Introduction of phosphine-gold(I) precursors into a Cys-modified enkephalin neuropeptide as part of solid phase peptide synthesis

Article abstract of DOI:10.1515/znb-2007-0321

The synthesis and full characterization of a series of (triphenylphosphine) gold complexes with thiol-containing amino acids and peptides is reported. Boc-Cys-Au(PPh₃) was prepared in solution by reaction of Boc-Cys-OH with (Ph₃P)AuCl. The related Ac-CyS-Au(PPh₃) and an Au derivative of the cysteine-modified neuropeptide enkephalin (Enk), [Cys] ⁵-Au(PPh₃)-Enk, were prepared on the resin, using the orthogonal trityl-protecting group on Cys. For comparison, the metal-free [Cys]⁵-Enk and an S-tert-butyl-protected [Cys]⁵-Enk were also prepared. Most noteworthy, gold complexation works best when carried out on the resin, and the gold thiolate survives cleavage from the resin under optimized conditions. The new conjugates were comprehensively characterized, including ID and 2D NMR spectroscopy, and mass spectrometry. Along with characteristic changes in the ¹H and ¹³C NMR spectra, ³¹P NMR spectra reveal a characteristic downfield shift of ca. 5 ppm upon complexation of the Au(PPh₃)-fragment to Cys, which is also pertinent in the Au-labelled [Cys]⁵-Enk.

Full text of DOI:10.1515/znb-2007-0321

Introduction of Phosphine-Gold(I) Precursors into a Cys-modified
Enkephalin Neuropeptide as Part of Solid Phase Peptide Synthesis
Judy Caddya, Ulrich Hoffmannsb, and Nils Metzler-Nolteb,c
a Project AuTEK, Mintek, Private Bag X 3015, Randburg 2125, South Africa
b Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg,
Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
c New address: Department of Chemistry and Biochemistry, Ruhr-Universita¨t Bochum,
Universita¨tsstraße 150, 44801 Bochum, Germany
Reprint requests to Prof. Dr. N. Metzler-Nolte. E-mail: Nils.Metzler-Nolte@ruhr-uni-bochum.de
Z. Naturforsch. 2007, 62b, 460 – 466; received August 30, 2006
Dedicated to Prof. Helgard G. Raubenheimer on the occasion of his 65 th birthday
The synthesis and full characterization of a series of (triphenylphosphine)gold complexes with
thiol-containing amino acids and peptides is reported. Boc-Cys-Au(PPh3) was prepared in solution
by reaction of Boc-Cys-OH with (Ph3P)AuCl. The related Ac-Cys-Au(PPh3) and an Au derivative of
the cysteine-modified neuropeptide enkephalin (Enk), [Cys]5-Au(PPh3)-Enk, were prepared on the
resin, using the orthogonal trityl-protecting group on Cys. For comparison, the metal-free [Cys]5-Enk
and an S-tert-butyl-protected [Cys]5-Enk were also prepared. Most noteworthy, gold complexation
works best when carried out on the resin, and the gold thiolate survives cleavage from the resin un-
der optimized conditions. The new conjugates were comprehensively characterized, including 1D
and 2D NMR spectroscopy, and mass spectrometry. Along with characteristic changes in the 1H and
13C NMR spectra, 31P NMR spectra reveal a characteristic downfield shift of ca. 5 ppm upon com-
plexation of the Au(PPh3)-fragment to Cys, which is also pertinent in the Au-labelled [Cys]5-Enk.
Key words: Enkephalin, Gold Compounds, Medicinal Inorganic Chemistry, Peptides
Introduction
nofin’s success, many other gold derivatives of carbo-
hydrates were prepared and tested for activity [2, 3].
In relation, far less gold derivatives of amino acids
were reported. Ligand exchange reactions of gold-
phosphine [4, 5] and gold-cyano [6, 7] complexes with
biologically relevant thiols, in particular the amino acid
cysteine and the tripeptide glutathione, were investi-
gated. Glutathion in particular is an abundant reductant
in cells and is thus an obvious target for gold(I) com-
pounds, as are the thiol groups of cysteine in proteins.
