Selective synthesis and structural elucidation of S-acyl- and N-acylcysteines

Article abstract of DOI:10.1021/jo900853s

(Chemical Equation Presented) N-(Acyl)-1H-benzotriazoles 6a-f react with L-cysteine 5 at 20°C to give exclusively (i) N-acyl-L-cysteines 8a-e in the presence of triethylamine in CH₃CN-H₂O (3:1), but (ii) S-acyl-L-cysteines 7a-e in CH

Full text of DOI:10.1021/jo900853s

pubs.acs.org/joc
Scheme 1, trans-thioesterification of 1 with 2 results in inter-
Selective Synthesis and Structural Elucidation of
S-Acyl- and N-Acylcysteines
mediate 3, after which “classical” S to N acyl transfer takes
place forming an amide bond to give the new peptide 4.2a-e
The S to N acyl transfer from 3 to 4 is due to proximity of the
amino group in 3 to the thioester functionality.
Alan R. Katritzky,*,§ Srinivasa R. Tala,§
Nader E. Abo-Dya,§,^ Kapil Gyanda,§
Bahaa El-Dien M. El-Gendy,z,§
S-Acyl- and S-benzyloxycarbonyl cysteines are useful
potential intermediates for the synthesis of cysteine and
oxytocin-like peptides. Aimoto et al.3 recently confirmed
generation of an S-peptide via an N to S acyl shift reaction in
TFA solution by a combination of 13C NMR spectroscopy,
reverse-phase HPLC, and MS analyses. In neat TFA, acyl
chlorides selectively S-acylate the cysteine residues of pep-
tides that lack serine and threonine residues without acylat-
ing amino groups.4 Mautner et al.5 selectively acylated thiol
groups with selenol esters which react with thiols at slightly
acidic pH where amino groups are protonated; lysine and
histidine residues remained unreactive.
Published routes to synthesize S-acylcysteine derivatives
have utilized (i) acyl chlorides,4,6a,6b (ii) acid anhydrides,6c
(iii) selenol esters,5 and (iv) acyl-CoAs6d and peptide thioes-
ters.2b,6e Some of these methods have involved complex
procedures and low yields.6a,6b We have now developed mild
and efficient methods to synthesize S-acylcysteines.
Zakaria K. Abdel-Samii,^ and Peter J. Steel‡
§Center for Heterocyclic Compounds, Department of
Chemistry, University of Florida, Gainesville, Florida 32611-
7200, ^Department of Pharmaceutical Organic Chemistry,
Faculty of Pharmacy, Zagazig University, Zagazig, Egypt,
zDepartment of Chemistry, Faculty of Science, Benha
University, Benha, Egypt, and ‡Chemistry Department,
University of Canterbury, Christchurch, New Zealand
katritzky@chem.ufl.edu
Received April 24, 2009
N-Acylcysteines are found in many useful peptides1a,1b
and proteins.1c They also show potential (i) as amino acid
antagonists in bacteria,7a (ii) for quantitative determination
of enantiomers of amino compounds,7b and (iii) as chiral
ligands.7c Published routes to synthesize N-acylcysteine de-
rivatives have also used (i) acyl chlorides7a,8a and (ii) acid
anhydrides8b as well as (iii) chemical ligation2b,2c,2e in meth-
ods which have involved complex procedures8a and protec-
tion of the SH group in cysteine.8b Thus, mild and efficient
methods to synthesize N-acylcysteines are desired.
N-(Acyl)-1H-benzotriazoles 6a-f react with L-cysteine 5
at 20 ꢀC to give exclusively (i) N-acyl-L-cysteines 8a-e in
the presence of triethylamine in CH3CN-H2O (3:1), but
(ii) S-acyl-L-cysteines 7a-e in CH3CN-H2O (5:1) in
the absence of base. Structures 7b, 7d and 8b, 8d are
supported by 2D NMR spectroscopic methods including
N-Acylbenzotriazoles are advantageous for N-, O-, C-,
and S-acylation,9a-i especially where the corresponding acid
(3) Nakamura, K.; Sumida, M.; Kawakami, T.; Vorherr, T.; Aimoto, S.
Bull. Chem. Soc. Jpn. 2006, 79, 1773–1780.
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1
gDQCOSY, gHMQC, gHMBC, and H-15N CIGAR-
343, 557–562.
(5) Makriyannis, A.; Gunther, W. H. H.; Mautner, H. G. J. Am. Chem.
Soc. 1973, 95, 8403–8406.
gHMBC experiments. The structure of compound 8d was
also supported by single-crystal X-ray diffraction.
€
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251–254.
