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

Dawadi, Prativa B. S.; Lugtenburg, Johan

doi:10.3390/molecules18010482

Open AccessReview

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

by

Prativa B. S. Dawadi

and

Johan Lugtenburg

^*

Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands

^*

Author to whom correspondence should be addressed.

Molecules 2013, 18(1), 482-519; https://doi.org/10.3390/molecules18010482

Submission received: 25 October 2012 / Revised: 10 December 2012 / Accepted: 24 December 2012 / Published: 2 January 2013

(This article belongs to the Special Issue Chemical Protein and Peptide Synthesis)

Abstract

:

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.

Keywords:

amino acids; isotope labelling; [5-¹³C]-leucine; [4-¹³C]-valine; ¹³C or ¹⁵N-enriched L-lysine; [¹⁸O]-benzylchloromethyl ether; [¹³C]-benzonitrile

1. 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, as mammals are incapable of synthesizing or of synthesizing them in sufficient amount to meet metabolic needs.

Figure 1. Genetically encoded amino acids (Gly (G), Ala (A), Ser (S), Cys (C), Sec, Thr (T), Asn (N), Asp (D), Pro (P), Val (V), Gln (Q), Glu (E), Met (M), Leu (L), Ile (I), Lys (K), His (H), Arg (R), Phe (F), Tyr (Y), Trp (W) and Pyl are listed based on the number of carbon atoms and amino acids with the same number of carbon atoms are listed based on increasing atomic mass.

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, ¹³C-, ¹⁵N-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-¹³C or U-¹⁵N or both U-¹³C and U-¹⁵N 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-¹³C, U-¹⁵N-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 ¹⁸O isotopes in amino acid residues has aided the use of mass spectrometry in the protein study [28].

2. Synthetic Schemes

A number of simple highly stable isotope (²H, ¹³C, and ¹⁵N), ¹⁷O (70%) and ¹⁸O (97%) enriched building blocks are commercially available. Except glycine all amino acids in Figure 1 exist in the L-form. This gives a (2S)-configuration except in Cys and Sec. These amino acids have a (2R)-configuration due to the presence of S and Se atoms, respectively. Ile and Thr have (2S, 3S)- and (2S, 3R)-configurations, respectively.

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 (Scheme 1,Scheme 2,Scheme 3,Scheme 4,Scheme 5,Scheme 6) are indicated in the specific Roman numbers whereas the synthesis of 22 amino acids (Schemes 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30) 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.

3. General Methods to Synthesize L-Amino Acids

3.1. Catalytic Reduction of 2,3-Dehydroamino Acids I

N-Acetyl-2,3-dehydroamino acid esters I are easily available via elimination reactions of β-substituted N-acetyl amino acid esters [29,30,31]. Asymmetric hydrogenation of 2,3-dehydroamino acid esters gives an access to prepare a wide range of amino acids. N-Acetyl-2,3-dehydroamino acid derivatives I have been treated with D₂ (²H₂) in the presence of a chiral Rh catalyst to afford (2R, 3R)-[²H₂]-L-amino acids II (Scheme 1) [32]. Using the [3-²H]-2,3-dehydroamino acid derivatives it is possible to obtain (3S)-[3-²H]-L-amino acids in highly enantiomeric pure form [33,34].

Scheme 1. N-Acetyl-2,3-dehydroamino acid derivatives I occur in the thermodynamically stable Z-form. Catalytic reduction with D₂ in the presence of chiral catalyst and subsequent hydrolysis gives (2R, 3R)-[²H₂]-L-amino acids II.

3.2. 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]. The synthetic method described in the Scheme 2 allows to the incorporation of [2-²H]-, [2-¹⁵N]- and [¹³C]-isotopes in the corresponding amino acids V.

Scheme 2. Enzymatic reductive amination of α-keto acids IV to afford [²H, ¹⁵N or ¹³C]-amino acids V.

3.3. 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].

Scheme 3. Preparation of D,L-mixtures of α-amino nitriles VII via the Strecker reaction.

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].

Scheme 4. Preparation of enantiomeric pure L-amino acids V via dynamic kinetic resolution of oxazol-5-(4H)-ones X.

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.

3.4. Alkylation of N-(Diphenylmethylene)glycine tert-Butyl Ester XIV (O’Donnell Method)

In Scheme 6 it is shown that the N-(diphenylmethylene)glycine tert-butyl ester XIV is converted into protected monoalkylated L-α-amino acid derivative XV in a high yield with a high enantiomeric excess. These derivatives are easily converted into the corresponding amino acids [42,43,44,45].

Scheme 6. Monoalkylation of N-(diphenylmethylene)glycine tert-butyl ester XIV with phase transfer catalyst O-allyl-N-(9-anthracenylmethyl)cinchonidinium bromide with primary halides to give the N-protected L-α-amino acid derivatives XV.

4. Synthesis of 22 Amino Acids

4.1. 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 (²H, ¹³C and ¹⁵N) at the α-carbon, the carboxylic acid and the amino group of all L-α-amino acids.

In Scheme 7 it is indicated that starting materials NH₄Cl, HCHO and KCN are used for the preparation of glycine and N-(diphenylmethylene)glycine tert-butyl ester XIV. The Strecker reaction of NH₄Cl (1), two equivalents of HCHO (2) and KCN (3) afforded the product hexahydro-1,3,5-triazine-1,3,5-tris(acetonitrile) (4) [46]. Treatment of compound 4 with acid in ethanol yielded 2-aminoacetonitrile (5) and diethoxymethane. Subsequent acid hydrolysis of the compound 5 gave glycine (6). Starting materials NH₄Cl (1), HCHO (2) and KCN (3) are commercially available in all possible stable isotope enriched forms.

Scheme 7. Synthesis of 2-aminoacetonitrile (5) and its conversion into N-(diphenylmethylene)-glycine tert-butyl ester XIV for the preparation of glycine in all possible isotope enriched forms.

The amino and carboxyl groups of glycine (6) are protected using benzyl chloroformate and tert-butyl bromide, respectively, to afford N-protected glycine tert-butyl ester 7 [47]. Hydrogenation of N-benzyloxycarbonyl glycine tert-butyl ester (7) with palladium on charcoal yielded the tert-butyl ester of glycine with a free amino group. Reaction with commercially available 1,1-diphenylmethyl-eneimine afforded N-(diphenylmethylene)glycine tert-butyl ester XIV [47].

The synthetic methods depicted in Scheme 8 show that glycine (6) can be converted into oxazol-5-(4H)-one X (R = H) via N-acetyl glycine IX (R = H). Treatment of compound X (R = H) with potassium cyanate afforded carbamide 8, that is refluxed in aqueous HCl to obtain 1-acetylimidazolidine-2,4-dione (9) [48]. Hydantoin derivative 9 is treated with benzaldehyde via a Knoevenagel reaction followed by hydrolysis of product 10 to afford the corresponding α-keto acid IVa or N-acetyl-2,3-dehydroamino acid derivative Ia [49,50].

Scheme 9 illustrates the conversion of acetic acid (11) into 2-bromoacetic acid via a Hell-Volhard-Zellinsky reaction. Esterification of 2-bromoacetic acid with tert-butanol afforded tert-butyl 2-bromoacetate (12) [47]. Treatment of ester 12 with NH₃ yielded the tert-butyl ester of glycine (6) [51]. Transimination of tert-butyl ester of glycine (6) with benzophenoneimine afforded N-(diphenylmethylene)glycine tert-butyl ester XIV.

Scheme 8. The conversion of glycine (6) into oxazol-5-(4H)-one X (R = H) and 1-acetylimidazolidine-2,4-dione (9).

