Dehydration of threonine esters during tosylation

Article abstract of DOI:10.1081/SCC-100107012

Tosylation of N-α-protected threonine methyl-and benzyl esters was investigated. Depending on the protecting groups, the composition of the reaction mixtures showed different ratio between O-tosyl-and dehydrothreonine derivatives. The latter was formed ex

Full text of DOI:10.1081/SCC-100107012

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DEHYDRATION OF THREONINE ESTERS DURING
TOSYLATION
Csaba Somlai a , Sándor Lovas b , Péter Forgó c , Richard F. Murphy b & Botond Penke a
a Department of Medical Chemistry , University of Szeged , Dóm tér 8, Szeged, H-6720,
Hungary
b Department of Biomedical Sciences , School of Medicine, Creighton University , 2500
California Plaza, Omaha, Nebraska, 68178, U.S.A.
c Department of Organic Chemistry , University of Szeged , Dóm tér 8, Szeged, H-6720,
Hungary
Published online: 16 Aug 2006.
To cite this article: Csaba Somlai , Sándor Lovas , Péter Forgó , Richard F. Murphy & Botond Penke (2001) DEHYDRATION
OF THREONINE ESTERS DURING TOSYLATION, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 31:23, 3633-3640, DOI: 10.1081/SCC-100107012
To link to this article: http://dx.doi.org/10.1081/SCC-100107012
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SYNTHETIC COMMUNICATIONS, 31(23), 3633–3640 (2001)
DEHYDRATION OF THREONINE
ESTERS DURING TOSYLATION
Csaba Somlai,1, Sandor Lovas,2 Peter Forgo,3
*
Richard F. Murphy,2 and Botond Penke1
1Department of Medical Chemistry, University
of Szeged, Dom ter 8, H-6720 Szeged, Hungary
2Department of Biomedical Sciences,
School of Medicine, Creighton University,
2500 California Plaza, Omaha,
Nebraska 68178, USA
3Department of Organic Chemistry,
University of Szeged, Dom ter 8,
H-6720 Szeged, Hungary
ABSTRACT
Tosylation of N-a-protected threonine methyl-and benzyl
esters was investigated. Depending on the protecting groups,
the composition of the reaction mixtures showed different
ratio between O-tosyl-and dehydrothreonine derivatives.
The latter was formed exclusively in case of N-a-Fmoc-threo-
nine benzyl ester.
*Corresponding author.
3633
Copyright & 2001 by Marcel Dekker, Inc.
www.dekker.com

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SOMLAI ET AL.
a,b-Dehydroamino acid units are frequently found in antibiotic and
phytotoxic peptides.1–3 Nisin, which contains both dehydroalanine- and
b-methyl-dehydroalanine residues displays antimalarial action and release
of lysosomal enzymes and lysis of erythrocytes.4 Terminamycin A, a cyclo-
dehydrodecapeptide with two dehydrodehydrodipeptide residues and seven
dehydroamino acid moieties has been isolated from a culture of
Streptomyces bernensis.5
Dehydroalanine is one of the constituents of the toxin alternariolide,
which responsible for necrotic brown spots of apple.6 The partially
recemized D-form of vinylglycine has been obtained from mushrooms7
and it is an intermediate in the enzymatic conversions of homoserine to
threonine and a-ketobutyrate. Various thiazole a-dehydroamino acids,
including ÁGlu(OMe), ÁLeu, ÁPhe, ÁThr and ÁVal and their dehydrodi-
and tripeptides were synthesized in 1992.8 Different 2-(1-amino)alkenyl-thia-
zoline-4-carboxylates–synthesized by Shin and associates9– are also import-
ant partial skeleton of many thiostrepton antibiotics such as thiomycin A10
and thiopeptin-Ba.11 These examples show that much interest has been
directed towards the synthesis and bioactivity of dehydropeptides contain-
ing a-dehydroamino acid residues.
a,b-Unsaturated amino acid derivatives and dehydropeptides can be
prepared by base catalyzed elimination of sulfonium derivatives12 and sulf-
oxides,13 furthermore by condensation reaction of 2-oxoalkanoic acid with
benzyl carbamate or p-toluenesulfonamide.14 The most frequently used
method for synthesis of a,b-dehydroamino acids is b-elimination reaction
of O-tosyl derivatives of 3-hydroxy amino acids described by I. Photaki.15
N-acylcrotonates have been reported by R.K. Olsen and associates via
b-chloro-and O-tosyl derivatives of the respective N-acyl-DL-threonine
methyl esters using 1,4-diazabicyclo[2,2,2]octane (Dabco) as base in ethyl
acetate.16 On the other hand, O-tosyl threonine as well as peptides contain-
ing O-tosyl threonine residue have been easily converted into aziridine
rather than a,b-dehydro compounds upon treatment with triethylamine.12,17
Direct tosylation of N-a-Fmoc-, N-a-Boc-and N-a-Z-protected threo-
nine benzyl-and methyl esters is presented here. The original aim was to
synthesize a new g-lactam constrained dipeptide analogue to stabilize b-turn
conformation in peptides.18 Elongation of the side chain of threonine with
one carbon atom by replacement the tosyl group by the nitrile group using
tetraethylammonium cyanide in dichloromethane was the key step of the
synthetic plan. For this purpose, O-tosyl derivative of N-protected threonine
ester as starting material was needed.
First, N-a-Fmoc-threonine benzyl ester was treated with excess
amount of p-toluenesulfonyl chloride in dry pyridine at ꢀ 5ꢁC for 2 h,
then allowed to stand overnight as usual for tosylation reactions. NMR

