Amino Acids

DOI 10.1007/s00726-010-0808-8

ORIGINAL ARTICLE

Synthesis of 4-thia-[6-13C]lysine from [2-13C]glycine:

access to site-directed isotopomers of 2-aminoethanol,
2-bromoethylamine and 4-thialysine
Amarendra Nath Maity • Ajam C. Shaikh •
Sankareswaran Srimurugan • Chi-Ju Wu •
Chinpiao Chen • Shyue-Chu Ke

Received: 18 August 2010 / Accepted: 2 November 2010

Ó Springer-Verlag 2010

Abstract 4-Thialysine (S-(2-aminoethyl)-L-cysteine) is an Introduction

analog of lysine. It has been used as an alternative substrate
for lysine in enzymatic reactions. Site-directed isotopomers Isotopomers have been extensively used in the study of
are often needed for elucidation of mechanism of reac- mechanism and structure determination. Labeling with
tions. 4-Thialysine can be synthesized by reacting cysteine radioisotopes finds extensive application for tracking mol-
with 2-bromoethylamine, an important reagent in chemical- ecule or molecular fragment in various chemical and bio-
modification rescue (CMR) of proteins. Here, we present the chemical processes. It has been widely used in enzymology
synthesis of 4-thia-[6-13C]lysine, one of the isotopomers of and molecular biology. Nevertheless, labeling with stable
4-thialysine, from commercially available starting material isotopes is also of great importance (Lloyd-Jones and
[2-13C]glycine via formation of five intermediates including Munoz 2007). Labeling proteins with stable isotopes is a
2-amino[2-13C]ethanol and 2-bromo[1-13C]ethylamine. The common practice to study protein structure, dynamics and
compounds were characterized using various spectroscopic quantification using various spectroscopic techniques such
techniques. Moreover, we discuss that our strategy would as mass (Tao and Aebersold 2003), vibrational (Decatur
provide access to site-directed isotopomers of 2-aminoeth- 2006; Ludlam et al. 1995) and magnetic resonance spec-
anol, 2-bromoethylamine and 4-thialysine. Biological troscopy (Macnaughtan et al. 2005). Isotopic substitution,
activity of 4-thia-[6-13C]lysine was tested in the enzymatic which usually results in substitution of nuclei with nuclei
reaction of lysine 5,6-aminomutase. having different spin multiplicity, has been extensively used
to simplify the spectra obtained from magnetic resonance
Keywords Amino acids  Carbon labels  spectroscopy to get more information. Recently, stereo-
Site-directed isotopomers  4-Thialysine  array isotope labeling approach, together with advanced
2-Bromoethylamine  2-Aminoethanol nuclear magnetic resonance (NMR), was employed for
determination of protein structure and hydrogen exchange
rates of hydroxyl groups of tyrosine residues in proteins
(Kainosho et al. 2006; Takeda et al. 2009). Electron para-
magnetic resonance (EPR) combined with specific isotope
Electronic supplementary material The online version of this labeling has been instrumental to study the mechanism of
article (doi:10.1007/s00726-010-0808-8) contains supplementary reactions involving radicals (Frey et al. 2006). For example,
material, which is available to authorized users. this approach has successfully been employed to elucidate
the mechanism of the reaction involving lysine aminomu-
A. N. Maity  C.-J. Wu  S.-C. Ke (&)
Department of Physics, National Dong Hwa University, tases (Behshad et al. 2006; Frey et al. 2006; Lees et al. 2006;
Hualien 974-01, Taiwan Wu et al. 1995). The synthesis of site-specific isotopomers
e-mail: ke@mail.ndhu.edu.tw of lysine was reported earlier (Raap et al. 1995; Siebum
et al. 2004a). 4-Thialysine (S-(2-aminoethyl)-L-cysteine) in
A. C. Shaikh  S. Srimurugan  C. Chen
Department of Chemistry, National Dong Hwa University, which 4-methylene group of lysine is replaced by a sulfur
Hualien 974-01, Taiwan atom is a very effective analog of lysine and has been