Scheme 9. The conversion of acetic acid (11) into N-(diphenylmethylene)glycine tert-butyl ester XIV and N-(methylphenylmethylene)glycine tert-butyl ester XIVa via tert-butyl glyoxalate (15). Conversion of ethyl glyoxalate (15a) into N-acetyl phosphonato glycine ethyl ester (20). Products XIV, XIVa, 15 and 20 can be obtained in any ¹³C and ¹⁵N position to enrich glycine part with stable isotopes.

tert-Butyl 2-bromoacetate (12) with triphenylphosphine afforded the corresponding phosphonium bromide that in the presence of base gave ylide 13. One equivalent of compound 13 with 0.5 equivalents of triphenyl phosphite ozonide (TPPO) yielded di-tert-butyl fumarate (14). tert-Butyl glyoxalate (15) is obtained via ozonolysis of ester 14 [52]. Imine 16 is obtained by the reaction of tert-butyl glyoxalate (15) with diphenylmethylamine. Upon base treatment of imine 16 N-(diphenylmethylene)glycine tert-butyl ester XIV is obtained that served as the starting material for the O’Donnell alkylation [53]. Similarly, reaction of glyoxalate 15 with racemic 1-phenylethylamine (prepared by reductive amination of acetophenone with ammonia) afforded the imine 17 which upon base treatment is converted into N-(methylphenylmethylene)glycine tert-butyl ester XIVa.

Ethyl glyoxalate (15a) with acetamide afforded N-acetyl 2-hydroxyglycine tert-butyl ester (18). Upon treatment with SOCl₂, the hydroxyl group is substituted by a chlorine atom to afford compound 19. An Arbuzov reaction with trimethyl phosphite yielded the ethyl ester of N-acetyl-2-dimethyl phosphonato glycine (20). 2,3-Dehydroamino acid derivatives I can be achieved via Wittig reactions of product 20 with appropriate aldehydes [54,55].

4.2. Alanine

In Scheme 10 it is indicated that treatment of CH₃I (21) under O’Donnell conditions with protected glycine gave a high yield of L-α-alanine [56].

Reaction of CH₃I (21) with tert-butyl 2-(triphenylphosphonium)acetate (13) in the presence of a base afforded the ylide tert-butyl 2-(triphenylphosphonium)propionate (22, Scheme 10). Pyruvic acid (23) is obtained by the ozonolysis of the ylide 22. Esterification of pyruvic acid in ethanol afforded ethyl pyruvate (23a). Reaction of ethyl pyruvate (23a) with 2-methylpropane-2-sulfinamide (24) afforded imine 25 that upon reaction with L-Selectride resulted in L-alanine ethyl ester. L-Alanine is obtained after saponification in 98% yield [57].

The Strecker reaction of acetaldehyde (26) with NH₄Cl (1) and KCN (3) yielded racemic 2-aminopropionitrile (27). Earlier, the conversion of 2-aminopropionitrile (27) into L-alanine has been discussed (Scheme 3). Furthermore, reaction of acetaldehyde (26) with isocyanides 28 followed by hydrolysis formed racemic α-hydroxy amides 29 [58]. Subsequent oxidation with KMnO₄ and hydrolysis afforded pyruvic acid (23) [59].

Acetyl bromide (30) is obtained by the reaction of acetic acid (11) with PBr₃ and subsequent treatment with CuCN (3a) gave pyruvic nitrile 31. Hydrolysis of nitrile 31 afforded pyruvic acid (23) in 50% yield [60,61]. Acetyl bromide (30) is reacted with isocyanides 28 to obtain imine derivative 32. Hydrolysis of the imine compound 32 afforded pyruvic acid (23). The preparation of ¹³C-labeled pyruvic acid (23) at any position and combinations of positions is possible via ¹³C-labeled isocyanides 28 [62,63]. ¹³C-Labeled isocyanides 28 are easily accessible via dehydration of formylamines. The conversion of pyruvic acid (23) into L-alanine has been discussed before (Scheme 2) [64,65,66]. The isotopic enrichment of all atoms that constitute L-alanine is accessible via the availability of highly isotope-enriched building blocks.

Scheme 10. The preparation of isotopically enriched L-alanine in all possible positions with isotopically labeled commercially available building blocks.

4.3. 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].

Scheme 11. The preparation of isotopically enriched serine.

In order to enrich the hydroxyl group with ¹⁸O (and ¹⁷O) isotope the reactions in the lower part of Scheme 11 are carried out. 4-Nitrofluorobenzene is treated with H₂¹⁸O (36) in tert-butanol with one equivalent potassium tert-butanolate to obtain [¹⁸O]-4-nitrophenolate 37. This phenolate is treated with benzyl bromide to give the [¹⁸O]-benzyl-4-nitrophenylether which upon treatment with KOH in ethylene glycol gave [¹⁸O]-benzyl alcohol 38 without any ¹⁸O loss or dilution. Treatment of the alcohol 38 with formaldehyde in the presence of HCl gave (benzyloxy)methanol that is treated with thionyl chloride to obtain [¹⁸O]-benzylchloromethyl ether 39 [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].

4.4. 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 Se₈ 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].

Scheme 12. Preparation of cysteine and selenocysteine starting from N-protected serine 40.

4.5. Threonine

Acetaldehyde (26) is converted into 1,1-dipropoxyethane by acid catalyzed reaction with 1-propanol. 1,1-Dipropoxyethane is mixed with D₂¹⁸O in the presence of HCl_(g) to afford ¹⁸O-acetaldehyde (26a) (Scheme 13).

Scheme 13. Preparation of stable isotope enriched L-threonine via the Schöllkopf method or stereo-induced oxazoline method.

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].

4.6. Asparagine and Aspartic Acid

In Scheme 14 it is indicated that N-Boc serine tert-butyl ester (40) reacted with triphenylphosphine and DMAD (dimethyl azodicarboxylate) to form the β-lactone 47. β-Lactone 47 is treated with KCN (3) to achieve 2-[(tert-butoxycarbonyl)amino]-3-cyanopropanoic acid (48) that upon acid catalyzed hydrolysis and deprotection afforded aspargine. Aspartic acid is obtained from aspargine after acid catalyzed hydrolysis of the amide group [71].

Scheme 14. Preparation of aspargine and aspartic acid via the N-Boc serine tert-butyl ester 40 and O’Donnell method, respectively.

O’Donnell coupling between the N-protected glycine tert-butyl ester XIV and methyl bromoacetate (12a) resulted in N-(diphenylmethylene)aspartic acid 1-tert-butyl-4-methyl ester (49) that upon deprotection followed by saponification afforded aspartic acid [43]. An enzymatic method has been described to convert di-tert-butyl fumarate (14, symmetrically enriched with stable isotope) into aspartic acid by treatment with ammonia in the presence of immobilized enzyme aspartase [72].

4.7. Proline

In Scheme 15 it is depicted that reaction of HCHO (2) and ethyl 2-diethylphosphonoacetate (13a) afforded ethyl acrylate (50) [73].

Scheme 15. Conversion of N-(diphenylmethylene)glutamic acid 1-tert-butyl-5-ethyl ester (51) into stable isotope labeled proline.

O’Donnell reaction of ethyl acrylate (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 NaBH₄/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].

4.8. 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 K₂CO₃. The phosphorane 53 is treated with H₂C¹³O (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 NH₃ 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 NaBH₄ 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.

Scheme 16. Synthesis of the carbon skeleton of valine from 2-methyl fumaric acid (55).

