure with 2D-Heteronuclear NMR techniques [6]. Somewhat later, methods for preparing either U-13 C or U-15 N or both U-13 C and U-15 N with 99% stable isotope incorporation in the genetically encoded amino acids became available. This has led to a quantum jump in the application of NMR techniques in the study of proteins. The NMR methods have been optimized by using U-13 C, U-15 N-amino acids in which only one proton is stereoselectively replaced by deuterium at a methylene group (stereo-array isotope labeling-SAIL) [7,8]. Many more important new NMR techniques for the study of proteins have been reported [9][10][11][12][13][14]. In addition to the NMR methods, IR techniques are also used to study the protein function [15]. In general the required isotopically labeled proteins are prepared by using genetic expression techniques; one of the optimal methods of this technique is the use of cell-free synthesis [16]. The preparation of proteins with isotopically enriched amino acids via genetic techniques has as a main drawback that all amino acid residues of one type in the protein chain will be enriched.
At the moment chemical total synthesis is an active and a fruitful research field that allows the site-directed stable isotope incorporation at any specific amino acid residue in a protein molecule [17].
The first native chemical ligation procedure has been developed based on the cystein residues. Later, a method was developed to make a new peptide bond via deselenization of piptidyl selenoester where sulfur atom from corresponding thioester of cystein residue is replaced with selenium atom [18][19][20]. Native chemical ligation of hydrophobic peptides that are insoluble in water has also been revealed [21]. A general method of chemoselective ligation involves decarboxylative condensation of an α-keto acid of a peptide and a hydroxyl amine function of another peptide to make a new amide bond of the expected peptide [22].
In order to study the site-directed stable isotopically labeled proteins with the known and newly developed isotope sensitive non-invasive techniques, the access to any stable isotopologue and stable isomer of the genetically introduced amino acids is essential. In this paper the synthetic and chemoenzymatic methods to get access to these systems will be discussed.
Known L-α-amino acids labeled with stable isotopes at specific positions have been reported [23,24]. Synthetic methods are optimized to resolve problems due to diastereotopic methyl groups, hydrogen atoms and additional chiral centers. The synthetic schemes discussed in this paper are easily simplified when these problems are not present for the isotopomer in question. The use of possible isotopologues of amino acids in a protein molecule will allow the use of mass spectral techniques that plays an important role in the field of metabolomics and proteomics [25][26][27]. In addition, the possibility of introducing 18 O isotopes in amino acid residues has aided the use of mass spectrometry in the protein study [28].

Synthetic Schemes
A number of simple highly stable isotope ( 2 H, 13  The synthetic schemes should start from achiral building blocks wherein high enantioselectivity is achieved by using chiral catalysts, chiral phase transfer catalysts and enzymes. The use of chiral templates that require additional chemical reaction steps should be avoided. The synthetic schemes should result in the product without isotopic loss, dilution or scrambling. The schemes should give well-defined synthetic methods in the case of the presence of diasteretopic methyl groups (Val, Leu) or diasteretopic hydrogen atoms (except Ala, Val and Thr).
The schemes discussed in this paper are optimized to meet these requirements for the synthesis of all possible isotopologues and isotopomers. General methods (Schemes 1-6) are indicated in the specific Roman numbers whereas the synthesis of 22 amino acids (Schemes 7-30) are indicated in Arabic numbers. When building blocks from the general schemes are used in the specific schemes they maintain their Roman numbers. Based on the required isotope enrichment in the system, many synthetic schemes can easily be simplified.

