The synthesis and reactivity of optically pure amino acids bearing side-chain thioamides

Article abstract of DOI:10.1039/b004688o

The synthesis and reactivity of fully protected thioamide analogues of asparagine and glutamine are described. A key feature of the synthetic strategies employed was the ability to perform selective thiations on multiple carbonyl-containing substrates. Al

Full text of DOI:10.1039/b004688o

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The synthesis and reactivity of optically pure amino acids bearing
side-chain thioamides
John M. Sanderson,a P. Singh,a Colin W. G. Fishwick*a and John B. C. Findlayb
1
a The University of Leeds, School of Chemistry, Leeds, UK LS2 9JT
b The University of Leeds, Research School of Biological Sciences, Leeds, UK LS2 9JT
Received (in Cambridge, UK) 13th June 2000, Accepted 4th July 2000
First published as an Advance Article on the web 14th September 2000
The synthesis and reactivity of fully protected thioamide analogues of asparagine and glutamine are described. A key
feature of the synthetic strategies employed was the ability to perform selective thiations on multiple carbonyl-
containing substrates. Also described are the preparations of thioamide derivatives of phenylalanine. The utility of
these amino acid derivatives for solid-phase peptide synthesis is discussed.
Introduction
One of the most important techniques for studying the inter-
action of small biologically active molecules with their recep-
tors is photoaffinity labelling.1 This methodology is particularly
important where high-resolution structures from X-ray or
NMR data are not available for the receptor, as is often the case
for large membrane proteins. During the course of our work
Fig. 1 Target amino acids incorporating a thiocarbonyl functional
group.
directed towards the synthesis of photoactivatable analogues of
peptides by solid-phase methods, it became apparent that all of
the photoactivatable moieties currently used are based around
carboxamide of the side chain was protected by a trityl group.
aromatic (phenylalanine) systems. Whilst this is acceptable for
This would have allowed direct access to a protected thioamide
peptides which naturally contain residues with aromatic side-
analogue of asparagine. Due to the acid lability of the trityl
chains, it is less than ideal for peptides which are highly polar in
group and the base lability of the Fmoc group, an orthogonal
nature, or contain no aromatic residues. We found it desirable
means of protecting the carboxy functionality of this derivative
therefore, to have access to a non-aromatic, more polar amino
prior to thiation was required. Thus reaction with TBDMS
acid that would be suitable as a photoaffinity tool and could
chloride furnished the pure silyl ester 5 in good yield following
be incorporated directly into peptides by solid-phase synthetic
recrystallisation. Reaction of ester 5 with phosphorus penta-
methods. We decided to explore the known photochemistry of
the thiocarbonyl group2 by synthesising thioamide derivatives
of asparagine 1 and glutamine 2 in a protected form that would
subsequently allow their incorporation into peptides by solid-
phase synthetic strategies. Additionally, we desired thioamide-
modified amino acids 3 and 4, analogues of phenylalanine, in
order to extend the range of photoactivatable groups available
for the preparation of peptide analogues (Fig. 1).
As part of our design strategy, protection of the primary
thioamide functionality present in 1 and 2 was considered
essential during peptide synthesis to avoid unwanted side-
reactions at the thioamide moiety, most notably acylation of
the sulfur atom during amino acid coupling. Our initial
protecting group of choice was the bulky, acid-labile trityl
group, by analogy with the side-chain protection strategy
employed for asparagine and glutamine. All of our synthetic
sulfide in THF under ultrasound irradiation, and in situ
strategies centred on selective thiations of enantiopure aspar-
deprotection of the silyl ester with wet tetra-n-butylammonium
agine or glutamine analogues, thereby avoiding the requirement
fluoride produced a material with spectroscopic characteristics
for an enantioselective step or resolution of a racemic mixture
consistent with the desired target compound 6. In spite of this,
in the synthetic route to the final compounds. In the case of
satisfactory elemental analyses and mass spectroscopic data
the aromatic thioamide analogues, N-thioacyl derivatives of
could not be obtained for this compound.3 Reaction of 5 with
(4-aminophenyl)alanine were prepared and used without
Lawesson’s reagent furnished phosphonothioate 7 as the sole
protection.
characterisable compound.
Attention was therefore directed towards an alternative
synthetic strategy to 1 and 2 based on Fmoc-protected aspar-
agine 8 and glutamine 9 respectively, with introduction of the
protecting group at the final stage of the syntheses (Scheme 1).