Cysteine-containing peptides are valuable intermediates
for the synthesis of more complex peptides1a,1b and proteins
using native chemical ligation (NCL).1c Native chemical
ligation (NCL)2a is widely applied for the synthesis of
proteins, uses a chemoselective reaction between two unpro-
tected fragments, a C-terminal thioester (peptide A) 1, and
N-terminal cysteine (peptide B) 2 (Scheme 1). As shown in
(8) (a) Martin, T. A. J. Med. Chem. 1969, 12, 950–953. (b) Martin, T. A.;
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S. J.; Offer, J.; Dawson, P. E. Bioorg. Med. Chem. 2001, 9, 2323–2328.
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1994, 266, 776–779. (b) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.;
Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684–11689.
(c) Yeo, D. S. Y.; Srinivasan, R.; Chen, G. Y. J.; Yao, S. Q. Chem.;Eur. J.
2004, 10, 4664–4672. (d) Macmillan, D. Angew. Chem., Int. Ed. 2006, 45,
7668–7672. (e) Pentelute, B. L.; Kent, S. B. H. Org. Lett. 2007, 9, 687–690.
402. (d) Katritzky, A. R.; Suzuki, K.; Singh, S. K.; He, H.-Y. J. Org. Chem.
2003, 68, 5720–5723. (e) Katritzky, A. R.; He, H.-Y.; Suzuki, K. J. Org.
Chem. 2000, 65, 8210–8213. (f) Katritzky, A. R.; Wang, M.; Yang, H.;
Zhang, S.; Akhmedov, N. G. ARKIVOC 2002, 8, 134–142. (g) Katritzky, A.
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DOI: 10.1021/jo900853s
r
Published on Web 08/21/2009
J. Org. Chem. 2009, 74, 7165–7167 7165
2009 American Chemical Society

Katritzky et al.
JOCNote
SCHEME 1. Native Chemical Ligation Mechanism
TABLE 2. Synthesis of N-Acylcysteine 8a-e
entry
6, R =
8, yield (%)a
mp (ꢀC)
1
2
3
4
5
6a, phenyl
8a, 68
8b, 67
8c, 86
8d, 85
8e, 51
oil
6b, 4-MeO-phenyl
6c, 4-NO2-phenyl
6d, 2-naphthyl
6f, palmityl
99-101
58-60
151-152
70-72
aIsolated yield
SCHEME 2. S- and N-Acylation of L-Cysteine 5
FIGURE 1. 1H, 13C, and 15N chemical shift assignments of 7b.
X-ray Crystal Structure of 8d. The structure of the
N-naphthoyl-L-cysteine 8d was unambiguously established
by single-crystal X-ray structure determination (Figure 39 in
the Supporting Information). The locations of the OH, NH,
and SH hydrogen atoms were established from difference
electron density maps. This is only the second example of an
X-ray structure of an acylcysteine, the other being N-acetyl-
L-cysteine.10 In the solid state the molecules are linked by
a complex system of hydrogen bonding involving interac-
tions between adjacent amide groups and chains of hydrogen
bonded carboxylic acid groups. Unfortunately, none of
the S-acyl derivatives furnished crystals suitable for X-ray
crystallography.
TABLE 1. Synthesis of S-Acylcysteines 7a-e
entry
6, R =
7, yield (%)a
mp (ꢀC)
1
2
3
4
5
6a, phenyl
7a, 66
7b, 80
7c, 81
7d, 77
7e, 85
136-139
166-168
158-160
167-168
160-161
6b, 4-MeO-phenyl
6c, 4-NO2-phenyl
6d, 2-naphthyl
2D NMR Studies. The 2D NMR spectra were recorded
at 500 MHz for 1H, 125 MHz for 13C, and 50 MHz for 15N,
equipped with a three-channel, 5-mm, indirect detection
probe, with z-axis gradients. The chemical shifts for 1H
and 13C were referenced to the residual solvent signal on
the tetramethylsilane scale. The chemical shifts for 15N were
referenced to a frequency ratio 10.1328898482, correspond-
ing to 0 for neat ammonia. 1H and 13C chemical shifts were
6e, 4-Me-phenyl
aIsolated yield
chlorides are unstable or difficult to prepare.9j,9k Surpris-
ingly, we located no X-ray crystal structural data for an
S-acylcysteine and only one for an N-acylcysteine,10 hence
our studies also aimed to elucidate structural information for
S-acylcysteines as well as N-acylcysteines. We now report the
use of N-acylbenzotriazoles for the selective synthesis of
S-acyl- and N-acylcysteines and the structural confirmation
of both classes using X-ray crystallography and 2D NMR
techniques.