Reductive Amination of α-Keto Acids IV
α-Keto acids IV of the corresponding amino acids are easily available from acid bromides III. Treatment of acid bromides III with copper (I) cyanide and subsequent hydrolysis followed by enzymatic reductive amination gives the corresponding enantiomeric pure amino acids V [35,36]

Hydrolysis of α-Amino Nitriles VII
In the Strecker reaction aldehydes VI are treated with ammonia in the presence of hydrocyanic acid to give D,L-mixtures of α-amino nitriles VII followed by hydrolysis to afford D,L-mixtures of amino acids VIII (Scheme 3) [37,38].  Treatment of the D,L-mixture of α-amino nitrile VII with an enzyme nitrilase gives a separable mixture of L-α-amino acid and D-α-amino nitrile. In general the presence of a chiral catalyst does not lead to an enantiomeric pure form except for the synthesis of valine [39]. In Scheme 4 it is shown that D,L-α-amino acid VIII can be converted into the oxazol-5-(4H)-ones X (azlactones) via N-acetylated glycine ester derivative IX. Following the dynamic kinetic resolution procedure the corresponding L-α-amino acid derivative V can be separated from D,L-α-amino acids VIII [40,41]. Another approach is shown in Scheme 5 for the conversion of D, L-α-amino nitriles VII into the corresponding D,L-α-amides XI. The final hydrolysis of the mixture with an enzyme amidase gives pure L-α-amino acids V and pure D-α-amino amides XII. D-α-Amino amides XII are simply racemized via an intermediate benzalimine to form a second batch of D, L-α-amino amides XI [36]. Scheme 5. The conversion of D,L-α-amino nitriles VII into D,L-α-amino amides XI to afford L-α-amino acids V.

Glycine
Glycine serves as a building block of peptides and proteins. Stable isotope enriched glycine derivatives function as the starting materials to introduce stable isotopes ( 2 H, 13 C and 15 N) at the α-carbon, the carboxylic acid and the amino group of all L-α-amino acids.

Alanine
In Scheme 10 it is indicated that treatment of CH 3 I (21) under O'Donnell conditions with protected glycine gave a high yield of L-α-alanine [56].

Serine
In Scheme 11 it is indicated that stable isotope enriched N-benzoylglycine ethyl ester 6a is treated with stable isotope enriched formate 33 to form the 2,3-didehydroderivative 34. Compound 34 is converted into the corresponding tert-butyldiphenyl silyl ether that is subsequently hydrogenated with a chiral rhodium catalyst to give stable isotope enriched serine derivative 35 [67].  [68]. The N-protected glycine ester XIV is treated with compound 39 in the presence of a base and phase transfer catalyst. The N-protecting benzophenoneimine group is removed by treatment with citric acid followed by a reaction using Boc-anhydride and triethylamine to afford N-Boc, O-benzyl serine tert-butyl ester. Catalytic hydrogenation of this compound yielded serine derivative 40 with a free hydroxyl group. Final deprotection of the amino group and ester hydrolysis afforded L-serine which is now accessible in any stable isotope-enriched form using commercially available building blocks [69].

Cysteine and Selenocysteine
In Scheme 12 it is indicated that the protected serine derivative 40 is converted via a Mitsonobu reaction into N-protected S-acetyl cysteine 41 that after base induced deacetylation and acid catalyzed N-deprotection afforded cysteine [69].
Treatment of N-protected serine derivative 40 with bromine and triphenylphosphine in the presence of imidazole afforded N-protected 3-bromo serine 40a. Reaction of N-protected 3-bromo serine 40a with Se 8 and hydrazine in the presence of NaOH yielded the diselenide derivative 42. After sodium borohydride reduction and acid catalyzed amino group deprotection selenocysteine is obtained [69]. Besides selenocysteine, upon catalytic reduction of N-protected 3-bromo serine derivative 40a stable isotope enriched alanine can be obtained [67].

Threonine
Acetaldehyde (26) is converted into 1,1-dipropoxyethane by acid catalyzed reaction with 1-propanol. 1,1-Dipropoxyethane is mixed with D 2 18 O in the presence of HCl (g) to afford 18  Next, the bislactimether of cyclo-(D-Val-Gly) is treated with n-BuLi at −78 °C in THF to obtain an anion of compound 43 that is treated with chlorotitaniumtris(diethylamide). To this mixture isotopically labeled acetaldehyde (26a) is added to afford the required bislactimether of cyclo-(D-Val-Thr) 44 via Schöllkopf method. Hydrolysis of the product 44 afforded methyl esters of D-valine and L-threonine. Removal of valine by cationic exchange chromatography and hydrolysis of the remaining product gave L-threonine and L-allo-threonine in a 15:1 ratio. These two compounds could be separated easily. It is gratifying that this method led to an optimal formation of the two chiral centers in one step [68].
Oxazolines 46 can be prepared via the reaction of aldehydes with methyl α-isocyanoacetate (45) in the presence of a chiral catalyst. Recently, a general method to prepare stereo-induced oxazolines 46 has been used to synthesize threonine [70].