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3635
measurements revealed that N-a-Fmoc-dehydrothreonine benzyl ester
was obtained, unexpectedly almost quantitatively, without formation of
the corresponding O-tosyl derivative. Direct b-elimination is known in the
literature. In 1981 C. Shin and associates reported the synthesis of several
dehydrodehydrodipeptides under mesylation by base catalyzed b-elimina-
tion.19 In order to examine the solvent-dependence of the tosylation,
the same reaction was repeated in other tertiary bases. Under the same
reaction conditions, conversion of the starting material didn’t result in
main product in triethylamine and many spots have been observed on
TLC. In 4-methylmorpholine a moderate amount of the appropriate dehydro-
threonine was obtained, but the reaction mixture contained a portion of the
starting material even after 2 days. The starting material in dry pyridine in
the absence of p-toluenesulfonyl chloride at room temperature was stabile
for after a week. Consequently, O-tosyl derivative as an intermediate has
to be formed first, which serves the appropriate dehydrothreonine derivative
by a fast b-elimination.
When N-a-Fmoc-threonine methyl ester was tosylated in dry pyridine,
the desired O-tosyl compound and the dehydrothreonine derivative
were isolated from the reaction mixture with 70% and 30% yield, respec-
tively. The effect of replacement of the N-a-Fmoc group by the N-a-Boc-and
the N-a-Z was also studied. Both the N-a-Boc and the N-a-Z-threonine

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SOMLAI ET AL.
Table 1. Conversion of Protected Threonine Esters to O-Tosyl-
and Dehydrothreonine Derivatives
X
R
O-Tosyl Yield (%)
Dehydro Yield (%)
Fmoc
Fmoc
Z
Z
Boc
Boc
Bzl
Me
Bzl
Me
Bzl
Me
0
70
90
100
100
100
100
30
10
0
0
0
methyl esters gave exclusively O-tosyl products, therefore these protecting
groups do not favour the formation of the a,b-unsaturated systems.
Similarly, the N-a-Boc-threonine benzyl ester yielded only the O-tosyl
derivative. On the other hand, both the O-tosyl- and the dehydrothreonine
were obtained under the conversion of N-a-Z-threonine benzyl ester with
90% and 10% yields, respectively (Table 1).
Deprotection of the benzyl ester group to obtain N-a-Fmoc-dehydro-
threonine (2) was achieved using 30% HBr in acetic acid solution.
The geometry of the double bond was investigated by one-dimensional
NOE experiments (Nuclear Overhauser Effect). The selective excitation of
the methyl group at 1.71 ppm in 1 and at 1.68 ppm in 2 was used to identify
the isomers in solution phase. The experiment provided enhanced signals
on the NH protons at 8.87 ppm in 1 and at 8.64 ppm in 2. These data
revealed that a single stereoisomer of dehydrothreonine was formed which
has a Z geometry in both cases. The existence of the Z isomer was proved
further as the saturation of the benzyl CH2 at 5.15 ppm in 1 in the NOE
experiment did not yield any enhancements on the methyl doublet at
1.71 ppm. However, an enhanced signal was observed on the olefinic quartet
at 6.60 ppm. Our observations are in a close correlation with formation
[16, 20]
of the more stable Z isomer of 2-acylaminocrotonates
.