The X-ray crystal structure of a gold(I) complex of hu-
man glutathione reductase (hGR) has recently been re-
ported and gives interesting insights into the biologi-
cal function of gold complexes [8]. Earlier on, the first
crystallographic investigation of a gold protein com-
plex revealed an unexpected binding of the Au(PEt3)
fragment to the Nε of a histidine residue in the pro-
tein cyclophilin-3 [9]. Decoration of gold nanoparti-
cles with other peptides has also been reported recently
[10 – 12]. However, to the best of our knowledge, no
Gold complexation to a range of biologically rel-
evant organic molecules is an active field of re-
search. Well known examples include the anti-arthritis
drugs gold-thiomalate and gold-thioglucose [1]. All
these first-generation drugs have polymeric structures
in the solid state and only low aqueous solubility,
thus limiting their oral applicability. These problems
are overcome with the second-generation drug Aura-
nofin, which is a well-defined molecular compound.
In Auranofin, a triethylphosphine-gold(I) fragment is
complexed to acetylated thioglucose. Following Aura-
Abbreviations: Bocb – tert-butoxycarbonyl; Fmoc – 9-fluor-
enylmethoxycarbonyl; WANG – p-hydroxybenzyl alcohol;
TBTU
–
2-(1H-benzotriazole-1-yl-)-1,1,3,3-tetramethyl-
uronium tetrafluoroborate; HOBt – hydroxybenzotriazole;
DIPEA – diisopropyl(ethyl)amine; DCM – dichloromethane;
TIS – triisopropylsilane; TFA – trifluoroacetic acid; HPLC –
high-performance liquid chromatography.
0932–0776 / 07 / 0300–0460 $ 06.00 © 2007 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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Scheme 1. Synthesis of Boc-Cys-
Au(PPh3) 3 in solution.
Scheme 2. Synthesis of Ac-Cys-Au(PPh3) 4 by solid phase synthesis. See Scheme 3 for reaction numbering order and ex-
perimental section for details.
fully characterized 1:1 adduct of biologically relevant Results and Discussion
peptides to a molecular gold complex fragment has
been reported to the present day.
Our studies were initiated using (triphenylphos-
phine)gold chloride 1 and Boc-cysteine 2, in order to
confirm the feasibility of the planned chemistry. The
desired Boc-cysteine-gold triphenylphosphine com-
plex 3 was obtained in fair yield according to methods
A and B, respectively (Scheme 1).
The main source for small peptides is solid phase
peptide synthesis (SPPS, also called Merrifield syn-
thesis), in which the peptide is built from suitable
amino acid building blocks in a successive manner
[13, 14]. The challenge of introducing metal com-
plexes during SPPS is that the complexation reaction
must be selective for a given amino acid. In addi-
tion, the thus formed complex must survive all sub-
sequent synthetic steps including cleavage of the fi-
nal product from the resin [15, 16]. In recent work,
we have demonstrated that introduction of metal com-
plexes in an SPPS scheme is possible even for rela-
tively sensitive organometallic compounds by a suit-
able choice of resin, linker and side chain protecting
groups [17 – 27]. Very stable Pt(II)-peptide conjugates
were also obtained by two Dutch groups [28– 32].
Much of our work has focussed on the neuropep-
tide enkephalin (Enk). Enkephalin is the natural lig-
and for opiate receptors in the central nervous sys-
tem [33– 39]. It is a pentapeptide with the sequence
H-Tyr-Gly-Gly-Phe-Aaa-OH. The fifth amino acid is
indeed variable, with Aaa being leucine in the natu-
rally most abundant example [Leu]5-Enk. Given the
known preference of gold(I) for thiol ligands, we set
Complexation was confirmed by a shift in the
31P NMR from 33.3 to 38.3 ppm [40] along with a
downfield shift of signals in both the 1H and 13C spec-
tra. The most notable shifts observed in the 13C spec-
trum were those of the ipso-carbon atoms on the triphe-
nylphosphine and the CH2 group of the cysteine from
29.9 to 30.2 ppm, as well as the CH2 signal of the cys-
1
teine becoming a better defined AB system in the H
spectrum. These results were further supported by ESI
and FAB mass spectrometric analysis.