Proline
In Scheme 15 it is depicted that reaction of HCHO (2) (50) with the N-(diphenylmethylene)glycine tert-butyl XIV afforded the N-(diphenylmethylene)glutamic acid 1-tert-butyl-5-ethyl ester (51). Removal of benzophenoneimine group, Boc protection of the amino group and subsequent NaBH 4 /LiCl reduction of the ethyl ester function yielded the alcohol derivative 52. Conversion of the primary alcohol function into the corresponding bromide is carried out with a mixture of triphenylphosphine and bromine in dichloromethane. The internal nucleophilic substitution of the free amino group led to ring closure to afford proline tert-butyl ester. Removal of the tert-butyl ester by hydrolysis with 10% TFA afforded L-proline [74].

Valine
In Scheme 16 it is shown that the phosphorane 53 is obtained by the alkylation of ethyl-(triphenylphosphoranylidene)acetate (13a) with ethyl 2-bromoacetate (12b) in the presence of solid K 2 CO 3 . The phosphorane 53 is treated with H 2 C 13 O (2a) to otain itaconic diester 54 via the Wittig reaction. Upon treatment with DBU and heating in the presence of concentrated HCl isomerization of the exo-double bond and hydrolysis of the ester bond are effected to give pure 2-methyl fumaric acid 55. Reaction of the product 55 with NH 3 and β-methyl aspartase afforded 3-methyl aspartic acid (56). The formation of the N-trifluoroacetamide of the succinic acid anhydride is achieved by the addition of trifluoroacetic anhydride in THF. The ring opening of the anhydride with 2-propanol afforded the product 57 with ester function at C-1 position. The mixed anhydride derivative of product 57 is reduced with NaBH 4 to afford the alcohol 58. Conversion of the primary alcohol function into the iodo compound 59 is effected by the treatment with triphenyl phosphite and iodine. The iodo function is removed by catalytic reduction and deprotection in the presence of a base to yield valine. The 13 C-isotope enriched [4-13 C]-valine is prepared to show that these synthetic methods allow the chiral discrimination between the two diastereotopic methyl groups. Using 13 C-formaldehyde (2a) (2S,3S)-[4-13 C]-valine with a trace of the other enantiomeric form is obtained [47].
In Scheme 17 it is indicated that the Wittig-Horner reaction of ethyl 2-(diethylphosphono)acetate [13a, prepared by the reaction of ethyl 2-bromoacetate (12b) and triethyl phosphite] with acetaldehyde (26) afforded ethyl crotonate (63) followed by DIBAL-H reduction to afford crotyl alcohol (64). These compounds are accessible in all possible stable isotopologues and isotopomers [76]. Sharpless asymmetric epoxidation of alcohol 64 gave the epoxide 65. The epoxide is treated with (Ph) 3 C-Cl (trityl chloride) to protect the primary alcohol group as trityl ether 66. The S N 2 reaction with trideuteromethyl-lithium copper complex gave the enantiomeric pure deuterated derivative 67. Mesitylation followed by reaction with the sodium azide afforded an azido derivative with trityl ether. The O-protection is removed by refluxing in acetic acid to obtain the azido alcohol derivative 68. Reduction of the azide function yielded (2S, 3S)-[4-CD 3 ]-valine [77].
Another alternative method of preparation of an α-keto acid 73 would be a Hell-Volhardt-Zellinsky reaction of the product 72 with PBr 3 . The corresponding bromide could be further reacted with triphenylphosphine to obtain the ylide followed by ozonolysis to afford the α-keto acid 73. Reductive amination of α-keto acid 73 affords valine (Scheme 2).