Clearly, 8 and 9 contain three different types of carbonyl group,
of which the amide function was required to undergo selective
Results and discussion
Preparation of thioamide analogues of asparagine and glutamine
Initial experiments centred on a commercially available Fmoc-
asparagine derivative for peptide synthesis, in which the
DOI: 10.1039/b004688o
J. Chem. Soc., Perkin Trans. 1, 2000, 3227–3231
This journal is © The Royal Society of Chemistry 2000
3227

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Scheme 2 Reagents and conditions: 9-hydroxyxanthene, acetic acid, ᭝,
60%.
found to be stable to all of the reagents encountered during
peptide synthesis and was smoothly deprotected to thioacet-
amide in 5 h using TFA.3
Preparation of thioamide analogues of phenylalanine
Phenylalanine derivatives 3 and 4 were prepared by manipu-
lation of the (S)-enantiomer of phenylalanine. All our attempts
to acylate the amino group of the Fmoc-protected 4-
aminophenyl substituted alanine 1610 in DMF at 50–60 ЊC
were unsuccessful due to in situ deprotection of the α-amino
group by the primary aromatic amine. This was supported
in one case by the isolation of (S)-2-acetylamino-3-(4-amino-
phenyl)propionic acid 17 following attempted acetylation using
Scheme 1 Reagents and conditions: i, ClCH2PhOMe, NaI, DMA, RT,
17 h, 93% (10), 93%) (11); ii, P4S10, THF, ultrasound, 88% (12), 88%
(13); iii, TFA, HS(CH2)2SH, PhSMe, 91% (1), 67% (2).
thiation. By first protecting the carboxy groups as their 4-
methoxybenzyl esters 10 and 11 however, using conditions
similar to those previously described4 for the attachment of
amino acids to resins as their 4-alkoxybenzyl esters, a highly
selective thiation could be achieved using phosphorus penta-
sulfide5 in THF under ultrasound irradiation in excellent
yield to give thioamides 12 and 13 respectively. Thiation of 10
with Lawesson’s reagent6 in toluene at 50 ЊC was less selective,
giving rise to unacceptable quantities of thiomaleimide 14
acetic anhydride. Reactions at lower temperatures yielded only
unreacted starting materials. As an alternative approach, we
considered thioacylation of the aromatic amino group using
dithioesters 1811b and 1911a as active thioacylating agents.
Model experiments clearly illustrated that 18 was capable of
thioacylating the amino group of 2-(4-aminophenyl)acetic acid
20 under mild conditions to produce the thioamide 21 in good
yield (Scheme 3). Repetition of this reaction with phenylalanine
(10%), which was absent from the phosphorus pentasulfide
thiation. Proof of the chemoselectivity of the thiations was
obtained via analysis using IR and 13C NMR spectroscopies. In
the IR spectra of 12 and 13, the carbonyl stretching vibrations
of the amide groups of 10 and 11 were clearly absent. The
13C NMR spectra of 13 showed a clear resonance for the thio-
carbonyl carbon at δ 209 ppm, with corresponding absence
of the amide carbonyl resonance (present at δ 159.9 ppm in
compound 11). Additional model thiation experiments with N-
Fmocbenzylamine illustrated that no reaction occurred with
the urethane carbonyl under these conditions. Deprotection of
the carboxy moiety was then readily achieved without alkyl-
ation of the thioamide by using TFA in the presence of thio-
anisole and ethane-1,2-dithiol as cation scavengers to yield
compounds 1 and 2. Previous experiments directed towards the
synthesis of thioasparagine analogues7 had illustrated the
susceptibility of the thioamide sulfur to alkylation by alkyl
cations generated during the strong acid cleavage of amine
protecting groups. These problems were not encountered by us.
As the final step of the syntheses, it was intended to
incorporate trityl protecting groups onto the thioamide nitro-
gens using trityl alcohol in the presence of sulfuric acid as
dehydrating agent, conditions previously described for the
N-alkylation of thioacetamide.8 Despite our best attempts
however, the N-alkylated product could not be isolated. We
therefore considered the xanthen-9-yl moiety9 as an alternative
protecting group, and in the case of asparagine, we were able
to incorporate this group successfully by heating 1 with two
equivalents of 9-hydroxyxanthene in acetic acid to produce 15
(Scheme 2). With thioglutamine 2, material giving suitable
elemental analysis could not be obtained. N-Xanthenyl thio-
acetamide9 was also prepared as a model compound, and was
Scheme 3 Reagents and conditions: 18, NaOH, H2O, 76%.
derivative 16 proceeded smoothly to the target compounds 3
and 4. The retention of the optical purity of these compounds
was illustrated by chiral TLC on the Fmoc-deprotected deriv-
atives, where no traces of the (R)-isomers were detectable.