The ¹³C-isotope enriched [4-¹³C]-valine is prepared to show that these synthetic methods allow the chiral discrimination between the two diastereotopic methyl groups. Using ¹³C-formaldehyde (2a) (2S,3S)-[4-¹³C]-valine with a trace of the other enantiomeric form is obtained [47].

On the lower line in Scheme 16 it is depicted that dimethyl N-benzyl-N-(9-phenyl-9H-fluoren-9-yl)-aspartate (60) reacted with lithium hexamethyldisilazide (LHMDS) to obtain the anion of the product 60, that subsequently underwent alkylation with ¹³C-methyl iodide (21a) to afford the N-protected ¹³C-methyl aspartic acid ester 61. Reaction of the product 61 with DIBAL-H afforded the alcohol 62, further protection of the hydroxyl group with mesityl chloride followed by iodine substitution, reduction and deprotection of the α-carboxylic acid ester resulted in (2S,3S)-[4-¹³C]-valine [75].

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].

Scheme 17. Synthesis of isotopically enriched valine starting from acetaldehyde 26 or acetic acid 11.

Sharpless asymmetric epoxidation of alcohol 64 gave the epoxide 65. The epoxide is treated with (Ph)₃C-Cl (trityl chloride) to protect the primary alcohol group as trityl ether 66. The S_N2 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-CD₃]-valine [77].

Another simple approach to enrich isotopes in valine is via the preparation of intermediate molecule (3S)-3-methyl-β-butyrolactone (70). β-Butyrolactone 70 is obtained by the reaction of acetyl bromide (69) [prepared from Hell-Volhardt-Zellinsky reaction of acetic acid (11) with PBr₃] with acetaldehyde (26) in the presence of a base and a chiral catalyst. The ring-opening of β-butyrolactone 70 with ethoxide anion resulted in the formation of (3S)-3-hydrobutyrate (71) which could be dehydrated to obtain ethyl crotonate (63). Reaction of β-butyrolactone 70 with the Grignard copper complex of CD₃I resulted in optically pure (3R)-3-(trideuteromethyl)butyric acid (72) [78].

Another alternative method of preparation of an α-keto acid 73 would be a Hell-Volhardt-Zellinsky reaction of the product 72 with PBr₃. 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).

4.9. 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 AgNO₂. 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 NaBH₄ in the presence of LiCl to obtain 2-cyanoethanol (79). Acrylonitrile (80) is obtained by treatment of the alcohol 79 with Ac₂O followed by the base catalyzed elimination of acetic acid [81].

4.10. 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 CH₃I yielded the methylthioether formation to give the required protected methionine that upon deprotection of the amino group with acid afforded methionine [69].

Scheme 19. Preparation of methionine from N-(diphenylmethylene)aspartic acid 1-tert-butyl-4-methyl ester 49.

4.11. Leucine

In Scheme 20 it is depicted that the pyroglutamic acid (77, Scheme 18) is converted into the N-tert-butoxycarbonyl pyroglutamic acid. The carboxylic acid of the product 77 is esterified with ClCO₂Et/Et₃N, followed by reduction with NaBH₄ to obtain the product alcohol which is protectetd with hydroxyl function with tert-butyldimethyl silyl chloride (TBDSCl) to afford the product 84. Strong base induced deprotonation and subsequent treatment with phenyl selenide chloride, followed by treatment with ¹³CH₃I (21a) afforded methylated product that upon reaction with H₂O₂ afforded [¹³C]-methylated unsaturated lactam 85. Treatment of the unsaturated lactam 85 with D₂ in the presence of catalyst PtO₂ resulted in the product 86 with stereospecific introduction of deuterium at positions 3 and 4. After removal of the silyl protecting group of the lactam 86 followed by oxidation of the resulting primary alcohol group with RuO₂/NaIO₄ the carboxyl function is introduced in the molecule. Treatment with dimethylformamide di-tert-butyl acetal [4-¹³C]-methyl-[3,4-D₂]-pyroglutamic acid tert-butyl ester (87) is formed. Base catalyzed ring opening of the amide function and NaBH₄ reduction yielded the alcohol derivative 88 which is finally converted into (2S,3S,4S)-[5-¹³C;3,4,5′,5′,5′,-D₅]-leucine (89) [82,83].

Scheme 20. Preparation of enantiomerically pure isotopically labeled leucine starting from pyroglutamic acid 77.

[2-¹³C]-Methyl propylbromide (90) has been prepared via the Evans template method. The ratio of distereomers (2R):(2S) is 13:1. The conversion of propyl bromide 90 into the α-keto ester 93 is effected by the reaction with 2-ethoxycarbonyl-1,3-dithiane (91) followed by the oxidative hydrolysis of the resulting product 92 with NBS. Upon reductive amination (2S,4R)-[5-¹³C]-leucine mixed with 7% of (2S,4S)-[5-¹³C]-leucine is obtained [84]. The conversion of (2S)-[¹³C]-1-iodo-2-methyl propane 90a into (2S,4R)-[5-¹³C]-leucine with a mixture of diasteromers (4R):(4S) in the ratio of 8.5:1, has been effected via O’Donnell method [47].

4.12. 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 K₂CO₃. 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].

Scheme 21. Preparation of isotopically enriched isoleucine.

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(OⁱPr)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].

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].

4.13. Lysine

In Scheme 22 it is indicated that ethyl bromoacetate (12b) reacted with KCN (3) to yield ethyl cyanoacetate (103) that is treated with a base and an additional equivalent of ethyl bromoacetate (12b) to give ethyl 2-cyanobutanedioate 104. Diester 104 (in the presence of catalytic amount of NaCl, H₂O in DMF) is converted into ethyl-4-cyanopropionate (105). Selective NaBH₄ reduction of the ester function into alcohol, subsequent conversion of hydroxyl function into a tosyl group and substitution with iodide ion afforded 4-iodobutyronitrile (106) [88].

Scheme 22. The preparation of stable isotope labeled lysine.

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].

In Scheme 23 it is indicated that deuterated lysine is prepared from deuterated glutamic acid. The conversion of (2S,3S,4R)-(3,4-²H₂;1,2,3,4,5-¹³C₅;2-¹⁵N]-glutamic acid into (2S,3S,4R)-(2,3,4-²H₃;1,2,3,4-¹³C₄;4-¹⁵N]-aminobutyric acid (108) is achieved by enzymatic decarboxylation with glutamic acid decarboxylase in D₂O. Subsequent protection of the free amino group into the phthaloyl group afforded the product 109. The free carboxyl group is converted into acid chloride and reductive deuteration with tributyltin deuteride afforded deuterated aldehyde 110. The condensation of aldehyde 110 with N-acetyl phosphonato glycine ethyl ester (20, Scheme 9) in the presence of DBU afforded product 111. Asymmetric hydrogenation with (+)-1,2-bis[(2S,5S)-2,5-diethylphospholano] benzene-(cyclooctadiene)-rhodium(I)-trifluoro-methanesulfonate [(S,S)-Et-DuPhos-Rh] and deprotection of the amino function by refluxing in HCl, followed by the hydrazine treatment afforded (2S,3R,4R,5S,6R)-[3,4,5,6-²H₄;1,2,3,4,5,6-¹³C₆;2,6-¹⁵N₂]-lysine [89].

Scheme 23. Preparation of deuterated lysine from deuterated glutamic acid.

4.14. 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 K₂CO₃ to form an intermediate imidazolidine ring. With the elimination of p-toluenesulfinic acid the imidazole ring is formed to afford the product 119.

Scheme 24. The synthesis of stable isotope enriched histidine from methylammonium chloride (112).