Glutamine and Glutamic Acid
In Scheme 18 it is indicated that ethyl 2-nitroacetate (74) is prepared by the reaction of ethyl bromoacetate (12b) with NaI and AgNO 2 . Michael addition of compound 74 with ethyl acrylate (50) in the presence of benzyltrimethyl ammonium hydroxide afforded diethyl 2-nitroglutarate (75). The anion of product 75 is ozonolyzed to obtain diethyl 2-oxoglutarate (76). The reductive amination with ammonia in the presence of L-glutarate dehydrogenase followed by base catalyzed saponification afforded glutamic acid [73]. Glutamic acid can easily be converted into pyroglutamic acid (5-oxoproline, 77) that offers an alternative building block for the synthesis of isotopically enriched proline [79,80].

Scheme 18.
Preparation of stable isotope enriched glutamic acid and glutamine from ethyl 2-bromoacetate 12b and 2-bromoacetic acid 12c, respectively. The reaction of acrylonitrile (80) with the N-protected glycine tert-butyl ester XIV under O'Donnell conditions afforded the expected product 81. N-Deprotection followed by the conversion of the nitrile function of product 81 into amide afforded glutamine [74]. A similar reaction of ethyl acrylate (50) with XIV yielded the N-protected ester derivative 51. N-Deprotection followed by hydrolysis of the ester function of product 51 into carboxylic acid afforded glutamic acid [74].
All possible isotopomers of acrylonitrile (80) are accessible from bromoacetic acid (12c). Cyanoacetic acid is obtained by the reaction of bromoacetic acid (12c) with KCN (3). Esterification of the carboxylic group with ethanol afforded ethyl cyanoacetate (78) followed by the reduction with NaBH 4 in the presence of LiCl to obtain 2-cyanoethanol (79). Acrylonitrile (80) is obtained by treatment of the alcohol 79 with Ac 2 O followed by the base catalyzed elimination of acetic acid [81].

Methionine
Synthetic methods are shown in Scheme 19 for the conversion of N-Boc aspartic acid tert-butyl ester 49 (Scheme 14) into the alcohol 82. The reaction steps necessary for this conversion have been described in the Scheme 15 (the conversion of product 51 into product 52). Mitsonubu reaction with thioacetic acid and DIAD afforded the N-Boc protected thioacetate derivative 83. Treatment of product 83 with a base in the presence of CH 3 I yielded the methylthioether formation to give the required protected methionine that upon deprotection of the amino group with acid afforded methionine [69].

Isoleucine
In Scheme 21 synthetic methods are shown to prepare isoleucine. The hydroxyl function of the valine derivative 62 (Scheme 16) is tosylated and then treated with lithium dimethyl copper to give the protected isoleucine derivative 94 which upon deprotection yielded isoleucine [75]. Acetaldehyde (26) reacted with phosphorane 53 to obtain 2-ethylidenebutanedioate (95) that upon treatment with DBU the exo-double bond is isomerized to afford 2-ethylbutanedioate 16 it is shown that phosphorane 53 can be obtained by the reaction of an ylide 13a with ethyl br (96). In Scheme omoacetate (12b) in the presence of solid K 2 CO 3 . The conversion of 2-ethyl-2-butenedioate (96) into the corresponding isoleucine is effected by following a procedure similar to the conversion of the lower homologue 2-methyl fumaric acid (55) into valine in Scheme 16 [47]. The N-(tert-butylphenylmethylene)glycine tert-butyl ester XIVb reacted with methyl crotonate (63a) on a chiral calcium complex (prepared by the reaction of Ca(O i Pr) 2 with a chiral catalyst) to give protected (2R,3R)-3-methyl glutamic acid (97) [85]. After saponification of the methyl ester and exchange of the nitrogen protection the N-Boc glutamic acid derivative 98 is obtained which after Barton radical decarboxylation afforded valine [47]. Reaction of glutamic acid derivative 98 with ethyl chloroformate yielded the protected alcohol 99. Isoleucine is obtained after reduction of the iodine function derived from the alcohol and removal of the N-protection group [86].