123
 A. N. Maity et al.

widely used to mimic the chemistry involving lysine. For 2-bromoethylamine have been utilized in the study of
instance, it was used in the study of feedback inhibition of chemical rescue by electrospray ionization Fourier transform
a-aminoadipate reductase, a key enzyme for biosynthesis of mass spectrometry (Hopkins et al. 2002) and NMR (Hopkins
lysine in yeast (Suvarna et al. 1998). In mammalian cells et al. 2005). 2-Bromoethylamine can be synthesized (Cortese
and human parasites of the genus Leishmania, 4-thialysine 1943) from 2-aminoethanol which is a synthon for synthe-
is the substrate for the enzyme thialysine Ne-acetyltrans- sis of many important molecules including natural products.
ferase (Coleman et al. 2004; Luersen 2005). 4-Thialysine So, there is a need to develop a strategy to synthesize
undergoes metabolism to form cyclic ketimine derivatives all possible isotopomers of 4-thialysine, ethanolamine and
found in mammalian tissues including brain and as urinary 2-bromoethylamine. In this report, we describe the synthe-
metabolites (Coleman et al. 2004). These metabolites can sis of 4-thia-[6-13C]lysine starting from [2-13C]glycine
undergo further reactions to form compounds which have through intermediate formation of 2-amino[2-13C]ethanol
been postulated to serve neurochemical roles or to have and 2-bromo-[1-13C]ethylamine. Additionally, we discuss
antioxidant properties. Interestingly, possible application of the strategy to access site-directed isotopomers of 4-thialy-
4-thialysine as potential chemotherapeutic agent for cancer sine, 2-aminoethanol and 2-bromoethylamine.
has been proposed on the basis of its observed cytotoxicity
toward human acute leukemia Jurkat T cells (Jun et al.
2003). Recently, 4-thialysine has been used (Maity et al. Materials and methods
2009; Tang et al. 2009) to elucidate the mechanism of lysine
5,6-aminomutase, an adenosylcobalamin (AdoCbl) and Materials
pyridoxal-50 -phosphate (PLP) dependent enzyme which
catalyzes reversible 1,2-shift of the e-amino group of All commercially available compounds were purchased
DL-lysine or L-b-lysine into 2,5- or 3,5-diaminohexanoic from Sigma–Aldrich, Across or Fluka. All chemicals were
acid. The reaction is proposed to follow a radical mecha- used without further purification. [2-13C]glycine ([98%
nism involving substrate–PLP radical (S), a cyclic aziridi- isotope-enriched) was purchased from Cambridge Isotope
nylcarbinyl–PLP radical (I) and product–PLP radical (P) Laboratories. HPLC-grade solvents were used.
(Scheme 1).
Uniform and partial labeling of 4-thialysine could not Methods
unambiguously identify the radical intermediate. The site-
1
directed labeling at carbon-5 and carbon-6 of 4-thialysine H and 13C NMR spectra were recorded using a Bruker
would help to identify the radical intermediate. Although Spectrospin NMR spectrometer. NMR chemical shifts were
some of the isotopomers have previously been synthesized, referenced to TMS (CDCl3) and TSP (D2O). Infrared spectra
the report of the synthesis of site-directed isotopomers at d were acquired by Perkin–Elmer spectrophotometer. Only
and e is absent. Several labeled 4-thialysine were prepared by the major bands in IR spectra are reported. Optical rotations
reacting labeled cysteine with 2-bromoethylamine following were measured using a Jasco P-1010 polarimeter. Elemental
the procedure which was first reported by Cavallini et al. analyses were performed on a Flash EA 1112 elemental
(1955). However, detailed characterizations of the com- analyzer. Mass spectra were recorded on Bruker Daltonic
pounds were not reported. 2-Bromoethylamine is an impor- autoflex MALDI-TOF mass spectrometer. The values of
tant reagent in chemical-modification rescue (CMR) of m/z were calculated using ChemDraw Ultra software.
proteins (Gloss and Kirsch 1995a, b; Hopkins et al. 2002). In
this process active site mutation of lysine to cysteine, fol- [2-13C]Glycine ethyl ester hydrochloride (1)
lowed by aminoethylation of cysteine residue to 4-thialysine
is correlated with loss and restoration of activity. CMR A suspension of [2-13C]glycine (2.00 g, 26.3 mmol) in
has been used to investigate the role of active site lysine ethanol (160 mL) was taken to ice bath. To this suspension
residues in enzymatic catalysis. Recently, isotopomers of was added SOCl2 (13.5 mL, 185.8 mmol) over a period of