The product 119 is treated with a mixture of potassium osmate (VI) dihydrate (K₂OsO₄·2H₂O) and sodium periodate (NaIO₄) 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 LiAlH₄ 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. ¹⁵N-Benzylamine has been prepared via the reaction of benzoyl chloride with ¹⁵NH₃, subsequent LiAlH₄ 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 NaNO₂ resulting in the ethyl ester of the protected imidazole compound. LiAlH₄ 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 POCl₃ to form the vinyl chloride chloroimidinium salt 126. Molecules analogous to 126 reacted with NH₄Cl and Na₂CO₃ under substitution of the chloride function to form the benzyl-5-carboethoxyimidazole which upon reaction with LiAlH₄ afforded the alcohol 125.

4.15. 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 H₂ in the presence of PtO₂ to afford the N-protected L-ornithine 127 [74].

Scheme 25. Synthesis of arginine from N-acetyl ornithinine tert-butyl ester 127.

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 NH₄Cl 1 with potassium thiocyanate (KSCN) followed by the S-methylation of thiourea with CH₃I.

4.16. 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 ²H, ¹³C incorporation in the benzene ring. In Scheme 26 a synthetic method is depicted that allows the isotopic enrichment in the benzene ring of phenylalanine.

Scheme 26. Preparation of stable isotope enriched phenylalanine.

Acetic acid (11) is treated with a four-fold excess of PBr₃ and one equivalent of Br₂. 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. 2,2-Diphenoxy ethanol (132) is achieved by the LiAlH₄ reduction of the ester function of the product 131. Subsequently Swern oxidation of the product 132 with oxalyl chloride in DMSO and triethyl amine afforded 2,2-diphenoxy aldehyde that upon reaction with the anion of ethyl diethylphosphonoacetate 13a afforded ethyl 4,4-diphenoxy-2-butenoate (133) via a Horner-Wadsworth-Emmons reaction.

Repeating the same sequence for the conversion of ester into alcohol followed by Swern oxidation of alcohol into aldehyde and final Wittig reaction with the ylide 13a afforded the unsaturated ester 135. Deprotection of the phenoxy groups in the product 135 afforded the aldehyde ester 136 in situ followed by subsequent Horner-Wadsworth-Emmons reaction with the anion of N-[(diphenylphosphoryl)methyl]-N-methylaniline (137) to afford ethyl 5-[methyl(phenyl)amino] penta-2,4-dienoate (138). The product 137 is easily obtainable via the the Mannich reaction between N-methylaniline and HCHO (2) in ethanol, and the resulting intermediate aminal reacted with chlorodiphenylphosphine via an Arbuzov type reaction.

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].

DIBAL-H reduction of benzonitrile (142) afforded benzaldehyde (143) that upon Horner-Wadsworth-Emmons coupling with 13a and subsequent saponification afforded cinnamic acid 144. Cinnamic acid (144) has been enzymatically converted in the presence of NH₃ into L-phenylalanine [36].

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 NaBH₄ 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].

4.17. 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 ¹³C 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 NaBH₄ 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 [1,2,3-¹³C₃]-labeled acetone (149) the ¹³C isotopes are introduced at the carbon positions 1, 2, 6 of p-nitrophenol (37). In this way tyrosine with ¹³C at positions 3′, 4′ and 5′ has been produced [94].

Scheme 27. Conversion of benzonitrile (142) into tyrosine.

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 Cu₂O/Co(NO₃)₂ afforded phenol (147). It is possible to obtain phenol (147) enriched with ¹³C 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 ¹⁷O and ¹⁸O nitrophenol (37) has been discussed in Scheme 11 [94,97].

The reactions discussed in Scheme 27 afford tyrosine with ¹⁷O or ¹⁸O in the phenolic OH group if necessary also in combinations with isotope incorporation in the aliphatic side chain. Schemes that allow to ¹⁷O or ¹⁸O 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 ¹⁷O or ¹⁸O exchange [98].

4.18. Tryptophan

In Scheme 28 it is indicated that crotyl alcohol 64 (Scheme 17) is converted into crotonaldehyde after MnO₂ oxidation and treated with propargyl amine 154 to form the imine 155.

Scheme 28. Access to isotope enrichment in indole and side chain of indole at any position and combinations of positions. The preparation of anthranilic acid enriched at any position and combinations of positions with stable isotopes.

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 ¹⁵N, ¹³C at positions 4, 5, 6, 7 and 8, and ²H at positions 4, 5, 6 and 7.

At this moment no scheme is available to enrich the isotopes in propargyl amine (154). Via an E. colimutant indole (158) can be reacted with serine to convert it into tryptophan. ¹⁵N-Anthranilic acid (163) can be incorporated into tryptophan residues of protein without ¹⁵N 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].

Scheme 29. Preparation of indole (158) from anthranilic ester 162.

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 D₂ 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 CH₃I to afford trimethylammonium iodide 168. This molecule is treated with the protected glycine under O’Donnell conditions to yield the protected tryptophan [109].

4.19. Pyrrolysine

Pyrrolysine is the 22nd genetically encoded amino acid [2]. It consists of a (4R,5R)-4-methyl-5-carboxypyrroline 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 (Scheme 22 and Scheme 23).

In Scheme 30 the preparation of the sensitive (4R,5R)-4-methylpyrroline-5-carboxylic acid is depicted. Base catalyzed 1,4-addition of the anion of N-(tert-butylphenylmethylidene)glycine tert-butyl ester XIVb and methyl crotonate (63a) in the presence of the optically active catalyst indanol bisoxazoline afforded N-(tert-butylphenylmethylene)-3-methylglutamic acid tert-butyl ester (169) with the required (2R,3R) structure in high enantiomeric excess [86].

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.

5. 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.

Acknowledgments

This paper is written with a great indebtedness to the investigators who have been involved in the generation of the synthetic schemes and the realization of the site directed stable isotope enriched genetically encoded amino acids. We dedicate this paper to the future investigators who will extend the noninvasive physical methods, who will unravel new fields in the vital field of protein research. We are thankful to Rob van der Steen (Buchem), Reinier van Nispen (Buchem), and Shrinkhala Dawadi (McGill University) for their great efforts to optimize the text of this paper.