Scheme 21. Preparation of isotopically enriched isoleucine.
Treatment of the N-p-methoxyphenyl protected α-imino ester 100 [accessible by the reaction of glyoxalate (15a) and 4-methoxyaniline] with 2-butanone in the presence of L-proline resulted in (2S,3S)-N-p-methoxyphenyl protected ester 101 in high yield. Reduction of the ketofunction and deprotection of product 101 afforded (2S,3R,4S)-4-hydroxyisoleucine (102). The alkene derivative is obtained upon removal of the hydroxyl function of product 102 and subsequent catalytic reduction of the double bond afforded isoleucine [87].

Lysine
In Scheme 22 it is indicated that ethyl bromoacetate (12b) reacted with KCN (3)   O'Donnell reaction of 4-iodobutyronitrile (106) with XIV yielded product 81 (Scheme 18) that upon deprotection and again the protection with reducing agent stable N-protecting Boc group afforded 107, that upon catalytic reduction of the nitrile function and deprotection afforded lysine [74].

Histidine
In Scheme 24 it is indicated that methylammonium chloride (112) after neutralization with sodium methanolate reacted with formic acid (113) in acetic anhydride to form N-methylformamide (114). Upon treatment with tosyl chloride and the base quinoline, methyl isocyanide is formed that is further treated with two equivalents of LDA and then reacted with tosyl fluoride to afford tosylmethyl isocyanide (115). Reaction of the product 115 with BuLi and subsequent reaction with trimethylsilyl chloride afforded trimethylsilyl tosylmethyl isocyanide (116). The anion of the product 116 is reacted in a Peterson olefination reaction with 3-phenylpropenal (cinnamaldehyde, 117) to afford the conjugated isocyanide 118. The isocyanide 118 reacted with benzyl amine (112a) and K 2 CO 3 to form an intermediate imidazolidine ring. With the elimination of p-toluenesulfinic acid the imidazole ring is formed to afford the product 119. The product 119 is treated with a mixture of potassium osmate (VI) dihydrate (K 2 OsO 4 ·2H 2 O) and sodium periodate (NaIO 4 ) which cleaved the exo-double bond to afford N-benzyl imidazole aldehyde 120. This molecule can be converted into the (Z)-2,3-didehydrohistidine derivative by reaction with the Wittig reagent triethyl phosphonoacetate (not shown in the scheme). The aldehyde function of the product 120 is reduced with LiAlH 4 and the resulting hydroxyl group is subsequently treated with thionyl chloride to convert it into a chloride. The imidazole group has a pKa of about 7 that afforded the product 121 as a HCl salt.
Originally, product 121 is treated with two eq of the anion 43 of bislactim ether (Schöllköpf method). This led to a loss of one equivalent of anion 43. The formation of the protected histidine worked well and histidine is isolated after reluxing in HCl and hydrogenation with Pd in cyclohexene [90]. Later the reaction is carried out under O'Donnell conditions. With this method a much milder base at lower pH is used and the formation of the histidine derivatives is smoothly effected [91].
Stable isotope incorporation in 3-phenylpropenal is easily effected by Horner-Wardsworth-Emmons reaction of diethyl phosphonoacetonitrile and benzaldehyde. Subsequent DIBAL reduction converted the nitrile function into the aldehyde function. Diethyl phosphonoacetonitrile can be isotopically labeled at any position via commercially available labeled acetonitrile. 15 N-Benzylamine has been prepared via the reaction of benzoyl chloride with 15 NH 3 , subsequent LiAlH 4 reduction of benzamide afforded benzyl amine.
Because of the large number of steps involved in the synthesis of the product 119 a new synthetic method is explored with fewer steps. Ethyl bromoacetate (12b) is treated with benzyl amine (112a) to form ethyl N-phenylglycine which upon treatment with formic acid (113) in acetic anhydride gave the glycine formamide (123). The product 123 underwent a base induced ester condensation with methyl formate to give the enolate of the C-formyl derivative. This molecule reacted with thiocyanate to afford 2-thio imidazolone derivative 124. Removal of the sulfur is effected by treating it with nitric acid in the presence of NaNO 2 resulting in the ethyl ester of the protected imidazole compound. LiAlH 4 reduction gave the imidazole alcohol 125. This is converted into the imidazole derivative 121 that has been easily converted into histidine in a more efficient way than the first approach [92].
It is to be expected that the scheme can be optimized by treating the C-formyl derivative 123; with POCl 3 to form the vinyl chloride chloroimidinium salt 126. Molecules analogous to 126 reacted with NH 4 Cl and Na 2 CO 3 under substitution of the chloride function to form the benzyl-5carboethoxyimidazole which upon reaction with LiAlH 4 afforded the alcohol 125.