Scheme 1 Radical
intermediates in the reaction of
D-lysine and 5,6-LAM

123
Synthesis of 4-thialysine from glycine

30 min. The reaction mixture was warmed to room tem- IR (KBr, cm-1): 3,352, 2,978, 2,933, 2,877, 1,690, 1,525,
perature and stirred for a further 12 h. The solvent was 1,456, 1,393, 1,367, 1,277, 1,252, 1,172, 1,140 (sh) 1,063,
evaporated under reduced pressure to produce the product 864, 781, 756, 720–580 (br). 1H NMR (400 MHz, CDCl3):
1 as white needle-like crystals (yield = 3.65 g, 99%). d 1.45 (s, 9H), 2.40 (bs, 1H), 3.28 (dt, 2H, 1JCH =
Found: C, 32.08; H, 6.89; N, 9.99. Anal. Calcd. for 137.1 Hz, 3JHH = 4.6 Hz), 3.70 (t, 2H, 3JHH = 5.3 Hz),
C133 CH10ClNO2: C, 34.88; H, 7.17; N, 9.96. IR (KBr, 5.01 (bs, 1H). 13C NMR (100 MHz, CDCl3) d 28.38, 43.21,
cm-1): 2,800–3,100 (br), 1,745, 1,576, 1,549, 1,501, 1,457, 62.65 (d, 1JCC = 42.0 Hz), 79.74, 156.89.
1,423, 1,404, 1,384, 1,250, 1,040, 900, 854. 1H NMR
(400 MHz, D2O): d 1.14 (t, 3H, 2JHH = 7.2 Hz), 3.75 (d, 2-Amino[2-13C]ethanol hydrochloride (4)
2H, 1JCH = 145.8 Hz), 4.14 (q, 2H, 2JHH = 7.2 Hz). 13C
NMR (100 MHz, D2O) d 13.13, 40.13, 63.24, 168.12 (d, HCl gas was bubbled through a solution of 3 (1.38 g,
1
JCC = 62.0 Hz). MALDI-TOF MS: m/z = 104.779 8.5 mmol) in CH2Cl2 for 30 min. The completion of
(calcd. for [M–Cl]? 105.075). reaction was indicated by the formation of large amount of
solid formation. The solid was filtered under vacuum and
Ethyl 2-(tert-butoxycarbonylamino)[2-13C]acetate (2) washed with CH2Cl2 (10 mL) to produce the product 4 as
white solid. (yield = 0.82 g, 98%). Found: C, 22.38; H,
Triethylamine (4.5 mL, 32.3 mmol) was added slowly to a 8.42; N, 12.90. Anal. Calcd for C13CH8ClNO: C, 25.39; H,
solution of 1 (1.80 g, 12.8 mmol) in methanol (11 mL) at 8.18; N, 14.21. IR (KBr, cm-1): 3,848, 3,741, 3,412, 3,070,
4°C. To this mixture di-tert-butyl dicarbonate (3.0 mL, 1,624, 1,502, 1,316, 1,262, 1,105, 1,056, 999, 863, 814,
13.1 mmol) was added slowly. The resultant mixture was 765, 708, 652, 615, 593, 554, 499. 1H NMR (400 MHz,
stirred under nitrogen for 24 h. The solvent was evaporated D2O): d 2.99 (dt, 2H, 1JCH = 144.1 Hz, 3JHH = 5.4 Hz),
in vacuo. Water (10 mL) and EtOAc (10 mL) were added 3.67 (t, 2H, 3JHH = 5.2 Hz). 13C NMR (100 MHz, D2O)
to the above residue, acidified with 1 N HCl solution (pH d 41.20, 57.51 (d, 1JCC = 38.0 Hz). MALDI-TOF MS:
5–6). The organic layer was separated and the aqueous m/z = 62.747 (calcd. for [M–Cl]? 63.064).
layer was extracted with EtOAc (10 mL). The combined
EtOAc extract was washed with 1 N HCl, 5% NaHCO3 and 2-Bromo[1-13C]ethylamine hydrobromide (5)
brine, respectively. The organic layer was then dried over