Preparation of peptides
In order to demonstrate the applicability of 15 for peptide
synthesis, we synthesised the peptide analogue Arg-Lys-Ile-
Cys-Gly-Lys-Asn* (where Asn* indicates γ-thioasparagine 15),
corresponding to the seven C-terminal residues of mast cell
degranulating peptide (MCDP),12 using conventional Fmoc
solid-phase coupling strategies. Analysis of the synthesis data
indicated that derivative 15 was efficiently coupled onto the
peptide chain using N,N-diisopropylcarbodiimide as the car-
boxy activating agent. All other couplings using both penta-
fluorophenyl esters and diimides, and all Fmoc deprotections,
occurred smoothly. Deprotection of the resin following syn-
thesis was performed using TFA in the presence of scavengers,
3228
J. Chem. Soc., Perkin Trans. 1, 2000, 3227–3231

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and the crude peptide was analysed by MALDI-MS. This
produced no ions corresponding to the desired peptide (m/z
833.1), but peaks of m/z 886.0 1.0, 818.8 0.9 and
704.1 0.9. A similar deprotection was performed with a
modified version of the cleavage reagent used above containing
an excess of thioacetamide as an additional scavenger. In this
case, a similar ion distribution was observed, with the exception
that the 2,2,5,7,8-pentamethylchroman-6-ylsulfonyl (Pmc) pro-
tecting group of arginine remained intact. Consistent with these
observations, we were able to propose a mechanism whereby the
thioamide moiety of thioasparagine reacts in an intramolecular
fashion with tert-butyl cations derived from the neighbouring
lysine during the cleavage reaction. The resulting S-tert-
butyliminothiolate is then either partially hydrolysed under
these conditions to the corresponding aspartate (m/z 818.8),
or rearranges to the more stable N-tert-butylthioamide
(m/z 889.5). Interestingly, the peak of m/z 704.1, lacking the
C-terminal thioasparagine residue, is best accounted for by
intramolecular reaction of the thioamide with the peptide
backbone, in a similar fashion to the process occurring during
Edman degradation13 (Scheme 4). Production of this peptide by
phase techniques, it was not possible to incoporate
a
thioasparagine-based system into peptides due to degradation
during the solid-phase synthesis. This work does, however, give
access to a new class of peptide analogue with potential as
photochemical probes.
Experimental
General
Ultrasound experiments were carried out in a Kerry PUL55
ultrasonic bath with a power input of 55 W. Chiral TLC was
performed using chiral TLC plates purchased from Aldrich,
with methanol–water–acetonitrile (1:1:4) as the mobile phase
and -phenylalanine as reference. Spot detection was per-
formed using ninhydrin. Melting points were recorded on a
Reichert hot stage and are uncorrected. All infra-red spectra
were recorded as Nujol mulls unless otherwise stated. 1H
NMR spectra were recorded with TMS as internal standard
unless otherwise stated. Coupling constants are in Hz. The
solvent signal was used as internal standard for 13C NMR
experiments.
Peptide synthesis and analysis
Peptide synthesis was performed on a Milligen 9050 peptide
synthesiser using the ‘Express’ software application. Standard
Fmoc-compatible coupling and deprotection cycles were
performed throughout. The side-chain functional groups of
arginine, lysine and cysteine were protected as their 2,2,5,
7,8-pentamethylchroman-6-ylsulfonyl (Pmc), Boc and trityl
derivatives, respectively. Capping of the resin following coup-
ling of compound 15 was carried out using N-acetylimidazole.
Peptide chain assembly was performed on Novasyn KR 100
amide resin (0.1 meq gϪ1), with final cleavage performed on 50
mg of resin using a mixture of TFA (7 ml), ethane-1,2-dithiol
(0.5 ml), thioanisole (0.75 ml), triisopropylsilane (0.5 ml),
phenol (0.5 g) and water (0.5 ml) for 4 to 5 h. A modified
mixture was also used containing thioacetamide (0.15 g, 2
mmol). The crude peptides were precipitated with diethyl ether
(15 ml) and analysed directly by MALDI-MS. Mass spectra of
peptides were determined on a VG Organic TofSpec MALDI
mass spectrometer using Opus 3.1 software. The matrix used
was α-cyano-4-hydroxycinnamic acid, with adrenocorticotropic
hormone (ACTH, fragment 1–13, m/z 1623.8) as external
standard. Desorption was performed using a pulsed nitrogen
laser at 337 nm.
Scheme 4 Proposed mechanism for cleavage of the peptide backbone
by the thioamide.
(S)-Nꢀ-(Fluoren-9-ylmethoxycarbonyl)-Nꢁ-tritylasparagine
tert-butyldimethylsilyl ester (5)
a failure of the coupling reaction during peptide synthesis was
ruled out by the feedback data from the synthesiser, and the
presence of a capping step before coupling of the second amino
acid.