Arginine
In Scheme 25 it is indicated that the reaction of N-protected glycine tert-butyl ester XIV reacted with acrylonitrile (80) to afford the nitrile derivative 81 (Scheme 22) that upon N-deprotection with acid and subsequent N-protection with acetyl chloride afforded the N-acetyl protected nitrile. The nitrile derivative is reduced by H 2 in the presence of PtO 2 to afford the N-protected L-ornithine 127 [74]. The N-protected arginine tert-butyl ester 129 is obtained by the reaction of N-acetyl ornithine tert-butyl ester (127) with the thiourea derivative (methyl carbamodithioate) 128 [93]. Methyl carbamodithioate (128) can be obtained in any stable isotope enriched form by the reaction of NH 4 Cl 1 with potassium thiocyanate (KSCN) followed by the S-methylation of thiourea with CH 3 I.

Phenylalanine
The most difficult part in the preparation of L-phenylalanine is the development of a synthetic scheme that suited for all possible combinations of 2 H, 13 C incorporation in the benzene ring. In Scheme 26 a synthetic method is depicted that allows the isotopic enrichment in the benzene ring of phenylalanine. Acetic acid (11) is treated with a four-fold excess of PBr 3 and one equivalent of Br 2 . This afforded 2,2-dibromoacetic acid that reacted with ethanol to give ethyl 2,2-dibromoacetate (130). Upon treatment with phenolate ion the bromine groups are substituted by phenoxy groups to afford the product 131. Repeating the reduction of ester group in the product 138, Swern oxidation of corresponding alcohol afforded penta-2,4-diene-1-al (139). Horner-Wadsworth-Emmons reaction with diethyl phosphonoacetonitrile (140) gave 1,6-disubstitued hexatriene system 141. Heating product 141 led to cyclization with the expulsion of N-methyl aniline yielding benzonitrile (142). In this scheme the building blocks have been used that are easily available in all possible stable isotope enriched forms. The final product benzonitrile (142) is therefore now accessible in all possible isotopomeric forms [49].
Another route is the Wittig reaction of the aldehyde 143 with ethyl ester of N-acetyl-2-dimethyl phosphonato glycine (20, Scheme 9) to afford 2,3-didehydrophenylalanine (145) that has been converted into phenylalanine by asymmetric hydrogenation. An alternative method is the reduction of benzaldehyde (143) with NaBH 4 to obtain benzyl alcohol that is treated with thionyl chloride to obtain benzyl chloride (146). This has been reacted with the N-protected glycine XIV under O'Donnell conditions to obtain protected phenylalanine [47].