anhydrous MgSO4 and concentrated to afford the product 2 Forty-eight percent hydrobromic acid (6.6 mL) was added
as colorless oil. (yield = 2.52 g, 96%). Found: C, 52.23; H, to 4 (788 mg, 8.0 mmol) in a 10-mL round bottom flask
8.56; N, 7.41. Anal. Calcd. for C13 8 CH17NO4: C, 53.42; H, attached to a distillation assembly and heated in an oil bath
8.39; N, 6.86. IR (KBr, cm-1): 3,375, 2,980, 2,935, 1,753, until 1.5 mL distillate was collected. Then the temperature
1,720, 1,702 (sh), 1,519, 1,455, 1,392, 1,368, 1,350, 1,283, was lowered and maintained in such a way so that it ceased
1,251, 1,203, 1,169, 1,052, 1,029, 945, 863, 784, 764, to distill and merely refluxed. After refluxing for 1 h,
530–620 (br). 1H NMR (400 MHz, CDCl3): d 1.26 (t, 3H, 1.4 mL more was distilled and the solution was heated
3
JHH = 7.1 Hz), 1.43 (s, 9H), 3.88 (dt, 2H, 1JCH = under reflux for 1 h. This procedure was repeated for 1,
145.7 Hz, 3JHH = 5.5 Hz), 4.19 (q, 2H, 3JHH = 7.2 Hz), 0.7, 0.3 and 0.2 mL, respectively. During all these opera-
5.01(br, 1H). 13C NMR (100 MHz, CDCl3) d 14.16, 28.31, tions, precaution should be taken so that the temperature of
42.29, 43.86, 61.34, 79.97, 155.72. the oil bath does not exceed 160°C as the compound
decomposes. The last three portions of distillate were dis-
2-(tert-Butoxycarbonylamino)[2-13C]ethanol (3) tilled under reduced pressure. The reaction mixture was
cooled to 70°C and acetone was added and stirred well.
To a solution of 2 (2.50 g, 12.3 mmol) in THF (13 mL) After standing overnight at 4°C, it was filtered to obtain the
were added sodium borohydride (1.39 g, 36.6 mmol), product as white solid which was washed with acetone and
lithium chloride (1.55 g, 36.6 mmol) and methanol air dried. Concentrating the filtrate to syrup, adding acetone
(25 mL). The mixture was stirred at room temperature for and standing overnight at 4°C, a second crop of material
28 h. 15 mL of 5% citric acid was added to the reaction was obtained. Compound 5 is hygroscopic. (yield =
mixture and extracted with CH2Cl2. The organic extract 1.01 g, 61%). Found: C, 11.58; H, 3.40; N, 6.75. Anal.
was washed with 5% NaHCO3 and brine, respectively, Calcd. for C13CH7Br2N: C, 12.15; H, 3.43; N, 6.80. IR
dried over anhydrous MgSO4 and concentrated to afford (KBr, cm-1): 3,427, 3,100–2,750 (br), 1,930–1,830 (br),
the crude product as colorless oil. The crude product was 1,580, 1,560, 1,504, 1,483, 1,432, 1,373, 1,320, 1,251,
used for the next step without further purification. 1,231, 1,088, 1,032, 935, 869, 819, 763, 708, 651, 570, 496.
1
(yield = 1.46 g, 73%). Found: C, 49.08; H, 9.49; N, 9.03. H NMR (400 MHz, D2O): d 3.15 (t, 1H, 3JHH = 6.1 Hz),
Anal. Calcd. for C13
6 CH15NO3: C, 52.45; H, 9.32; N, 8.64. 3.50–3.57 (m, 3H). 13C NMR (100 MHz, D2O) d 27.84