References

Stryer, L. Biochemistry, 3rd ed; W.H. Freeman, and Company: New York, NY, USA, 1989. [Google Scholar]
Blaskovich, M.A. Handbook on Syntheses of Amino Acids. General Routes to Amino Acids; Oxford University Press: New York, NY, USA, 2010. [Google Scholar]
Hayashi, T.; Hamachi, I. Traceless affinity labeling of endogenous proteins for functional analysis in living cells. Acc. Chem. Res. 2012, 45, 1460–1469. [Google Scholar] [CrossRef]
Bertini, I.; Felli, I.C.; Gonnelli, L.; Kumar, M.V.; Pierattelli, R. ¹³C Direct-detection biomolecular NMR Spectroscopy in living cells. Angew. Chem. Int. Ed. 2011, 50, 2339–2341. [Google Scholar]
Serber, Z.; Straub, W.; Corsini, L.; Nomura, A.M.; Shimba, N.; Craik, C.S.; Ortiz de Montellano, P.; Dötsch, V. Methyl groups as probes for proteins and complexes in in-cell NMR experiments. J. Am. Chem. Soc. 2004, 126, 7119–7125. [Google Scholar]
Westler, W.M.; Stockman, B.J.; Hosoya, Y.; Miyake, Y.; Kainosho, M.; Markley, J.M. Correlation of carbon-13 and nitrogen-15 chemical shifts in selectively and uniformly labeled proteins by heteronuclear two-dimensional NMR spectroscopy. J. Am. Chem. Soc. 1988, 110, 6256–6258. [Google Scholar] [CrossRef]
Kainosho, M.; Torizawa, T.; Iwashita, Y.; Terauchi, T.; Ono, A.M.; Güntert, P. Optimal isotope labeling for NMR protein structure determinations. Nature 2006, 440, 52–57. [Google Scholar] [CrossRef]
Takeda, M.; Ikeya, T.; Güntert, P.; Kainosho, M. Automated structure determination of proteins with the SAIL-FLYA NMR method. Nat. Protoc. 2007, 2, 2896–2902. [Google Scholar]
Guo, C.; Godoy-Ruiz, R.; Tugarinov, V. High resolution measurement of methyl ¹³C(m)-¹³C and ¹H(m)-¹³C(m) residual dipolar couplings in large proteins. J. Am. Chem. Soc. 2010, 132, 13984–13987. [Google Scholar] [CrossRef]
Otten, R.; Chu, B.; Krewulak, K.D.; Vogel, H.J.; Mulder, F.A.A. Comprehensive and cost-effective NMR Spectroscopy of methyl groups in large proteins. J. Am. Chem. Soc. 2010, 132, 2952–2960. [Google Scholar] [CrossRef]
Ayala, I.; Hamelin, O.; Amero, C.; Pessey, O.; Plevin, M.J.; Gans, P.; Boisbouvier, J. An optimized isotopic labelling strategy of isoleucine-γ2 methyl groups for solution NMR studies of high molecular weight proteins. Chem. Commun. 2012, 48, 1434–1436. [Google Scholar]
Pandey, M.K.; Vivekanandan, S.; Ahuja, S.; Pichumani, K.; Im, S.C.; Waskell, L.; Ramamoorthy, A. Determination of ¹⁵N chemical shift anisotropy from a membrane-bound protein by NMR Spectroscopy. J. Phys. Chem. B 2012, 116, 7181–7189. [Google Scholar]
Elavarasi, S.B.; Kumari, A.; Dorai, K. Using the Chemical Shift Anisotropy Tensor of Carbonyl Backbone Nuclei as a Probe of Secondary Structure in Proteins. J. Phys. Chem. A 2010, 114, 5830–5837. [Google Scholar] [CrossRef]
Zhu, J.; Ye, E.; Terskikh, V.; Wu, G. Solid-state ¹⁷O-NMR spectroscopy of large protein-ligand complexes. Angew. Chem. Int. Ed. 2010, 49, 8399–8402. [Google Scholar] [CrossRef]
Yu, W.; Dawson, P.E.; Zimmermann, J.; Romesberg, F.E. Carbon-deuterium bonds as probes of protein thermal unfolding. J. Phys. Chem. B 2012, 116, 6397–6403. [Google Scholar] [CrossRef]
Torizawa, T.; Shimizu, M.; Taoka, M.; Miyano, H.; Kainosho, M. Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol. J. Biomol. NMR 2004, 30, 311–325. [Google Scholar] [CrossRef]
Kent, S.B.H. Total chemical synthesis of proteins. Chem. Soc. Rev. 2009, 38, 338–351. [Google Scholar] [CrossRef]
Metanis, N.; Keinan, E.; Dawson, P.E. Traceless ligation of cysteine peptides using selective deselenization. Angew. Chem. Int. Ed. 2010, 49, 7049–7053. [Google Scholar] [CrossRef]
Durek, T.; Alewood, P.F. Preformed selenoesters enable rapid native chemical ligation at intractable sites. Angew. Chem. Int. Ed. 2011, 50, 12042–12045. [Google Scholar] [CrossRef]
Shang, S.; Tan, Z.; Danishefsky, S.J. Application of the logic of cysteine-free native chemical ligation to the synthesis of Human Parathyroid Hormone (hPTH). Proc. Natl. Acad. Sci. USA 2011, 108, 5986–5989. [Google Scholar]
Dittmann, M.; Sauermann, J.; Seidel, R.; Zimmermann, W.; Engelhard, M. Native chemical ligation of hydrophobic peptides in organic solvents. J. Peptide Sci. 2010, 16, 558–562. [Google Scholar] [CrossRef]
Bode, J.W.; Fox, R.M.; Baucom, K.D. Chemoselective amide ligations by decarboxylative condensations of N-alkylhydroxylamines and α-ketoacids. Angew. Chem. Int. Ed. 2006, 45, 1248–1252. [Google Scholar] [CrossRef]
Winkler, F.J.; Kühnl, K.; Medina, R.; Schwarz-Kaske, R.; Schmidt, H.L. Principles and results of stable isotope mnmn7mn7ulabelling of L-α-aminoacids by combined chemical and enzymatic methods. Isot. Environ. Health Stud. 1995, 31, 161–190. [Google Scholar] [CrossRef]
Reid, C.M.; Sutherland, A. Synthesis of isotopically labeled α-amino Acids. In Amino Acids, Peptides and Proteins in Organic Chemistry: Origins and Synthesis of Amino Acid; Hughes, A.B., Ed.; Wiley-VCH Verlag GmbH and Co. KGaA: Weinheim, Germany, 2009; Volume 1, pp. 473–494. [Google Scholar]
Hiller, K.; Metallo, C.M.; Kelleher, J.K.; Stephanopoulos, G. Nontargeted elucidation of metabolic pathways using stable-isotope tracers and mass spectrometry. Anal. Chem. 2010, 82, 6621–6628. [Google Scholar]
Weber, R.J.M.; Southam, A.D.; Sommer, U.; Viant, M.R. Characterization of Isotopic abundance measurements in high resolution FT-ICR and Orbitrap Mass Spectra for improved confidence of metabolite identification. Anal. Chem. 2011, 83, 3737–3743. [Google Scholar] [CrossRef]
Svatoš, A. Single-cell metabolomics comes of age: New developments in mass spectrometry profiling and imaging. Anal. Chem. 2011, 83, 5037–5044. [Google Scholar] [CrossRef]
Kozmin, Y.P.; Manoilov, A.V.; Serebryakova, M.V.; Mirgorodskaya, O.A. A direct introduction of ¹⁸O isotopes into peptides and proteins for quantitative mass spectroscopy analysis. Russ. J. Bioorg. Chem. 2011, 37, 719–731. [Google Scholar] [CrossRef]