The combination of solid phase synthesis and gold
complexation began by using a trityl protected cys-
teine precursor, Fmoc-Cys(Trt)-Wang resin 4. The de-
sired acetyl-cysteine-gold triphenylphosphine 5 was
obtained as a white powder, after cleavage and wash-
ing (Scheme 2). Spectroscopic data for 5 were sim-
ilar to that of 3, thus confirming the integrity of the
phosphorus-gold-sulfur unit during cleavage.
Encouraged by the successful preparation of the
out to prepare a [Cys]5-Enk derivative for labelling small molecules 3 and 5, we set out to prepare the tar-
with phosphine-gold(I) complexes by SPPS. In here, get molecule [Cys]5-Enk. Suitable choice of protect-
we describe the first preparation of a gold-labelled ing groups is crucial for successful SPPS [41]. There-
cysteine-enkephalinevia solid phase peptide chemistry fore, standard Fmoc-solid phase synthesis was carried
(SPPS), in which gold-labelling is carried out on the out via two different starting materials, namely Fmoc-
resin.
Cys(Trt)-Wang resin 4 and Fmoc-Cys(tert-butyl)-
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J. Caddy et al. · Introduction of Phosphine-Gold(I) Precursors into a Cys-modified Enkephalin Neuropeptide
Scheme 3. Synthesis of 7 – 9 by solid phase synthesis. a) Fmoc deprotection; b) Coupling of amino acid; c) N-Terminal
acetylation; d) Cleavage from the resin; e) Trt deprotection; f) Gold complexation. See experimental section for details.
Wang resin 6, the only difference of the two being resin was effected using 5 % TFA and 5 % TIS in
the protecting group of the cysteine. Successful prepa- DCM, after which the resin was washed several times
ration of the desired target molecule [Cys]5-Enk was with dichloromethane and dried. The resin beads were
confirmed in both cases (Scheme 3). However, HPLC transferred to a Schlenk tube and the complexation
analysis of the product 8 obtained from 4 displayed was carried out under an argon atmosphere with (triph-
several side products. These were identified to be the enylphosphine)gold chloride and DIPEA in DCM. The
result of the free cysteine undergoing disulfide forma- complexation was allowed to take place overnight
tion. Preparation of the product 7 derived from 6 was followed by several dichloromethane washings and
of > 95 % purity. This finding is in accordance with cleavage. Phenol was added to the cleavage solution
the different stabilities of sulphur protecting groups. as a precaution against oxidation. Analytical HPLC
Whereas the trityl group may be removed by as little showed > 90 % purity of the product. Unfortunately
as 5 % TFA in DCM, the tert-butyl group remains in- subjection to preparative HPLC resulted in decompo-
tact even in the presence of 85 % TFA as shown in the sition of the product. Therefore, all further characteri-
case of 7.
zation was carried out without HPLC purification. The
structure of 9 was confirmed by NMR analysis and
mass spectrometry. 31P NMR showed a single broad
peak at 37.8 ppm. In addition 1H and 13C NMR spec-
tra displayed similar signatures as for 3. A peak for the
molecular ion at m/z = 1046 was observed in both FAB
and ESI MS (positive ion detection). Furthermore, the
Summarizing all of the above findings, the op-
timal synthesis requires incorporation of the gold
complex on the resin from a trityl protected cys-
teine. The desired gold cysteine-enkephaline com-
plex 9 was prepared by standard Fmoc-solid phase
chemistry (Scheme 3) [14]. Trityl deprotection on the
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J. Caddy et al. · Introduction of Phosphine-Gold(I) Precursors into a Cys-modified Enkephalin Neuropeptide
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ESI MS showed a peak at m/z = 610 for the metal-free 0.167 mmol) (1) and a solution of sodium hydroxide in water
[Cys]5-Enk 7.