To a solution of Nα-(fluoren-9-ylmethoxycarbonyl)-Nγ-trityl-
asparagine (1.00 g, 1.68 mmol), N,N-diisopropylethylamine
(320 µl, 1.84 mmol) and 4-dimethylaminopyridine (0.02 g, 0.2
mmol) under argon in dichloromethane (7.5 ml) at 0 ЊC, was
added tert-butyldimethylsilyl chloride (0.278 g, 1.84 mmol)
portion-wise with stirring. Following the addition, the mixture
was stirred at 0 ЊC for 15 min and then at room temperature for
70 min before being diluted with dichloromethane (10 ml) and
washed with water (2 × 10 ml). Following drying of the organic
layer (MgSO4) and removal of the drying agent by filtration, the
solvent was removed in vacuo to give the crude product as a
colourless solid. Recrystallisation from chloroform–pentane
gave the title compound 5 (1.13 g, 95%) as a colourless solid,
mp 108–112 ЊC; [α]D30 ϩ12 (c 0.6 in chloroform) (Found: C, 74.4;
H, 6.55; N, 4.2. C44H46N2O5Si requires: C, 74.3; H, 6.52; N,
3.9%); νmax/cmϪ1 3640, 3430, 2930, 1715s, 1490s, 1210s, 835;
δH(300 MHz, CDCl3) 0.21 and 0.24 (6 H, s, Si-CH3), 0.89 (9 H,
s, Si-C(CH3)3), 2.84 (1 H, dd, J 11.0 and 2.8, CHCHHC(O)-
NH(Trt)), 3.09 (1 H, dd, J 11.0 and 2.8, CHCHHC(O)-
NH(Trt)), 4.15–4.21 (1 H, m, -CH2CH Fmoc), 4.26–4.58 (2 H,
m, -CH2CH Fmoc), 4.54 (1 H, m, CHCH2C(O)NH(Trt)), 6.15
For the aromatic-based thioamide systems 3 and 4, the
applicability of these in solid-phase peptide synthesis was
demonstrated by their efficient incorporation into the neuro-
kinin A (NKA) peptide sequence to yield photolabile analogues
of NKA having the sequence His-Phe*-Lys-Thr-Asp-Ser-
Phe-Val-Gly-Leu-Met-NH2 (where Phe* corresponds to the
position of incorporation of 3 and 4 respectively).14 The NKA
system is a naturally occurring hormone and has been impli-
cated in a large number of important biological processes.15 The
coupling efficiency of both of the amino acids 3 and 4 was in
excess of 97%. Following purification using reverse-phase
HPLC, the purified peptides were characterised using mass
spectrometry and were both found to have the correct molec-
ular weights of 1415.1 and 1455.7 respectively.14
In conclusion, we have developed efficient methods for the
preparation of thiated amino acids based on both aromatic and
non-aromatic systems. Although the aromatic-based systems
are efficiently incorporated into peptides using standard solid-
J. Chem. Soc., Perkin Trans. 1, 2000, 3227–3231
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(1 H, d, J 8.8, NH Fmoc), 6.66 (1 H, s, C(O)NH(Trt)),
7.14–7.32 (17 H, m, Ar), 7.33–7.43 (2 H, m, Ar), 7.59 (2 H, d, J
7.8, Ar), 7.76 (2 H, d, J 8.1, Ar); m/z (EI) 710 (Mϩ, 1.5%), 532
(5), 243 (100).
(S)-Nꢀ-(Fluoren-9-ylmethoxycarbonyl)-ꢁ-thioasparagine
4-methoxybenzyl ester (12)
A mixture of amide 10 (0.20 g, 0.4 mmol) and phosphorus
pentasulfide (0.08 g, 0.2 mmol) in tetrahydrofuran (10 ml) was
irradiated with ultrasound under an argon atmosphere for 1 h,
care being taken to maintain the temperature at ≤40 ЊC. Follow-
ing concentration to a volume of approximately 2 ml in vacuo,
the mixture was filtered to remove any precipitate. The filtrant
was washed with small portions of hot chloroform, which were
combined with the tetrahydrofuran filtrate and purified directly
by flash column chromatography (CH2Cl2–pentane–EtOAc;
3:3:2) to give the title compound (0.18 g, 88%) as a colourless
solid, mp 136–137 ЊC; [α]D30 Ϫ7 (c 0.3 in chloroform) (Found: C,
66.05; H, 5.4; N, 5.7; S, 6.6. C27H26N2O5S requires: C, 66.11; H,
Bis(tert-butyldimethylsilyl) 4-methoxyphenylthiophosphonate (7)
A solution of ester 5 (0.62 g, 0.88 mmol) and Lawesson’s
reagent (0.21 g, 0.52 mmol) in toluene (4 ml) was stirred under
argon at 45 ЊC for 17 h before the solvent was removed in vacuo.
Purification by flash column chromatography (CH2Cl2–hexane;
1:1) gave the title compound 7 (0.33 g, 87%) as a yellow oil
(Found: C, 52.75; H, 8.7; S, 7.8. C19H37O3PSSi2 requires: C,
52.74; H, 8.6; S, 7.4%); νmax(film)/cmϪ1 2920s, 1590, 1470, 1250s,
1110s, 825s, 715; δH(300 MHz, CDCl3) 0.10 and 0.31 (12 H, s, Si-
(CH3)2), 0.91 (18 H, s, SiC(CH3)3), 3.86 (3 H, s, CH3OPh), 6.93
(2 H, dd, J 8.3 and 1.8, Ar), 7.85 (2 H, dd, J 8.3 and 13.9, Ar);
m/z (EI) 432 (Mϩ, 2%), 417 (5), 375 (100), 319 (18), 73 (25).