123
 A. N. Maity et al.

(d, 1JCC = 37.0 Hz), 41.00. MALDI-TOF MS: m/z 124.711, of the scheme. Isotopomers of cysteine are commercially
126.714 (calcd. for [M–Br]? 124.980, 126.977). available while isotopomers of 2-bromoethylamine are not.
However, it can be synthesized easily from 2-aminoethanol
4-Thia-[6-13C]lysine hydrochloride (6) (Cortese 1943). Although some of the isotopomers of
2-aminoethanol are commercially available, they are expen-
KOH (442 mg, 7.8 mmol) and cysteine hydrochloride sive, specially the site-directed ones. On the contrary, it can
(368 mg, 2.3 mmol) were taken in a 25-mL Schlenk flask be synthesized (Babior 1969) from relatively inexpensive
and evacuated and purged with nitrogen. Oxygen-free starting material, glycine. So, we chose glycine as the
water (two cycles of freeze–pump–thaw) was added to the starting material. Before starting with the labeled com-
flask under nitrogen atmosphere and heated in an oil bath at pound, we tried to standardize all the required steps with
70°C. Compound 5 (515 mg, 2.5 mmol) was added slowly unlabelled compounds. The synthesis of 2-aminoethanol,
with stirring to the solution under nitrogen atmosphere for following the reported procedure (Babior 1969), by directly
10 min. The mixture was kept at 70°C for another 10 min. reducing the ethyl glycinate with LiAlH4 and continuous
It was brought to room temperature under nitrogen atmo- extraction for 2 days resulted in poor yield. In the second
sphere and kept for 7 h. Then it was neutralized with 48% attempt following another literature report (Verhoeven
HBr. 2 mL of EtOH added and precipitate appeared. It was et al. 2004), protection of amino group by benzaldehyde
dissolved by adding water dropwise. Little EtOH was followed by reduction with LiAlH4 and subsequent
added to make it little turbid and kept at 4°C for 12 h. Then reduction with H2 and 10% Pd/C produced mixture of
it was filtered through filter paper (Advantec 5B) and the products in our hands. Moreover, N-benzylidene-glycine
filtrate was passed through a column of cation exchanger ethyl ester, obtained by reacting glycine ethyl ester with
(Dowex 50w 9 8 in H? form), washed with water and benzaldehyde, slowly decomposes in air. Then, another
eluted with 1 N ammonium hydroxide. The fractions which attempt by blocking amino group with benzoyl chloride,
had shown positive ninhydrin test were collected. The reduction with LiAlH4 and subsequent reduction with H2
collected fractions were evaporated to dryness using rotary and Pd/C also produced mixture of products. Therefore, we
evaporator. The oily residue was dissolved in minimum planned a new strategy that produced satisfactory yields for
amount of water and neutralized to slight acidity (pH *6) all the steps. Then, we followed the strategy starting with
by conc. HCl. Then EtOH (2 mL) and of acetone (3 mL) [2-13C]glycine (Scheme 2).
was added and kept at 4°C for 12 h. It was filtered In this method we synthesized ethyl [2-13C]glycinate (1)
to produce the crude product which was recrystallized by reacting thionyl chloride (Stocking et al. 2001) with
twice from water, ethanol and acetone mixture to produce [2-13C]glycine in ethanol. This method appeared to be better
needle-like crystals. (yield = 276 mg, 55%). [a]19D = -3.9° than the esterification with HCl gas as the reaction is
(c 1.0, H2O); Found: C, 29.03; H, 6.39; N, 13.91. Anal. incomplete in the latter case if the reaction mixture is not
Calcd for C134 CH13ClN2O2S: C, 30.27; H, 6.50; N, 13.89. saturated with HCl. Ethyl [2-13C]glycinate was then reacted
IR (KBr, cm-1): 3,436, 3,200–2,750 (br), 2,676, 2,558, with di-tert-butyl dicarbonate (Boc2O) (Kim et al. 2007) to
1,998, 1,634, 1,567, 1,488, 1,461, 1,414, 1,396, 1,385, get ethyl 2-(tert-butoxycarbonylamino)[2-13C]acetate (2)
1,351, 1,327, 1,313, 1,140, 1,121, 1,089, 1,072, 1,047, 960, which was reduced by NaBH4 to produce 2-(tert-butoxy-
933, 882, 844, 773, 550, 466. 1H NMR (400 MHz, D2O): carbonylamino)[2-13C]ethanol (3). The reduction with
d 2.72–2.77 (2H, m), 2.96 (2H, t, 3JHH = 4.3 Hz), 3.09 (2H, NaBH4 was initially performed in ethanol following the lit-
dt, 1JCH = 145.3 Hz, 3JHH = 6.6 Hz), 3.82 (1H, t, 3JHH = erature procedure (Iizuka et al. 1990). Very little product was
5.4 Hz). 13C NMR (100 MHz, D2O) d 28.43, 31.51, 38.15, obtained after stirring at room temperature. Despite refluxing
53.40, 172.60. MALDI-TOF MS: m/z = 165.845 (calcd. overnight, the reaction was found to be incomplete. The use
for [M–Cl]? 166.073). of methanol instead of ethanol resulted in almost quantitative
yield at room temperature. As ethanol is one of the byprod-
ucts of the reaction, the presence of bulk amount of ethanol as
Results and discussion solvent probably moves the reaction equilibrium towards
left. 3 was converted to 2-amino[2-13C]ethanol hydrochlo-
In order to synthesize the desired isotopomers of 4-thial- ride (4) by reacting with HCl gas. Bromination of 4 with HBr
ysine, we need to design a strategy keeping in mind some produced 2-bromo[1-13C]ethylamine hydrobromide (5). The
factors such as commercial availability and cost effec- proton NMR spectrum of 5 shows a triplet and a multiplet
tiveness of corresponding labeled starting material, inter- instead of the expected single triplet due to coupling from
mediates that can be isolated easily and reactions with high two protons and a doublet of triplets due to couplings
yield. As we mentioned earlier, the reaction between cys- from two protons and one 13C nucleus (see supplementary
teine and 2-bromoethylamine can be used as the final step Fig. S1). However, the integrals revealed that the multiplet is