Tyrosine
In Scheme 27 the synthetic route for the preparation of tyrosine starting from benzonitrile (142) is shown. The compound 142 is treated with methyllithium, followed by acid catalyzed hydrolysis to obtain acetophenone that upon reaction with m-chloroperbenzoic acid in water afforded the product phenyl acetate via Bayer-Villiger oxidation. Phenol (147) is obtained by hydrolysis of phenyl acetate [49]. Phenol 147 underwent an enzyme catalyzed reaction with serine to give a high yield of tyrosine [36,94].
An alternative route is the conversion of phenol (147) into anisole by the reaction with diazomethane. A subsequent Gatterman synthesis with Zn(CN) 2 in the presence of HCl afforded almost quantitatively p-methoxybenzaldehyde (148) [95]. Condensation of the aldehyde 148 with oxazol-5-(4H)-one X (R = H) (Scheme 8) and subsequent ring opening afforded 2,3-didehydrotyrosine that upon asymmetric catalytic hydrogenation gave the methyl ether of tyrosine. Final step is the HBr induced removal of the ether function to obtain tyrosine [96].
The schemes discussed so far that allow isotopic enrichment in tyrosine are rather lengthy. For a limited number of 13 C isotopes in the aromatic ring the reactions in lower line in Scheme 27 have been described. The condensation of acetone (149) with 2-nitromalonaldehyde (150) under basic conditions afforded p-nitrophenol (37) in a good yield. Reduction with NaBH 4 and hydrolysis of p-nitrophenol (37) yielded aminophenol (151). Diazotization with sodium nitrite and reduction of the diazonium ion with hypophosphite resulted in phenol (147). Phenol (147) is treated with serine in the presence of the enzyme to afford a high yield of tyrosine [94]. Using [1,2,3-13 C 3 ]-labeled acetone (149) the 13 C isotopes are introduced at the carbon positions 1, 2, 6 of p-nitrophenol (37). In this way tyrosine with 13 C at positions 3′, 4′ and 5′ has been produced [94]. Treatment of the product 37 in the presence of 5-chloro-1-phenyltetrazole (152) with potassium carbonate gave the product ether 153. Hydrogenolysis of the product 153 cleaved the ether bond and simultaneously reduced the nitro function to an amine resulting in aniline which upon diazotization and hydrolysis in water in the presence of Cu 2 O/Co(NO 3 ) 2 afforded phenol (147). It is possible to obtain phenol (147) enriched with 13 C isotopes at positions 3′, 4′ and 5′ using this method. The protons ortho to the phenolic hydroxyl function can easily be exchanged for deuterons [97].The preparation of 17 O and 18 O nitrophenol (37) has been discussed in Scheme 11 [94,97].
The reactions discussed in Scheme 27 afford tyrosine with 17 O or 18 O in the phenolic OH group if necessary also in combinations with isotope incorporation in the aliphatic side chain. Schemes that allow to 17 O or 18 O incorporation with stable isotope incorporation in the aromatic ring have not been reported. Deuteration at positions 3′ and 5′ in the ring is easily achieved by acid catalyzed deuterium exchange under these conditions without 17 O or 18 O exchange [98].

Tryptophan
In Scheme 28 it is indicated that crotyl alcohol 64 (Scheme 17) is converted into crotonaldehyde after MnO 2 oxidation and treated with propargyl amine 154 to form the imine 155.

157
Reaction of product 154 with ethyl chloroformate converted it into the ethyl carbamate derivative 155 that is treated with HCHO (2) in the presence of catalytic amounts of CuBr and diisopropyl amine, the alkyne 155 is converted into the alkene 156. Product 156 is converted into the tetrahydroindole ester 157 by heating at 160 °C. This molecule is oxidized with two equivalents of dichlorodicyanoquinone (DDQ) to afford the indole ester followed by a base catalyzed saponification to obtain indole 158. This synthetic method allows for the introduction of isotopes 15  At this moment no scheme is available to enrich the isotopes in propargyl amine (154). Via an E. coli mutant indole (158) can be reacted with serine to convert it into tryptophan. 15 N-Anthranilic acid (163) can be incorporated into tryptophan residues of protein without 15 N scrambling or isotope dilution [99][100][101].
The synthetic route for the prepration of anthranilic acid (163) is shown in the third line in Scheme 28. Acetaldehyde (26) is treated with diethyl phosphonoacetonitrile (140) in a Horner-Wadsworth-Emmons reaction to obtain crotononitrile (159). Upon reaction with two equivalents of LDA and two equivalents of triisopropylsilyl chloride, the bis-(triisopropylsilyl)-imine 160 is obtained, reacting this molecule with ethyl acrylate (50) underwent a Diels-Alder reaction to form the dihydroanthranilic ester derivative. Upon treatment with dichlorodicyanobenzoquinone N-silyl substituted anthranilic ester derivative 161 is obtained. Removal of the triisopropylsilyl group to achieve the ester function 162 and subsequent hydrolysis of the ester group afforded the anthranilic acid (163), the molecule is now accessible in any stable isotope enrich form [102].
A synthetic method for the conversion of anthranilic ester (162) into indole (158) has been depicted In Scheme 29. Anthranilic ester (162) is treated with ethyl bromoacetate (12b) in the presence of sodium ethanoalate. First, the amino group is alkylated followed by an intramolecular ester condensation to obtain 2-carbethoxy-β-hydroxy indole (164) [103]. Treatment of the product 164 with aq. KOH and subsequent acid induced decarboxylation afforded the hydroxy indole which is subsequently reduced to achieve indole (158) [104,105]. (158)