5.3; N, 5.7; S, 6.5%); νmax/cmϪ1 3330, 3200, 1730s (C᎐O ester),
᎐
1690s (C᎐O Fmoc), 1610, 1510, 750, 730; δ (300 MHz, CDCl )
᎐
H
3
3.20–3.23 (2 H, m, CHCH2C(S)NH2), 3.76 (3 H, s, CH3OPh),
4.17 (1 H, t, J 6.7, CH Fmoc), 4.37 (2 H, d, J 6.5, CH2 Fmoc),
4.65–4.73 (1 H, m, CHCH2C(S)NH2), 5.12 (2 H, s, CH3OPh-
CH2), 6.13 (1 H, d, J 7.0, NH Fmoc), 6.83 (2 H, d, J 8.5, Ar),
7.26–7.32 (4 H, m, Ar), 7.40 (2 H, t, J 7.3, Ar), 7.57 (2 H, d,
J 7.3, Ar), 7.76 (2 H, d, J 7.4, Ar); m/z (EI) 490 (Mϩ, 0.03%),
352 (0.3), 178 (100), 165 (28), 121 (30).
(S)-Nꢀ-(Fluoren-9-ylmethoxycarbonyl)asparagine 4-methoxy-
benzyl ester (10)
A solution of Nα-(fluoren-9-ylmethoxycarbonyl)asparagine 8
(4.90 g, 13.8 mmol), 4-methoxybenzyl chloride (2.80 ml, 16.4
mmol), sodium iodide (3.11 g, 20.1 mmol) and N,N-diiso-
propylethylamine (5.0 ml, 28 mmol) in N,N-dimethylacetamide
(40 ml) was stirred under argon at room temperature for 17 h
before being diluted with chloroform (500 ml) and washed with
water (2 × 500 ml). Following drying of the organic solution
(MgSO4), the drying agent was removed by filtration and the
solvent removed in vacuo to give the crude product as a yellow
oil. This oil was triturated with diethyl ether (100 ml) and left
for 1 h, producing a colourless precipitate. The solid was col-
lected by filtration, washed with diethyl ether (2 × 100 ml) and
recrystallised from chloroform–hexane to give the title com-
pound (6.10 g, 93%) as a colourless solid, mp 172–174 ЊC; [α]D30
Ϫ11 (c 0.2 in N,N-dimethylformamide) (Found: C, 68.05; H,
5.25; N, 6.0. C27H26N2O6 requires: C, 68.34; H, 5.52; N, 5.9%);
νmax/cmϪ1 3400, 3300s, 3200, 1740s (C᎐O ester), 1685s (C᎐O
(S)-Nꢀ-(Fluoren-9-ylmethoxycarbonyl)-ꢂ-thioglutamine
4-methoxybenzyl ester (13)
This was prepared from ester 11 (1.0 g, 2.05 mmol) using the
method described above for the preparation of thioamide 12,
yielding the title compound (0.86 g, 88%) as a colourless solid,
mp 134–136 ЊC (Found: C, 66.6; H, 5.5; N, 5.5; S, 6.4.
C28H28N2O5S requires: C, 66.7; H, 5.6; N, 5.6; S, 6.4%); νmax
/
cmϪ1 3330, 3200, 1730s (C᎐O ester), 1690s (C᎐O Fmoc), 1610,
᎐
᎐
1510, 750, 730; δH(300 MHz, CDCl3) 2.05–2.01 (1 H, m,
CHC(H)HCH2C(O)NH2), 2.39–2.35 (1 H, m, CHC(H)HCH2-
C(O)NH2), 2.60–2.57 (1 H, m, CHCH2C(H)HC(O)NH2), 2.67–
2.63 (1 H, m, CHCH2C(H)HC(O)NH2), 3.79 (3 H, s, CH3OPh),
4.19 (1 H, m, CH Fmoc), 4.47 (3 H, m, CHCH2CH2C(O)NH2
and CH2 Fmoc), 5.12 (2 H, s, CH3OPhCH2), 5.59 (1 H, d, J 7.5,
NH Fmoc), 6.87 (2 H, d, J 8.5, Ar), 7.50–7.25 (6 H, m, Ar), 7.57
(3 H, m, NH2 and Ar), 7.76 (2 H, d, J 7.5, Ar), 7.94 (1 H, m,
NH2); δC(100 MHz, CDCl3) 32.9, 40.7, 47.1, 52.9, 55.3, 67.1,
67.5, 114.0, 120.0, 124.9, 127.05, 127.1, 127.8, 130.3, 141.2,
143.7, 146.6, 159.8 (C᎐O ester), (C᎐O Fmoc), 171.5, 209.0
᎐
᎐
Fmoc), 1645s (C᎐O amide), 1505, 730; δ (300 MHz, CDCl )
᎐
H
3
2.77 (1 H, dd, J 4.1 and 15.4, CHCH2C(O)NH2) and 2.97 (1 H,
dd, J 4.1 and 15.5, CHCH2C(O)NH2), 3.77 (3 H, s, CH3Ph),
4.19–4.22 (1 H, m), 4.28–4.34 (1 H, m), 4.38–4.42 (1 H, m),
4.44–4.63 (1 H, m, CHCH2C(O)NH2), 5.14 (2 H, s, CH3OPh-
CH2), 5.37 and 5.51 (2 H, br s, CONH2), 6.07 (1 H, d, J 7.9, NH
Fmoc), 6.84 (2 H, d, J 7.6, Ar), 7.26–7.32 (4 H, m, Ar), 7.40
(2 H, t, J 7.5, Ar), 7.58 (2 H, d, J 7.1 Ar), 7.76 (2 H, d, J 7.4 Ar);
m/z (EI) 473 (Mϩ Ϫ H, 0.02%), 217 (2), 196 (2), 178 (100),
165 (19), 121 (37).