123
Synthesis of 4-thialysine from glycine

Scheme 2 Reagents and H2

conditions: a SOCl2, EtOH; 13 O H2 O H2
C a O
b Boc2O, Et3N, CH3OH; H2N 13
C b 13 O
OH NH2.HCl O N C
c NaBH4, LiCl, MeOH; d HCl H
gas, CH2Cl2; e 48% HBr; f (1) O O
cysteine, KOH, H2O, (2) HCl 1 2

H2 O H2
d
HO 13
C 13
C OH
NH2.HCl O N
H
4
3
e

H2 f
Br 13
C HO2C NH2.HCl
NH2.HBr S 13
C
NH2 H2

5 6

three times than the triplet. This means one triplet corre- Biophysical study
sponding to the doublet of triplets is merged with the other
triplet resulting in a multiplet. The reaction of 5 with cysteine 4-Thia-[6-13C]lysine has successfully mimicked the reac-
produced 4-thia-[6-13C]lysine hydrochloride (6) which was tion of unlabeled 4-thialysine with lysine-5,6-aminomutase.
purified by cation exchange chromatography followed by The EPR spectra of the radical intermediates after 10 s of
repeated crystallization. Specific rotation of -3.9° was reaction of 4-thialysine, 4-thia-[6-13C]lysine and 4-thia-
observed for the 1% solution of 4-thia-[6-13C]lysine in water. [5-13C]lysine with 5,6-LAM in the presence of [40 -2H]
In the literature there have been reports of two different pyridoxal-50 -phosphate are shown in Fig. 1. Absence of
values for unlabelled 4-thialysine. Cavallini et al. (1955) detectable line broadening in the case of 4-thia-
reported the value as ?7.2°, while Lindley (1959) reported it [6-13C]lysine with respect to that of 4-thialysine indicates
as -4.4°. Therefore, we measured the commercially avail- that the spin is not present on carbon-6. On the contrary, the
able unlabelled compound and found that the value is iden- spectrum of 4-thia-[5-13C]lysine showed observable line
tical with that of 4-thia-[6-13C]lysine.
We developed a scheme where all the steps have high
yields. Interestingly, the intermediates, which are stable,
can be easily purified avoiding cumbersome techniques
like chromatography or continuous extraction. In this
scheme, most of the reactions are performed in room
temperature and milder reagents were used avoiding
pyrophoric reagents like Pd/C or LiAlH4. Moreover, pre-
vious report of preparation of 2-aminoethanol from glycine
had the yield of 50%. We obtained the overall yield of
68%. This scheme essentially gives access to all possible
isotopomers of 4-thialysine in which any of the stable
isotopes such as 13C, 15N, 17O, 18O and 2H or radioisotopes
such as 14C, 11C, 3H and 35S can be incorporated in a site-
directed way. Either starting with appropriate isotopomer 2600 2800 3000 3200 3400 3600 3800 4000
of glycine or reacting with appropriate reagent such as Gauss
NaB2H4, NaB3H4 or site-specific isotopomer of cysteine
(Siebum et al. 2004b) would produce desired site-directed Fig. 1 X-band EPR spectra of radical intermediates in 5,6-LAM
reaction with 4-thialysine (top), 4-thia-[6-13C]lysine (middle) and
isotopomer of 4-thialysine. It is evident that the scheme 4-thia-[5-13C]lysine (bottom) in the presence of [40 -2H]pyridoxal-50 -
also gives easy access to site-directed isotopomers of phosphate. Experimental: microwave frequency, 9.536 GHz; power,
2-aminoethanol and 2-bromoethylamine. 20 mW; modulation, 4 G at 100 kHz; T = 80 K