Poisel, H.; Schmidt, U. Dehydroaminosaüren aus amino saüren. Chem. Ber. 1975, 108, 2547–2553. [Google Scholar] [CrossRef]
Schmidt, U. α-Mercapto-α-amino acids and dehydro amino acids-syntheses, relationships and interconversions. Pure Appl. Chem. 1977, 49, 163–168. [Google Scholar] [CrossRef]
Stohlmeyer, M.M.; Tanaka, H.; Wandless, T.J. A stereospecific elimination to form dehydroamino acids: Synthesis of the phomopsin tripeptide side chain. J. Am. Chem. Soc. 1999, 121, 6100–6101. [Google Scholar] [CrossRef]
Fryzuk, M.D.; Bosnich, B. Asymmetric synthesis. Production of optically active amino acids by catalytic hydrogenation. J. Am. Chem. Soc. 1977, 99, 6262–6267. [Google Scholar] [CrossRef]
Duthaler, R.O. Recent developments in the stereoselective synthesis of α-amino acids. Tetrahedron 1994, 50, 1539–1650. [Google Scholar] [CrossRef]
Corey, E.J.; Kürti, L. Enantioselective Chemical Synthesis, 1st ed; Direct Book Publishing, LLC: Dallas, TX, USA, 2010. [Google Scholar]
Winkler, F.J.; Kühnl, K.; Medina, R.; Schwarz Kaske, R.; Schmidt, H.L. Principles and results of stable isotope labelling of L-α-Aminoacids by combined chemical and enzymatic methods. Isot. Environ. Health Stud. 1995, 31, 161–190. [Google Scholar] [CrossRef]
Sonke, T.; Kaptein, B.; Schoemaker, H.E. Use of enzymes in the synthesis of amino acids. In Amino Acids, Peptides and Proteins in Organic Chemistry: Origins and Synthesis of Amino Acid; Hughes, A.B., Ed.; Wiley-VCH Verlag GmbH and Co. KGaA: Weinheim, Germany, 2009; pp. 79–117. [Google Scholar]
Chen, Y.; Goldberg, S.L.; Hanson, R.L.; Parker, W.L.; Gill, I.; Tully, T.P.; Montana, M.A.; Goswami, A.; Patel, R.N. Enzymatic preparation of an (S)-amino acid from a racemic amino acid. Org. Process Res. Dev. 2011, 15, 241–248. [Google Scholar] [CrossRef]
Kürti, L.; Czakó, B. Strategic Applications of Named Reactions in Organic Synthesis; Elsevier Academic Press: Amsterdam, The Netherlands, 2005. [Google Scholar]
Vachal, P.; Jacobsen, E.N. Structure-based analysis and optimization of a highly enantioselective catalyst for the Strecker Reaction. J. Am. Chem. Soc. 2002, 124, 10012–10014. [Google Scholar] [CrossRef]
Pellissier, H. Recent developments in dynamic kinetic resolution. Tetrahedron 2011, 67, 3769–3802. [Google Scholar] [CrossRef]
Liu, P.; Yang, X.; Birman, V.B.; Houk, K.N. Origin of enantioselectivity in benzotetramisole-catalyzed dynamic kinetic resolution of azlactones. Org. Lett. 2012, 14, 3288–3291. [Google Scholar] [CrossRef]
O’Donnell, M.J.; Polt, R.L.J. A mild and efficient route to Schiff base derivatives of amino acids. J. Org. Chem. 1982, 47, 2663–2666. [Google Scholar] [CrossRef]
Park, H.G.; Jeong, B.S. Cinchona-catalyzed nucleophilic alpha-substitution of carbonyl derivatives. In Cinchona Alkaloids in Synthesis and Catalysis Ligands, Immobilization and Organocatalysi; Choong Eui, S., Ed.; Wiley-VCH Verlag GmbH and Co. KGaA: Weinheim, Germany, 2009; Chapter 6. [Google Scholar]
Yeboah, E.M.O.; Yeboah, S.O.; Singh, G.S. Recent applications of cinchona alkaloids and their derivatives as catalysts in metal-free asymmetric synthesis. Tetrahedron 2011, 67, 1725–1762. [Google Scholar] [CrossRef]
Marcelli, T.; Hiemstra, H. Cinchona alkaloids in asymmetric organocatalysis. Synthesis 2010, 1229–1279. [Google Scholar] [CrossRef]
Dawadi, P.B.S.; Schulten, E.A.M.; Lugtenburg, J. Synthesis of [3-¹³C]-, [4-¹³C]- and [11-¹³C]-porphobilinogen. J. Labelled Compd. Radiopharm. 2009, 52, 341–349. [Google Scholar] [CrossRef]
Siebum, A.H.G.; Woo, W.S.; Lugtenburg, J. Preparation and characterization of [5-¹³C]-(2S, 4R)-leucine and [4-¹³C]-(2S, 3S)-valine- Establishing synthetic schemes to prepare any site-directed isotopomer of L-leucine, L-isoleucine and L-valine. Eur. J. Org. Chem. 2003, 4664–4678. [Google Scholar]
Patching, S.G. Efficient syntheses of ¹³C- and ¹⁴C-labelled 5-benzyl and 5-indolylmethyl L-hydantoins. J. Labelled Compd. Radiopharm. 2011, 54, 110–114. [Google Scholar]
Raap, J.; Nieuwenhuis, S.; Creemers, A.; Hexspoor, S.; Kragl, U.; Lugtenburg, J. Synthesis of isotopically labelled L-phenylalanine and L-tyrosine. Eur. J. Org. Chem. 1999, 2609–2621. [Google Scholar]
Ingersoll, A.W.; Babcock, S.H. Hippuric acid. Org. Synth. 1943, 12, 40. [Google Scholar]
Nieuwenhuis, S.A.M.; Mul, C.; Van Belle, N.J.; Lugtenburg, J.; Raap, J. Synthesis of stereospecifically β-deuterated L-tyrosine. In Photosynthesis, From Light to Biosphere; Mathis, P., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995; Volume II, pp. 313–316. [Google Scholar]
Heinen, W.; Rosenmoller, C.H.; Wenzel, C.B.; De Groot, H.J.M.; Lugtenburg, J.; van Duin, M. ¹³C-NMR study of the grafting of maleic anhydride onto polyethane, polypropene and ethene-propene copolymers. Macromolecules 1996, 29, 115–1157. [Google Scholar]
Hyett, D.J.; Mink, D.; Brvntermann, G.B.; Zeegens, H.M.G. WO 2004/078702 A2, 2004.
Schmidt, U.; Lieberknecht, A.; Wild, J. Amino acids and peptides; XLIII1. Dehydroamino acids; XVIII2. Synthesis of dehydroamino acids and amino Acids from N-acyl-2-(dialkyloxyphosphinyl)-glycin esters; II. Synthesis 1984, 53-61. [Google Scholar]
Schmidt, U.; Griesser, H.; Leitenberger, V.; Lieberknecht, A.; Mangold, R.; Meyer, R.; Riedl, B. Diastereoselective formation of (Z)-didehydroamino acid esters. Synthesis 1992, 487–490. [Google Scholar]
O’Donnell, M.J. The enantioselective synthesis of α-amino acids by phase-transfer catalysis with achiral Schiff base esters. Acc. Chem. Res. 2004, 37, 506–517. [Google Scholar] [CrossRef]
Reddy, L.R.; Gupta, A.P.; Liu, Y. Asymmetric synthesis of α-amino acids by reduction of N-tert-butanesulfinyl ketimine esters. J. Org. Chem. 2011, 76, 3409–3415. [Google Scholar] [CrossRef]
Takahiro Soeta, T.; Kojima, Y.; Yutaka Ukaji, Y.; Inomata, K. Borinic acid catalyzed α-addition to isocyanide with aldehyde and water. Tetrahedron Lett. 2011, 52, 2557–2559. [Google Scholar] [CrossRef]
Corson, B.B.; Dodge, R.A.; Harris, S.A.; Hazen, R.K. Ethyl benzoylformate. Org. Synth. 1941, 1, 241–243. [Google Scholar]