᎐
᎐
(C᎐S); m/z (FAB): 504 (Mϩ, 0.03%), 383 (1), 326 (2), 265 (21),
᎐
178 (66), 121 (100).
(S)-Nꢀ-(Fluoren-9-ylmethoxycarbonyl)-ꢁ-thioasparagine (1)
Thioamide 12 (0.158 g, 0.32 mmol) was treated with a mixture
of thioanisole (0.1 ml) and ethane-1,2-dithiol (0.1 ml) in tri-
fluoroacetic acid (1 ml) for 10 min at room temperature. Follow-
ing the addition of a diethyl ether–pentane mixture (1:1; 15
ml), the mixture was left to stand for 3.5 h at 0 ЊC and then
centrifuged at 3000 rpm for 5 min. After removal of the super-
natant by decantation, the remaining colourless precipitate was
washed with ice-cold diethyl ether (2 × 2 ml) and recrystallised
from ethyl acetate–petrol (bp 80–100 ЊC) to give the title com-
pound (0.108 g, 91%) as a colourless solid, mp 156–159 ЊC; [α]D30
Ϫ25 (c 0.1 in ethanol) (Found: C, 61.35; H, 4.7; N, 7.3.
C19H18N2O4S requires: C, 61.61; H, 4.9; N, 7.6%); λmax (EtOH)/
nm 318 (log10ε 1.86), 299 (3.77), 286 (3.87), 264 (4.42); νmax/cmϪ1
3300, 3140, 1690s, 1525s, 1260s, 1055, 755, 740s; δH(300 MHz,
(CD3)2SO) 2.79 (1 H, dd, J 5.0 and 14.0, CHC(H)HC(S)NH2),
2.90 (1 H, dd, J 5.0 and 14.2, CHC(H)HC(S)NH2), 4.21–4.27
(3 H, m, CH and CH2 Fmoc), 4.63–4.65 (1 H, m, CHCH2C-
(S)NH2), 7.32 (2 H, t, J 6.8, Ar), 7.41 (2 H, t, J 7.3, Ar), 7.62
(1 H, d, J 8.1, NH Fmoc), 7.69 (2 H, d, J 7.0, Ar), 7.88 (2 H,
d, J 7.5, Ar), 9.16 and 9.52 (2 H, br s, NH2); m/z (EI)
371 (Mϩ ϩ H, 50%), 332 (4), 191 (9), 179 (100), 121 (30).
(S)-Nꢀ-(Fluoren-9-ylmethoxycarbonyl)glutamine 4-methoxy-
benzyl ester (11)
This was prepared from Nα-(fluoren-9-ylmethoxycarbonyl)-
glutamine 9 (5.0 g, 13.6 mmol) using the procedure outlined for
the preparation of ester 10, to give the title compound (6.10 g,
93%) as a colourless solid, mp 172–174 ЊC (Found: C, 68.6; H,
5.7; N, 5.8. C28H28N2O6 requires: C, 68.8; H, 5.8; N, 5.7%); νmax
/
cmϪ1 3490, 3420, 3200, 1790s (C᎐O ester), 1685s (C᎐O Fmoc),
᎐
᎐
1645s (C᎐O amide), 1505, 1170, 750, 730; δ (400 MHz, CDCl )
᎐
H
3
2.01 (1 H, m, CHC(H)CH2C(O)NH2), 2.20 (3 H, m, CHC(H)-
HCH2C(O)NH2), 3.78 (3 H, s, CH3OPh), 4.20 (1 H, m, CH
Fmoc), 4.40 (3 H, m, CHCH2CH2C(O)NH2 and CH2 Fmoc),
5.11 (2 H, s, CH3OPhCH2), 5.66 (1 H, d, J 7.5, NH-Fmoc), 5.70
(1 H, m, NH2), 6.86 (2 H, d, J 8.5, Ar), 7.32–7.26 (4 H, m, Ar),
7.48 (2 H, t, J 7.5, Ar), 7.57 (2 H, d, J 7.5, Ar), 7.76 (2 H, d,
J 7.5, Ar); δC(100 MHz, CDCl3) 28.4, 31.6, 47.2, 53.4, 55.3,
67.0, 67.2, 114.0, 119.9, 125.0, 127.0, 127.3, 127.7, 130.2, 141.3,
143.6, 143.8, 156.7 (C᎐O ester), 159.9 (C᎐O amide), 171.7 (C᎐O
᎐
᎐
᎐
urethane); m/z (FAB) 488 (Mϩ Ϫ H, 0.02%), 350 (1), 275 (2),
196 (100), 178 (96), 165 (40), 121 (65).