N-Acylbenzotriazoles 6a-f were prepared in 82-90%
yield by the reaction of the corresponding carboxylic acid
with 4 equiv of 1H-benzotriazole and 1 equiv of SOCl2
in THF at 20 ꢀC for 2 h.9b Treatment of L-cysteine 5 with
N-acylbenzotriazoles 6a-e at room temperature in MeCN-
H2O (5:1) for 12 h gave exclusively S-acylcysteines 7a-e in
yields of 66-85% as the only products isolated (Scheme 2,
Table 1). Thus, at slightly acidic pH we isolated exclusively
S-acylcysteines 7a-e.
1
assigned based on the H-1H, one-bond and long-range
1H-13C couplings, seen in the gDQCOSY, gHMQC, and
1
gHMBC spectra. H-15N CIGAR-gHMBC spectra were
acquired with a pulse sequence optimized for 15N as des-
cribed in ref 11. The methylene protons in 7b, 7d and 8b, 8d
were not stereo chemically assigned.
The spectra for S-(4-methoxybenzoyl)-L-cysteine 7b were
recorded in TFA-d at room temperature. This compound
was fully assigned based on different 2D NMR experiments
(Figure 1). The gHMBC (Figures 7 and 8 in the Supporting
Information) experiment shows that both diastereotopic
protons at 3.82 and 4.07 ppm have three-bond correlations
with the S-CO group at 196.4 ppm. Also, the two aromatic
protons at the ortho positions (8.05 ppm) and at the meta
positions (7.13 ppm) show three and four-bond correlations
respectively to the S-CO. The nitrogen chemical shift of
39.5 ppm was recorded in a CIGAR-gHMBC experiment
(Figure 9 in the Supporting Information). This chemical shift
was revealed through the three-bond correlation with the
two diastereotopic protons at 3.82 and 4.07 ppm. The two
N-Acylcysteines 8a-e (Scheme 2, Table 2) were synthe-
sized by reacting L-cysteine 5 with 1 equiv of N-acylbenzo-
triazoles 6a-d,f in the presence of 1 equiv of triethylamine in
MeCN-H2O (3:1) in yields of 51-86%, using our previously
reported method.9b Under such basic pH conditions we
isolated exclusively N-acylcysteines 8a-e.
(10) Takusagawa, F.; Koetzle, T. F.; Kou, W. W. H.; Parthasarathy, R.
Acta Crystallogr., Sect. B 1981, 37, 1591–1596.
(11) Kline, M.; Cheatham, S. Magn. Reson. Chem. 2003, 41, 307–314.
7166 J. Org. Chem. Vol. 74, No. 18, 2009

Katritzky et al.
JOCNote
assigned through a two-bond correlation with the proton at
4.76 ppm and the three-bond correlations with the two
protons at 3.65 and 3.88 ppm.
In conclusion, we have developed a methodology for
selective synthesis of S-acyl- and N-acylcysteines using
N-acylbenzotriazoles under mild reaction conditions in
good yields. At lower pH we isolated S-acylcysteines and
at higher pH we isolated N-acylcysteines. Structural elucida-
tion of S-acyl- and N-acylcysteines was achieved by using
X-ray crystallography and 2D NMR techniques. 15N NMR
was very useful in distinguishing between the N- and
S-acylcysteines because of the big difference in chemical shift
between the free NH2 and the amidic NH (75.0 ppm on
average).
FIGURE 2. 1H, 13C, and 15N chemical shift assignments of 8b.
Experimental Section
General Procedure for Preparation of S-Acylcysteines 7a-e.
To a solution of L-cysteine 5 (2 mmol) in H2O (3 mL) was added
a solution of the corresponding N-acylbenzotriazole 6 (2 mmol)
in CH3CN (15 mL). The heterogeneous mixture was then stirred
at room temperature for 12 h. The solid was filtered, washed
with water (3 Â 10 mL), ethyl acetate (3 Â 10 mL), and diethyl
ether (3 Â 10 mL), and dried in a desiccator under vacuum to
give S-acylcysteines 7a-e.
FIGURE 3. 1H, 13C, and 15N chemical shift assignments of 7d.
NH2 protons and the adjacent proton at 4.93 ppm were too
broad to show any correlation to the nitrogen atom. The
nitrogen chemical shift appears in the expected region of free
NH2 reported in the literature.12 These correlations further
confirm that the compound exists exclusively in the S-acyl
form.
S-(Benzoyl)-L-cysteine (7a): yield 66%; white microcrystals;
mp 136.0-139.0 ꢀC (lit.6a mp 141.0-142.0 ꢀC); [R]23 -31.2
D
(c 1.0, MeOH-TFA (9:1)); 1H NMR (300 MHz, TFA-d) δ 4.47
(dd, J = 15.5, 6.5 Hz, 1H), 4.73 (dd, J = 15.7, 3.4 Hz, 1H),
5.56-5.57 (m, 1H), 8.21 (t, J = 8.1 Hz, 2H), 8.41 (t, J = 7.6 Hz,
1H), 8.65-8.68 (m, 2H); 13C NMR (75 MHz, TFA-d) δ 30.9,
57.5, 130.3, 131.9, 137.3, 138.7, 173.3, 200.8.