Roth, P.; Hädener, A.; Tamm, C. Further studies on the biosynthesis of tabtoxin (wildfire toxin): Incorporation of [2,3-¹³C₂]-pyruvate into the β-lactam moiety. Helv. Chim. Acta 1990, 73, 476–482. [Google Scholar] [CrossRef]
Larsen, B.D.; Eggert, H.; Harrit, N.; Holm, A. Photolysis of 1,2,3-thiadiazole. Formation of thiirene by secondary photolysis of thioketene. Acta Chem. Scand. 1992, 46, 482–486. [Google Scholar] [CrossRef]
Lee, C.H.; Westling, M.; Livinghouse, T.; Williams, A.C. Acylnitrilium Ion Initiated Heteroannulations in Alkaloid Synthesis. An efficient, stereocontrolled, total synthesis of the Orchidaceae alkaloid (±)-dendrobine. J. Am. Chem. Soc. 1992, 114, 4089–4095. [Google Scholar] [CrossRef]
Ugi, I.; Werner, B.; Dömling, A. The chemistry of isocyanides, their multi-component reactions and their libraries. Molecules 2003, 8, 53–66. [Google Scholar]
Xiao, X.; Xie, Y.; Su, C.; Liu, M.; Shi, Y. Organocatalytic asymmetric biomimetic transamination: From α-keto esters to optically active α-amino acid derivatives. J. Am. Chem. Soc. 2011, 133, 12914–12917. [Google Scholar] [CrossRef]
Chen, Y.; Goldberg, S.L.; Hanson, R.L.; Parker, W.L.; Gill, I.; Tully, T.P.; Montana, M.A.; Goswami, A.; Patel, R.N. Enzymatic preparation of an (S)-amino acid from a racemic amino acid. Org. Process. Res. Dev. 2011, 15, 241–248. [Google Scholar] [CrossRef]
Kelly, N.M.; O’Neill, B.C.; Probert, J.; Reid, G.; Stephen, R.; Wang, T.; Willis, C.L.; Winton, P. Chemo-enzymatic syntheses of isotopically labelled L-amino acids. Tetrahedron Lett. 1994, 35, 6533–6536. [Google Scholar]
Terauchi, T.; Kobayashi, K.; Okuma, K.; Oba, M.; Nishiyama, K.; Kainosho, M. Stereoselective synthesis of triply isotope-labeled Ser, Cys, and Ala: Amino Acids for stereoarray isotope labeling technology. Org. Lett. 2008, 10, 2785–2787. [Google Scholar]
Karstens, W.F.J.; Berger, H.J.F.F.; Van Haren, E.R.; Lugtenburg, J.; Raap, J. Enantioselective synthesis of isotopically labelled L-α-amino acids preparation of ¹³C-, ¹⁸O-and ²H-labelled L-serines and L-threonines. J. Label. Compd. Radiopharm. 1995, 36, 1077–1096. [Google Scholar] [CrossRef]
Siebum, A.H.G.; Woo, W.S.; Raap, J.; Lugtenburg, J. Access to any site-directed isotopomer of methionine, selenomethionine, cysteine, and selenocysteine- Use of simple, efficient modular synthetic reaction schemes for isotope ncorporation. Eur. J. Org. Chem. 2004, 2905–2913. [Google Scholar]
Kim, H.Y.; Oh, K. Highly diastereo- and enantioselective aldol reaction of methyl α-isocyanoacetate: A cooperative catalysis approach. Org. Lett. 2011, 13, 1306–1309. [Google Scholar] [CrossRef]
Lodwig, S.N.; Unkefer, C.J. Stereoselective synthesis of stable isotope labeled L-α-amino acids: Synthesis of L-[4-¹³C] and L-[3,4-¹³C₂] Aspartic Acid. J. Label. Compd. Radiopharm. 1992, 31, 95–102. [Google Scholar] [CrossRef]
Lee, K.M.; Ramalingam, K.; Son, J.K.; Woodard, R.W. A highly efficient and large-scale synthesis of (2S,3S)-[2,3-²H₂]- and (2S,3R)-[3-²H]-aspartic acids via an immobilized aspartase-containing microbial cell system. J. Org. Chem. 1989, 54, 3195–3198. [Google Scholar] [CrossRef]
Cappon, J.J.; Baart, J.; van der Walk, G.A.M.; Raap, J.; Lugtenburg, J. Chemo-enzymatic synthesis of specifically stable-isotope labeled L-glutamic acid and 2-oxoglutaric acid. Recl. Trav. Chim. Pays-Bas. 1991, 110, 158–166. [Google Scholar]
Siebum, A.H.G.; Tsang, R.K.F.; Steen, R.; Raap, J.; Lugtenburg, J. Synthesis of (Ïµ-¹³C-,Ïµ-¹⁵N)-Enriched L-Lysine- Establishing schemes for the preparation of all possible ¹³C and ¹⁵N isotopomers of L-lysine, L-ornithine, and L-proline. Eur. J. Org. Chem. 2004, 4391–4396. [Google Scholar]
Humphrey, J.M.; Hart, J.A.; Chamberlin, A.R. Efficient syntheses of diastereoselectively labeled (3S)-[4-¹³C]-L-valine, and regioselectively labeled [3-¹³CH₃]-L-isoleucine hydrochlorides. Bioorg. Med. Chem. Lett. 1995, 5, 1315–1320. [Google Scholar] [CrossRef]
Van den Berg, E.M.M.; Van Liemt, W.B.S.; Heemskerk, B.; Lugtenburg, J. Synthesis of indoles and L-tryptophans specifically ²H- or ¹³C-labelled in the six-membered ring. Recl. Trav. Chim. Pays-Bas. 1989, 108, 304–313. [Google Scholar]
Shattuck, J.C.; Meinwald, J. The preparation of L-(2S, 3S)-4,4,4-[²H₃]-valine. Tetrahedron Lett. 1997, 38, 8461–8464. [Google Scholar] [CrossRef]
Nelson, S.G.; Wan, Z.; Stan, M.A. S_N2 Ring opening of β-lactones:An Alternative to catalytic asymmetric conjugate additions. J. Org. Chem. 2002, 67, 4680–4683. [Google Scholar] [CrossRef]
Cappon, J.J.; Van der Walle, G.A.M.; Verdegem, P.J.E.; Raap, J.; Lugtenburg, J. Synthesis of specifically stable-isotope-labeled L-proline via L-glutamic acid. Recl. Trav. Chim. Pays-Bas. 1992, 111, 517–523. [Google Scholar]
Oba, M.; Miyakawa, A.; Nishiyama, K.; Terauchi, T.; Kainosho, M. Stereodivergent synthesis of (2S,3S,4R,5R)- and (2S,3S,4R,5S)-[3,4,5-D₃]-proline depending on the substituent of the γ-lactam ring. J. Org. Chem. 1999, 64, 9275–9278. [Google Scholar] [CrossRef]
Van den Berg, E.M.M.; Richardson, E.E.; Lugtenburg, J.; Jenneskens, L.W. Convenient syntheses for selectively isotopically labelled acrylonitriles. Synth. Commun. 1987, 17, 1189–1198. [Google Scholar] [CrossRef]
Oba, M.; Kobayashi, M.; Oikawa, F.; Nishiyama, K. Synthesis of ¹³C/D doubly labeled L-Leucines: Probes for conformational analysis of the leucine side-chain. J. Org. Chem. 2001, 66, 5919–5922. [Google Scholar] [CrossRef]
Ohfune, Y.; Tomita, M. Total synthesis of (−)-domoic acid. A revision of the original structure. J. Am. Chem. Soc. 1982, 104, 3511–3513. [Google Scholar] [CrossRef]
Fletcher, M.D.; Harding, J.R.; Hughes, R.A.; Kelly, N.M.; Schmalz, H.; Sutherland, A.; Willis, C.L. Three approaches to the synthesis of L-leucine selectively labelled with carbon-13 or deuterium in either diastereotopic methyl group. J. Chem. Soc. Perkin Trans. 1 2000, 43–51. [Google Scholar]