3230
J. Chem. Soc., Perkin Trans. 1, 2000, 3227–3231

View Article Online
(S)-Nꢀ-(Fluoren-9-ylmethoxycarbonyl)-ꢂ-thioglutamine (2)
(S)-2-(Fluoren-9-ylmethoxycarbonylamino)-3-(4-thiobenzoyl-
aminophenyl)propionic acid (3)
This was prepared from ester 13 (1.20 g, 2.4 mmol) using the
method described above for the preparation of thioamide 1
to give the title compound (0.89 g, 67%) as a colourless,
amorphous solid, mp 152–154 ЊC (Found: C, 62.5; H, 5.4; N,
7.3. C20H20N2O4S requires: C, 62.5; H, 5.2; N, 7.3%); νmax/cmϪ1
This was prepared from S-carboxymethyl dithiobenzoate
1811b (0.052 g, 0.25 mmol) and 3-(4-aminophenyl)-2-(fluoren-
9-ylmethoxycarbonylamino)propionic acid 1610 (0.1 g, 0.25
mmol) using the procedure described above for the preparation
of thioamide 21 to give the title compound 0.86 g (74%) as
a bright yellow solid, mp 145–146 ЊC. Full physical data have
been previously described.14
3300, 3140, 1690s (C᎐O Fmoc), 1525s, 1325s, 1260s, 1055, 755,
᎐
740s; δH(300 MHz, (CDCl3) 2.10–2.02 (1 H, m, CHC(H)-
HCH2C(O)NH2), 2.41–2.36 (1 H, m, CHC(H)HCH2C(O)-
NH2), 2.61–2.57 (1 H, m, CHCH2C(H)HC(O)NH2), 2.69–2.65
(1 H, m, CHCH2C(H)HC(O)NH2), 4.19 (1 H, m, CH-Fmoc),
4.71–4.3 (3 H, m, CHCH2CH2(O)NH2 and CH2 Fmoc), 7.76–
7.25 (8H, m, Ar-Fmoc); m/z (FAB) 384 (Mϩ, 14%), 352 (14),
298 (21), 178 (100), 166 (90), 121 (82).
(S)-2-(Fluoren-9-ylmethoxycarbonylamino)-3-[4-(2-phenylthio-
acetylamino)phenyl]propionic acid (4)
This was prepared from S-carboxymethyl phenyldithioacetate
1911a (0.056 g, 0.25 mmol) and 3-(4-aminophenyl)-2-(fluoren-
9-ylmethoxycarbonylamino)propionic acid 1610 (0.1 g, 0.25
mmol) using the procedure described above for the preparation
of thioamide 21 to give the title compound 0.86 g (74%) as
a light yellow solid, mp 193 ЊC. Full physical data have been
previously described.14
(S)-Nꢀ-(Fluoren-9-ylmethoxycarbonyl)-Nꢁ-xanthen-9-yl-ꢁ-
thioasparagine (15)
A solution of thioamide 1 (0.72 g, 1.9 mmol) and 9-
hydroxyxanthene (0.77 g, 3.9 mmol) in acetic acid (25 ml) was
heated under argon at 85 ЊC for 110 min. Following removal
of the solvent in vacuo, the residue was triturated with
diethyl ether (45 ml) and left for 30 min at room temperature,
producing the crude product as a colourless precipitate.
Following removal of the diethyl ether by decantation, the
residue was washed with diethyl ether (3 × 20 ml) and re-
crystallised from chloroform to give the title compound
(0.63 g, 60%) as a colourless solid, mp 157–159 ЊC; [α]D30 Ϫ5
(c 0.3 in N,N-dimethylformamide) (Found: C, 70.04; H, 5.01;
Acknowledgements
The authors would like to thank the Medical Research Council
and Wyeth Research (UK) for a studentship to J. M. S.
References
1 H. Bayley, Photogenerated Reagents in Biochemistry and Molecular
Biology, in Laboratory Techniques in Biochemistry and Molecular
Biology, vol. 12, eds. T. S. Work and R. H. Burdon, Elsevier, 1983;
G. Dorman and G. D. Prestwich, Trends Biotechnol., 2000, 18, 64;
A. R. Safa, Methods Enzymol., 1998, 292, 289; Y. Hatanaka and
Y. Kanaoka, Heterocycles, 1998, 47, 625.
2 J. D. Coyle, Tetrahedron, 1985, 41, 5393; S. B. Renwick, A. D.
Critchley, C. J. Adams, S. M. Kelly, N. C. Price and P. G. Stockley,
Biochem. J., 1995, 308, 447; A. Favre, C. Saintome, J. L. Fourrey,
P. Clivio and P. Laugaa, J. Photochem. Photobiol., B, 1998, 42, 109.
3 J. M. Sanderson, PhD Thesis, University of Leeds, 1996.
4 M. Mergler, R. Nyfeler, J. Gosteli and R. Tanner, Tetrahedron Lett.,
1989, 30, 447.