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The catalytic hydrogenation of N-a-Fmoc-dehydrothreonine benzyl
ester (1) with Pd/C simultaneously cleaved the benzyl ester and saturated
the a,b-olefin bond yielding racemic mixture of N-a-Fmoc-norvaline (10) in
the ratio of 1 : 1 (the product showed no optical rotation).
N-a-Fmoc-threonine benzyl ester can be converted into N-a-Fmoc-
dehydrothreonine benzyl ester almost quantitatively in direct tosylation
reaction in dry pyridine by b-elimination. After deprotection of the benzyl
ester group, N-a-Fmoc-dehydrothreonine was obtained, which can
be used as a building block for peptide synthesis and for tritium labelling
of peptides.
This research was supported by grants from the Scientific Council of
Ministry of Health of Hungary (ETT, No. 009/97) and from Cancer
and Smoking Related Diseases Research Program (LB595) of the State of
Nebraska, USA. The authors gratefully acknowledge the excellent technical
assistance of E. Menyharth and I. Nogradi.
EXPERIMENTAL
All reagents and solvents were of reagent grade and purchased from
commercial sources. Melting points were determined using a PAMK VEB
apparatus and are uncorrected. TLC was performed on silica gel precoated
glass plates 60 F254 (Merck). 1H NMR spectra were obtained using a Bruker
DRX 500 MHz spectrometer. Micoanalysis were made on a CHN Analyser
(Prague).
General Procedure for Tosylation
The N-a-protected-threonine esters (10–15 mmol, 65 mmol for
N-aFmoc-threonine benzyl ester) were dissolved in dry pyridine and
cooled to ꢀ 5ꢁC, then p-toluenesulfonyl chloride (1.5 equiv.) was added in
a small portions. The reaction mixtures were kept at ꢀ 5ꢁC, allowed to stand
overnight in fridge, then poured onto ice-water, acidified with 10% NaHSO4
solution. The precipitates were collected by filtration, washed with water to
neutral pH and dried.
N-a-Fmoc-dehydrothreonine Benzyl Ester (1)
Mp.: 137–138ꢁC from ethyl acetate/n-hexane; 95% yield; Rf : 0.48
(n-hexane/ethyl acetate, 3 : 2); 1H NMR (CDCl3), d ¼ 1.78 ppm (d, 3H, CH3),