Kobayashi, S.; Tsubogo, T.; Saito, S.; Yamashita, Y. Calcium-catalyzed diastereo- and enantioselective 1,4-addition of glycine derivatives to α,β-unsaturated esters. Org. Lett. 2008, 10, 807–809. [Google Scholar] [CrossRef]
Wong, M.L.; Guzei, I.A.; Kiessling, L.L. An asymmetric synthesis of L-pyrrolysine. Org. Lett. 2012, 14, 1378–1381. [Google Scholar] [CrossRef]
De Lamo Marin, S.; Catala, C.; Kumar, S.R.; Valleix, A.; Wagner, A.; Mioskowski, C. A practical and efficient total synthesis of potent insulinotropic (2S,3R,4S)-4-hydroxyisoleucine through a chiral N-protected γ-keto-α-aminoester. Eur. J. Org. Chem. 2010, 3985–3989. [Google Scholar]
Raap, J.; Wolthuis, W.N.E.; Hehenkamp, J.J.J.; Lugtenburg, J. Enantioselective syntheses of isotopically labeled α-amino acids. Preparation of specifically¹³C-labelled L-lysines. Amino Acids 1995, 8, 171–186. [Google Scholar]
Terauchi, T.; Kamikawai, T.; Vinogradov, M.G.; Starodubtseva, E.V.; Takeda, M.; Kainosho, M. Synthesis of stereoarray isotope labeled (SAIL) lysine via the “Head-to-Tail” conversion of SAIL glutamic Acid. Org. Lett. 2011, 13, 161–163. [Google Scholar]
Cappon, J.J.; Witters, K.D.; Baart, J.; Verdegem, P.J.E.; Hoek, A.C.; Luiten, R.J.H.; Raap, J.; Lugtenburg, J. Synthesis of L-histidine specifically labelled with stable isotopes. Recl. Trav. Chim. Pays-Bas. 1994, 113, 318–328. [Google Scholar]
Talab, S.; Lugtenburg, J. Unpublished results, Leiden University: The Netherlands, 2009.
Soede-Huijbregts, C.; Van Laren, M.; Hulsbergen, F.B.; Raap, J.; Lugtenburg, J. Improved specific synthesis of [1′-¹⁵N]- and [3′-¹⁵N]L-histidine. J. Label. Compd. Radiopharm. 2001, 44, 831–841. [Google Scholar] [CrossRef]
O’Donovan, D.H.; Roza, I. A concise synthesis of asymmetrical N,N′-disubstituted guanidines. Tetrahedron Lett. 2011, 52, 4117–4119. [Google Scholar] [CrossRef]
Walker, T.E.; Matheny, C.; Storm, C.B.; Hayden, H. An efficient chemomicrobiological synthesis of stable isotope-labeled L-tyrosine and L-phenylalanine. J. Org. Chem. 1986, 51, 1175–1179. [Google Scholar] [CrossRef]
Truce, W. The Gatterman synthesis of aldehydes. In Organic Reactions; Wiley: New York, NY, USA, 1957; Chapter 2. [Google Scholar]
Winkel, C.; Aarts, M.W.M.M.; Van der Heide, F.R.; Buitenhuis, E.G.; Lugtenburg, J. Synthesis and NMR spectroscopy of stable isotope-labelled phenols and L-tyrosines. Recl. Trav. Chim. Pays-Bas. 1989, 108, 139–146. [Google Scholar]
Nishiyama, K.; Oba, M.; Ueno, R.; Morita, A.; Nakamura, Y.; Kainosho, M. Synthesis of phenylalanines regiospecifically labelled with deuterium in the aromatic ring. J. Label. Compd. Radiopharm. 1994, 34, 831–837. [Google Scholar] [CrossRef]
Nieuwenhuis, S.A.M.; Hulsebosch, R.J.; Raap, J.; Gast, P.; Lugtenburg, J.; Hoff, A.J. Structure of the Y_D tyrosine radical in photosystem II. Determination of the orientation of the phenoxyl ring by enantioselective deuteration of the methylene group. J. Am. Chem. Soc. 1998, 120, 829–830. [Google Scholar]
Van den Berg, E.M.M.; Van Liemt, W.B.S.; Heemskerk, B.; Lugtenburg, J. Synthesis of indoles and L-tryptophans specifically ²H- or ¹³C-labelled in the six-membered ring. Recl. Trav. Chim. Pays-Bas. 1989, 108, 304–313. [Google Scholar]
Van den Berg, E.M.M.; Baldew, A.U.; De Goede, A.T.J.W.; Raap, J.; Lugtenburg, J. Synthesis of three isotopomers of L-tryptophan via a combination of organic synthesis and biotechnology. Recl. Trav. Chim. Pays-Bas. 1988, 107, 73–81. [Google Scholar]
Raap, J.; Winkel, C.; De Wit, A.H.M.; Van Houten, A.H.H.; Hoff, A.J.; Lugtenburg, J. Mass spectrometric determination of isotopically labeled tyrosines and tryptophans in photosynthetic reaction centers of Rhodobacter sphaeroides R-26. Anal. Biochem. 1990, 19, 19–15. [Google Scholar]
Van Liemt, W.B.S.; Van Henegouwen, W.G.B.; Van Rijn, A.; Lugtenburg, J. Synthesis and spectroscopic characterisation of ¹³C-labelled anthranilic acid. Recl. Trav. Chim. Pays-Bas. 1996, 115, 431–437. [Google Scholar]
Vorländer, D.; Von Schilling, R. Darstellung von indoxylsäureestern. Justus Liebigs Ann. Chem. 1898, 301, 349–352. [Google Scholar] [CrossRef]
Vorländer, D.; Apelt, O. Darstellung von indol aus indoxyl. Ber. Dtsch. Chem. Ges. 1904, 37, 1134–1135. [Google Scholar] [CrossRef]
Jessing, M.; Baran, P.S. Oxidative coupling of indoles with 3-oxindoles. Heterocycles 2010, 82, 1739–1745. [Google Scholar] [CrossRef]
Noriya, T.; Yoneda, N. Synthesis of methyl 2-isocyano-3-[3(¹H)-indolyl] acrylate and related compounds from 3-(aminomethylene)-3H-indoles. Chem. Pharm. Bull. 1982, 30, 158–166. [Google Scholar] [CrossRef]
Kaur, H.; Heapy, A.M.; Brimble, M.A. The synthesis of dehydrotryptophan and dehydrotryptophan-containing peptides. Org. Biomol. Chem. 2011, 9, 5897–5907. [Google Scholar] [CrossRef]
Vogel, A.I. Vogel's Textbook of Practical Organic Chemistry, 5th ed; Longman: London, UK, 1989. [Google Scholar]
Kainosho, M.; Tekauchi, T. Aromatic amino acids labeled with stable isotope methods for incorporating the same into target protein and method for analyzing protein structure using NMR. JSTA Patent Application 20100056799.

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Dawadi, P.B.S.; Lugtenburg, J. Access to Any Site Directed Stable Isotope (²H, ¹³C, ¹⁵N, ¹⁷O and ¹⁸O) in Genetically Encoded Amino Acids. Molecules 2013, 18, 482-519. https://doi.org/10.3390/molecules18010482

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Dawadi PBS, Lugtenburg J. Access to Any Site Directed Stable Isotope (²H, ¹³C, ¹⁵N, ¹⁷O and ¹⁸O) in Genetically Encoded Amino Acids. Molecules. 2013; 18(1):482-519. https://doi.org/10.3390/molecules18010482

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Dawadi, Prativa B. S., and Johan Lugtenburg. 2013. "Access to Any Site Directed Stable Isotope (²H, ¹³C, ¹⁵N, ¹⁷O and ¹⁸O) in Genetically Encoded Amino Acids" Molecules 18, no. 1: 482-519. https://doi.org/10.3390/molecules18010482

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