5 R. N. Hurd and G. de la Mater, Chem. Rev., 1961, 61, 45.
6 M. P. Cava and M. I. Levinson, Tetrahedron, 1985, 41, 5061.
7 C. Ressler and S. N. Banerjee, J. Org. Chem., 1976, 41, 1336.
8 R. N. Hurd, G. de la Mater and J. P. McDermott, J. Org. Chem.,
1962, 27, 269.
9 W. Walter and J. Krohn, Chem. Ber., 1969, 102, 3786; W. Walter and
J. Voss, The Chemistry of Thioamides, in The Chemistry of Amides,
ed. J. Zabicky, series ed. S. Patai, John Wiley and Sons, London,
1970.
N, 4.8. C32H26N2O5S requires: C, 69.80; H, 4.76; N, 5.1%); νmax
/
cmϪ1 3360, 3310, 1730s, 1690s, 1450s, 1260s, 1140, 755, 745;
δH(300 MHz, (CD3)2SO) 2.90–2.99 (1 H, m, CHC(H)HC-
(O)NH), 3.12–3.21 (1 H, m, CHC(H)HC(O)NH), 4.02–4.08
(1 H, m, CH Fmoc), 4.16–4.25 (2 H, m, CH2 Fmoc), 4.73–4.76
(1 H, m, CHCH2C(O)NH), 7.00–7.41 (13 H, m, Ar, NH Fmoc
and NH amide), 7.56 (2 H, d, J 8.1, Ar), 7.70 (2 H, d, J 7.3, Ar),
7.88 (2 H, d, J 7.3, Ar), 11.23 (1 H, br, CO2H); m/z (EI) 550
(Mϩ, 0.04%), 334 (4), 309 (4), 181 (100), 165 (26).
2-(4-Thiobenzoylaminophenyl)acetic acid (21)
S-Carboxymethyl dithiobenzoate 1811b (2.12 g, 1.0 mmol) was
added to a solution of 2-(4-aminophenyl)acetic acid (1.5 g, 1.0
mmol) in aqueous sodium hydroxide (1 M, 5 ml) and the
mixture stirred at room temperature for 6 h. The mixture was
then acidified to pH 3 by the careful addition of concentrated
hydrochloric acid, and extracted with ethyl acetate (2 × 20 ml).
The combined organic extracts were dried (MgSO4), filtered,
and concentrated in vacuo. Purification of the crude product by
flash column chromatography (CHCl3–EtOAc–EtOH; 6:2:2)
gave the title compound (1.85 g, 70%) as a yellow solid, mp
171–172 ЊC (Found: C, 66.3; H, 4.7; N, 5.2; S, 11.9. C15H13NO2S
requires: C, 66.4; H, 4.8; N, 5.2; S, 11.8%); λmax (MeOH)/nm 320
(ε/dm3 molϪ1 cmϪ1 121), 290 (95); νmax/cmϪ1 3200–2800, 1700s
10 J. S. Davies and K. A. Mohammed, J. Chem. Soc., Perkin Trans. 2,
1984, 1723.
11 (a) A. Kjaer, Acta Chem. Scand., 1950, 4, 1347; (b) A. Kjaer, Acta
Chem. Scand., 1952, 6, 327.
12 E. M. Dotimas, K. R. Hamid, R. C. Hider and U. Ragnarsson,
Biochim. Biophys. Acta, 1987, 911, 285; M. Reza Ziai, S. Russek,
H.-C. Wang, B. Beer and A. J. Blume, J. Pharm. Pharmacol., 1990,
42, 457.
(C᎐O), 1520, 860s; δ (400 MHz, (CD ) SO) 3.72 (2 H, s, CH ),
᎐
H
3
2
2
13 J. Kyte, Structure in Protein Chemistry, Garland Publishing,
London, 1995.
14 P. Singh, C. R. Hurrell, J. B. C. Findlay and C. W. G. Fishwick,
Bioorg. Med. Chem. Lett., 1997, 7, 715.
15 J. M. Hall, The Tachykinin Receptors, ed. S. H. Buck, Humana Press,
Totowa, New Jersey, pp. 515–580.
7.44 (2 H, d, J 8.5, Ar), 7.59 (2 H, t, J 7.0, Ar), 7.64 (1 H, m,
Ar), 7.88 (2 H, d, J 8.5, Ar), 7.95 (2 H, d, J 7.0, Ar), 11.82 (1 H,
br s, NH); δC(100 MHz, (CD3)2SO) 44.26, 127.9, 131.3, 131.9,
133.4, 135.8, 136.9, 142.5, 146.6, 174.5 (C᎐O), 201.4 (C᎐S);
᎐
᎐
m/z 271 (Mϩ, 48%), 238 (30), 224 (27), 168 (8), 121 (100).
J. Chem. Soc., Perkin Trans. 1, 2000, 3227–3231
3231