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SOMLAI ET AL.
4.20 ppm (t, 1H, CH), 4.41 ppm (d, 2H, CH2), 5.20 ppm (s, 2H, CH2),
6.83 ppm (q, 1H, CH), 7.20–7.80 ppm (m, 13Haromatic); C26H23NO4
(413.45), calcd. C (75.53%), H (5.61%), N (3.39%), found C (75.35%), H
(5.49%), N (3.20%).
N-a-Fmoc-dehydrothreonine (2)
N-a-Fmoc-dehydrothreonine benzyl ester (1, 1 g, 2.43 mmol) was dis-
solved in 30 wt.% HBr solution in acetic acid (10 ml) and stirred for 1 h
at room temperature. The reaction mixture was concentrated in vacuo to
dryness, the residue was suspended in distilled water, filtered, washed with
water to neutral pH and dried, 600 mg (77%). The crude product was pur-
ified by column chromatography on silica gel (eluent CH2Cl2/MeOH, 95 : 5).
The appropriate fractions were combined, evaporated in vacuo to dryness.
The residue was triturated with diethyl ether to give the product as white
crystalline powder, 300 mg (39%), mp.: 220–222ꢁC, Rf : 0.30 (dichloro-
1
methane/methanol, 9 : 1); H NMR (CDCl3), d ¼ 1.69 ppm (d, 3H, CH3),
4.24–4.33 ppm (m, 3H, CH2 and CH), 6.56 ppm (q, 1H, CHb),
7.31–7.95 ppm (m, 8Haromatic); C19H17NO4 (323.33), calcd. C (69.04%),
H (5.30%), N (4.33%), found C (68.91%), H (5.13%), N (4.21%).
N-a-Fmoc-norvaline (10)
N-a-Fmoc-dehydrothreonine benzyl ester (1, 2.07 g, 5 mmol) was dis-
solved in dry tetrahydrofuran (60 ml) and hydrogenated over 10% Pd/char-
coal for 2 h. The catalyst was filtered and the filtrate was concentrated in
vacuo to dryness. The crude product (1.4 g, 88%) was recrystallized from
ethyl acetate and n-hexane solvent mixture, mp.: 161–163ꢁC, Rf : 0.22
(dichloromethane/methanol, 9 : 1); 1H NMR (DMSO), d ¼ 0.87 ppm (t,
3H, CH3), 1.63 and 1.75 ppm (m, 2H, CH2), 3.89 ppm (m, 1H, CHa),
4.21–4.29 ppm (m, 3H, CH2 and CH), 7.11–7.89 ppm (m, 8Haromatic);
C19H19NO4 (325.35), calcd. C (70.16%), H (5.88%), N (4.30%), found
C (69.85%), H (5.53%), N (4.19%).
N-a-Boc-O-tosyl-threonine Benzyl Ester (3)
Mp.: 128–130ꢁC from methanol/water; 70% yield; Rf : 0.55 (n-hexane/
ethyl acetate, 3 : 2); 1H NMR (CDCl3) d ¼ 1.33 ppm (d, 3H, CH3), 1.43 ppm
(s, 9H, CH3), 2.42 ppm (s, 3H, CH3), 4.46ppm (d, 1H, CH), 4.92 and

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5.12 ppm (d, 2H, CH2,benzyl), 5.19 ppm (m, 1H, CH), 5.20 ppm (d, 1H, NH),
7.20–7.70 ppm (m, 9Haromatic); C23H29NO7S (463.50), calcd. C (59.60%), H
(6.31%), N (3.02%), found C (59.37%), H (6.10%), N (2.85%).
N-a-Boc-O-tosyl-threonine Methyl Ester (4)
Oil; 80% yield; Rf : 0.45 (n-hexane/ethyl acetate, 3 : 2); 1H NMR
(CDCl3), d ¼ 1.36ppm (d, 3H, CH 3), 1.44 ppm (s, 9H, CH3), 2.44 ppm (s,
3H, CH3), 3.57 ppm (s, 3H, OCH3), 4.69 ppm (d, 1H, CH), 5.13 ppm (m, 1H,
CH), 5.22 ppm (d, 1H, NH), 7.34–7.80 ppm (m, 4Haromatic); C17H25NO7S
(387.50), calcd. C (52.69%), H (6.50%), N (3.62%), found C (52.40%),
H (6.31%), N (3.49%).
N-a-Z-O-tosyl-threonine Methyl Ester (7)
Mp.: 70–72ꢁC from methanol/water; 90% yield; Rf : 0.30 (n-hexane/
ethyl acetate, 3 : 2); 1H NMR (CDCl3), d ¼ 1.35 ppm (d, 3H, CH3), 2.43 ppm
(s, 3H, CH3), 3.59 ppm (s, 3H, OCH3), 4.48 ppm (d, 1H, CH), 5.11 ppm (s,
2H, CH2), 5.16ppm (m, 1H, CH), 5.46ppm (d, 1H, NH), 7.30–7.70 ppm (m,
9Haromatic); C20H23NO7S (421.50), calcd. C (56.99%), H (5.50%), N (3.32%),
found C (56.77%), H (5.45%), N (3.23%).
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Received in the UK October 26, 2000

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