123
 A. N. Maity et al.

broadening which means the spin is on the carbon-5. Taken inhibitors designed from angiotensinogen transition state. Chem
together EPR results suggest that radical intermediate is the Pharm Bull 38:2487–2493
Jun DY, Rue SW, Han KH, Taub D, Lee YS, Bae YS, Kim YH (2003)
thia-analog of substrate–PLP radical (S). Mechanism underlying cytotoxicity of thialysine, lysine analog,
toward human acute leukemia Jurkat T cells. Biochem Pharma-
col 66:2291–2300
Conclusion Kainosho M, Torizawa T, Iwashita Y, Terauchi T, Mei Ono A,
Guntert P (2006) Optimal isotope labelling for NMR protein
structure determinations. Nature 440:52–57
In summary, we presented the synthesis of 4-thia- Kim SG, Lee SH, Park TH (2007) Novel stereoselective synthesis of
[6-13C]lysine, a site-directed isotopomer of 4-thia analog of all four diastereomers of 3a-methyl-pyrrolo[3,4-c]piperidine
lysine. We also demonstrated that the same synthetic strategy from glycine ethyl ester. Tetrahedron Lett 48:5023–5026
Lees NS, Chen DW, Walsby CJ, Behshad E, Frey PA, Hoffman BM
would give access to all possible site-directed isotopomers of (2006) How an enzyme tames reactive intermediates: positioning
2-aminoethanol, 2-bromoethylamine and 4-thialysine. of the active-site components of lysine 2,3-aminomutase during
enzymatic turnover as determined by ENDOR spectroscopy.
Acknowledgments We thank Dr. Amiya Kumar Medda for useful J Am Chem Soc 128:10145–10154
discussion on synthetic strategy. We also thank Kau-Chun Kao and Lindley H (1959) The preparation of compounds related to S-2-
Prof. Yen-Peng Ho for measuring mass spectra. This research was aminoethyl-L-cysteine. Aust J Chem 12:296–298
supported by the National Science Council Grant NSC97-2627-M- Lloyd-Jones GC, Munoz MP (2007) Isotopic labelling in the study of
259-001, Taiwan. organic and organometallic mechanism and structure: an
account. J Label Compd Radiopharm 50:1072–1087
Ludlam CF, Sonar S, Lee CP, Coleman M, Herzfeld J, Raj Bhandary
UL, Rothschild KJ (1995) Site-directed isotope labeling and
ATR-FTIR difference spectroscopy of bacteriorhodopsin: the
References peptide carbonyl group of Tyr 185 is structurally active during
the bR?N transition. Biochemistry 34:2–6
Babior BM (1969) The mechanism of action of ethanolamine Luersen K (2005) Leishmania major thialysine Ne-acetyltransferase:
deaminase. I. Studies with isotopic hydrogen and oxygen. identification of amino acid residues crucial for substrate
J Biol Chem 244:449–456 binding. FEBS Lett 579:5347–5352
Behshad E, Ruzicka FJ, Mansoorabadi SO, Chen D, Reed GH, Macnaughtan MA, Kane AM, Prestegard JH (2005) Mass spectrom-
Frey PA (2006) Enantiomeric free radicals and enzymatic etry assisted assignment of NMR resonances in reductively
13
control of stereochemistry in a radical mechanism: the case of C-methylated proteins. J Am Chem Soc 127:17626–17627
lysine 2,3-aminomutases. Biochemistry 45:12639–12646 Maity AN, Hsieh CP, Huang MH, Chen YH, Tang KH, Behshad E,
Cavallini D, De Marco C, Mondovi B, Azzone GF (1955) A new Frey PA, Ke SC (2009) Evidence for conformational movement
synthetic sulfur-containing amino acid: S-aminoethylcysteine. and radical mechanism in the reaction of 4-thia-L-lysine with
Experientia 11:61–62 lysine 5,6-aminomutase. J Phys Chem B 113:12161–12163
Coleman CS, Stanley BA, Jones AD, Pegg AE (2004) Spermidine/ Raap J, Wolthuis WNE, Hehenkamp JJJ, Lugtenburg J (1995)
spermine-N1-acetyltransferase-2 (SSAT2) acetylates thialysine Enantioselective syntheses of isotopically labeled a-amino-