The NMR spectra for N-(4-methoxybenzoyl)-L-cysteine
8b (Figure 2) were recorded in DMSO-d6 at room tempera-
ture. The NH proton at 8.49 ppm couples with the adjacent
tertiary proton at 4.51 ppm and it appears as a doublet in
the 1H NMR. In gHMBC (Figure 14 in the Supporting
Information), the amino proton (8.49 ppm) shows two-bond
correlations with the carbonyl group at 166.7 ppm. This
carbonyl group shows further correlation with the aromatic
protons at 7.89 ppm, which confirms the N-acyl configura-
tion. In a CIGAR-gHMBC experiment, the NH proton at
8.49 ppm shows one-bond correlation to the nitrogen atom
at 114.2 ppm (Figure 15 in the Supporting Information).
This chemical shift value is in agreement with the previously
reported values for amide nitrogens.12 This proton appeared
as a doublet with a typical one-bond coupling constant of
90.3 Hz. This nitrogen shows three other correlations, two-
bond correlation to the proton at 4.51 ppm and three-bond
correlations to the protons at 2.89 and 2.99 ppm.
Compound 7d exists exclusively in the S-acyl form
(Figure 3) and that was easily confirmed by using different
2D NMR techniques. In a gHMBC experiment (Figures 23
and 24 in the Supporting Information), there is a three-bond
correlation between the carbonyl group attached to sulfur
(C at 199.2 ppm) and the aromatic proton at 7.99 ppm.
Moreover, the two protons at 3.65 and 3.88 ppm have three-
bond correlations with the same carbon. The assignment is
confirmed by the nitrogen chemical shift (39.0 ppm) ob-
served in a CIGAR-gHMBC experiment (Figures 25 and
26 in the Supporting Information), which falls within the
range of free NH2 reported in the literature.12 This nitrogen
has a slightly upfield value because of protonation since
the spectra were taken in TFA-d. This chemical shift was
General Procedure for Preparation of N-Acylcysteines 8a-e.
To a solution of L-cysteine 5 (2 mmol) and triethylamine
(2 mmol) in CH3CN-H2O (3:1, 8 mL) was added the corres-
ponding N-(acylbenzotriazole) 6 (2 mmol). The mixture was
stirred at room temperature for 2 h. Solvent was removed under
reduced pressure and ethyl acetate (10 mL) was added. The
organic layer was washed with 2 N HCl and brine. Solvent
evaporation and recrystalization (AcOEt:hexanes = 3:1) gave
N-acylcysteines 8a-e.
N-(Benzoyl)-L-cysteine (8a):8a yield 68%; colorless oil; [R]23
D
-31.2 (c 3.16, MeOH); IR (neat) 3333.04, 2926.6, 1733.0,
1705.5, 1641.8, 1526.0 cm-1 1H NMR (300 MHz, CDCl3)
;
δ 1.51 (t, J = 9.0 Hz, 1H), 3.10-3.26 (m, 2H), 5.05-5.13 (m,
1H), 7.20 (d, J = 6.9 Hz, 1H), 7.40-7.57 (m, 3H), 7.79-7.86 (m,
2H), 8.59 (br s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.8, 54.2,
127.5, 127.6, 127.7, 128.9, 129.0, 132.5, 168.3, 173.1. Anal. Calcd
for C10H11NO3S 1/2H2O: C, 51.27; H, 5.16; N, 5.98. Found: C,
3
51.64; H, 5.42; N, 6.31.
Acknowledgment. We thank Dr. C. D. Hall for helpful
suggestions and Mr. K. Pandya for some preliminary
experiments.
Supporting Information Available: Materials and Methods
section, characterization data for compounds 7b-e and
8b-e, 1H NMR, gDQCOSY, gHMQC, gHMBC, and CI-
1
GAR-gHMBC spectra for compounds 7b, 7d, 8b, and 8d, H
and 13C NMR spectra of compounds 7a, 7c, 7e, 8a, 8c, and 8e
and IR spectra of compounds 7c, 7d, and 8a, crystal data and
structure refinement for 8d and additional tables for 8d, and
crystallographic information file (CIFs) for 8d. This material is
available free of charge via the Internet at http://pubs.acs.org.
(12) Marek, R.; Lycka, A. Curr. Org. Chem. 2002, 6, 35–66.
J. Org. Chem. Vol. 74, No. 18, 2009 7167