168
Indole reacts efficiently with electrophilic reagents. The Vielsmeier-Haack reaction of indole (158) with dimethyl formamide (165) afforded the indole derivative 166 that is reacted with methyl isocyanoacetate (45,Scheme 13) to obtain the isocyano derivative of indole 167. Mild acid treatment and catalytic asymmetric reduction with D 2 gives the access to prepare tryptophan specifically deuterated in the aliphatic side chain [106,107]. It is also possible to obtain tryptophan via Mannich reaction of indole (158) [108]. 3-Dimethylamino methyl indole is obtained by the treatment of the indole (158) with formaldehyde and dimethylamine, followed by the reaction with CH 3 I to afford trimethylammonium iodide 168. This molecule is treated with the protected glycine under O'Donnell conditions to yield the protected tryptophan [109].

Pyrrolysine
Pyrrolysine is the 22nd genetically encoded amino acid [2]. It consists of a (4R,5R)-4-methyl-5carboxypyrroline ring linked to the ε-nitrogen of L-lysine. The access to any stable isotopologue of lysine has been discussed in the paragraph in lysine (Schemes 22 and 23).

Scheme 30.
The access to stable isotope labeled (4R,5R)-4-methyl pyrroline-5-carboxylic acid and its conversion into pyrrolysine. The acid derivative 170 is obtained after removal of the amino and carboxylic acid protecting groups. This step is followed by protection of the amino function with a Boc group and reduction of the acid into the alcohol function and protection of the hydroxyl group with tert-butyldiphenylsilyl chloride to afford 171. Hydrolysis of the tert-butyl ester and removal of the N-Boc protection is achieved by the treatment of the product 171 with trifluoroacetic acid. Treatment of this free amine with triflyl azide under diazo transfer conditions afforded the azide 172. The azide function is reacted with N-trifluoroacetamidyl lysine O-methyl ether that reacted with the free ε-amino group of the protected lysine to give an amide bond. Removal of the alcohol protection and subsequent Swern oxidation of the hydroxyl group led to the azide aldehyde derivative 173. Staudinger reduction of 173 with triphenylphosphine and intramolecular Aza-Wittig reaction afforded pyrrololysine with the protection in the lysine side chain which is removed by treatment with LiOH in methanol to obtain lithium salt of pyrrolysine 174.

Conclusions
In this paper the known synthetic schemes to access stable isotope enrichment in the genetically encoded amino acids is reported, together with the stable isotope enrichment of the building blocks. These building blocks are synthesized from the commercially available isotopically labeled starting materials. An essential fact in the syntheses of stable isotope enriched amino acids is that depending on the isotopologues and isotopomers of the required amino acid these schemes can be simplified and the number of synthetic steps can be minimized in a rational way.
With the availability of the full set of isotopomers of the proteinogenic amino acids, all peptides and proteins composed of these amino acids can be labeled at any position or combinations of positions. The isotopically enriched amino acids in the protein will greatly facilitate the study of intra-protein distances, torsion, bond angles and aliphatic-aromatic interactions. With the development of new and better synthetic schemes in the near future to enrich proteins with stable isotopes in an efficient way this will be a preeminent technique in the process of translating structural and functional, biological information etc. at the atomic level of the protein coded by genome into spectroscopic information.