and is not involved in polyamine metabolism. Biochem J acids—preparation of specifically 13C-labeled L-lysines. Amino
384:139–148 Acids 8:171–186
Cortese F (1943) b-Bromoethylamine hydrochloride. In: Cortese F Siebum AHG, Tsang RKF, van der Steen R, Raap J, Lugtenburg J
(ed) Organic syntheses, collect, vol 2. Wiley, New York, (2004a) Synthesis of (e-13C-, e-15N)-enriched L-lysine—estab-
pp 91–92 lishing schemes for the preparation of all possible 13C and 15N
Decatur SM (2006) Elucidation of residue-level structure and isotopomers of L-lysine, L-ornithine, and L-proline. Eur J Org
dynamics of polypeptides via isotope-edited infrared spectros- Chem 2004:4391–4396
copy. Acc Chem Res 39:169–175 Siebum AHG, Woo WS, Raap J, Lugtenburg J (2004b) Access to any
Frey PA, Hegeman AD, Reed GH (2006) Free radical mechanisms in site-directed isotopomer of methionine, selenomethionine, cys-
enzymology. Chem Rev 106:3302–3316 teine, and selenocysteine—use of simple, efficient modular
Gloss LM, Kirsch JF (1995a) Decreasing the basicity of the active site synthetic reaction schemes for isotope incorporation. Eur J Org
base, Lys-258, of Escherichia coli aspartate aminotransferase by Chem 2004:2905–2913
replacement with c-thialysine. Biochemistry 34:3990–3998 Stocking EM, Sanz-Cervera JF, Williams RM (2001) Studies on the
Gloss LM, Kirsch JF (1995b) Use of site-directed mutagenesis and biosynthesis of paraherquamide: synthesis and incorporation of a
alternative substrates to assign the prototropic groups important hexacyclic indole derivative as an advanced metabolite. Angew
to catalysis by Escherichia coli aspartate aminotransferase. Chem Int Ed 40:1296–1298
Biochemistry 34:3999–4007 Suvarna K, Seah L, Bhattacherjee V, Bhattacharjee JK (1998)
Hopkins CE, O’Connor PB, Allen KN, Costello CE, Tolan DR (2002) Molecular analysis of the LYS2 gene of Candida albicans:
Chemical-modification rescue assessed by mass spectrometry Homology to peptide antibiotic synthetases and the regulation of
demonstrates that c-thia-lysine yields the same activity as lysine the a-aminoadipate reductase. Curr Genet 33:268–275
in aldolase. Protein Sci 11:1591–1599 Takeda M, Jee J, Ono AM, Terauchi T, Kainosho M (2009) Hydrogen
Hopkins CE, Hernandez G, Lee JP, Tolan DR (2005) Aminoethyla- exchange rate of tyrosine hydroxyl groups in proteins as studied
tion in model peptides reveals conditions for maximizing thiol by the deuterium isotope effect on Cf chemical shifts. J Am
specificity. Arch Biochem Biophys 443:1–10 Chem Soc 131:18556–18562
Iizuka K, Kamijo T, Harada H, Akahane K, Kubota T, Etoh Y, Tang KH, Mansoorabadi SO, Reed GH, Frey PA (2009) Radical triplets
Shimaoka I, Tsubaki A, Murakami M, Yamaguchi T et al (1990) and suicide inhibition in reactions of 4-thia-D-and 4-thia-L-lysine
Synthesis and structure–activity relationships of human renin with lysine 5,6-aminomutase. Biochemistry 48:8151–8160

123
Synthesis of 4-thialysine from glycine

Tao WA, Aebersold R (2003) Advances in quantitative proteomics Wu WM, Lieder KW, Reed GH, Frey PA (1995) Observation of a 2nd
via stable isotope tagging and mass spectrometry. Curr Opin substrate radical intermediate in the reaction of lysine 2,3-
Biotech 14:110–118 aminomutase—a radical centered on the b-carbon of the
Verhoeven A, Williamson PT, Zimmermann H, Ernst M, Meier BH alternative substrate, 4-thia-L-lysine. Biochemistry 34:10532–
(2004) Rotational-resonance distance measurements in multi- 10537
spin systems. J Magn Reson 168:314–326

123