(6.6 mg in minimal water), under argon. The reaction mix-
ture was allowed to stir overnight. The solvent was removed
in vacuo to obtain a sticky white crude product, which ap-
peared to oxidize if exposed to air for extended periods of
Conclusions
The synthesis of a cysteine-derived enkephalin was time. The reaction was repeated in dichloromethane, with
DIPEA as a base to yield similar results. Analytical Data
for C26H29NO4SAuP (679.53). – MS (FAB, pos): m/z =
721 [Ph3P–Au–PPh3]+, 579 [M + H – Boc]+, 459 [Au–
PPh3]+. – MS (ESI neg., MeOH): m/z = 678 [M – H]−. –
1H NMR (CDCl3, 360.1 MHz): δ = 8.26 (br s, 1H, NH),
7.69 – 7.15 (m, 15H, PPh3), 5.77 (br s, 1H, OH), 4.44 (br
s, 1H, Cα−CysH), 3.51 (unres. dd, 1H, Cβ−CysH), 3.27 (un-
achieved by solid phase Fmoc-chemistry from a Wang
resin starting material. Gold complexation to Cys-Enk
was successfully achieved as part of the solid phase
synthesis scheme by careful selection of protecting
groups and linkers. Unambiguous identification of the
[Cys]5-Enk-gold triphenylphosphine conjugate 9 was
greatly assisted by data obtained for a series of related
smaller molecules. To the best of our knowledge, com-
pound 9 is the first example of a gold peptide conju-
gate prepared by solid phase synthesis. Further studies
of related molecules are underway in our laboratories.
res. dd, 1H, Cβ−CysH), 1.36 (s, 9H, C(CH3)3). – 13C NMR
(CDCl3, 90.6 MHz): δ = 174.4 (COOH), 155.7 (OCNH),
134.2 (d, J = 13.8 Hz, ortho), 131.6 (d, J = 2.17 Hz,
para), 129.5 (d, J = 56.2 Hz, ipso), 129.2 (d, J = 11.4 Hz,
meta), 79.6 (C(CH3)3), 56.9 (Cα-Cys), 30.1 (Cβ -Cys), 29.2
(C(CH3)3). – 31P NMR (CDCl3, 101.26 MHz): δ = 38.3.
Experimental Section
Preparation of (triphenylphosphine)gold-acetyl-cysteine-
thiolate 5
All peptide synthesis reactions were carried out in ordi-
nary glassware and solvents. Chemicals were obtained from
Aldrich, Iris, or Novabiochem. DMF for peptide synthe-
sis (amine-free) was obtained from Roth. Enantiomerically
pure L-amino acids were used throughout. (Triphenylphos-
phine)gold chloride (Ph3PAuCl, 1) was prepared by a stan-
dard procedure [42].
The synthesis was performed manually in a syringe
equipped with a porous filter (10 mL, MultiSynTech) us-
ing Fmoc-protected Cys(Trt)-Wang resin (50 mg, loading
0.7 mmol/g, Iris Biotech) (4). Synthesis: (a) deprotection
using two times about 3 mL of a 20 % piperidine solution
in DMF, first at 5 min and then at 10 min, without wash-
ing in between; washing 5 times with about 6 mL of DMF;
(c) acetylation (acetic anhydride/2,6-lutidine/N-methyl im-
idazole/THF = 1/1/1/7, 1 mL, 1 h); washing 5 times with
about 6 mL of DMF; (e) trityl-deprotection 2 min of man-
ual washing with 5 % TFA, 5 % TIS in DCM, followed
by a further six 2 min washes on the shaker; washing 5
times with about 6 mL of DCM; (f) gold complexation:
The resin was dried and gold complexation initiated by in-
jection of a solution of (triphenylphosphine)gold chloride
(86 mg, 0.015 mmol) and DIPEA (23 mg, 5 equivalents)
in DCM, under argon. The reaction was allowed to take
place overnight, followed by three DCM washing sessions
of 2 min each; (d) cleavage: After the resin was successively
rinsed with DMF and DCM and dried under reduced pres-
sure (1 h, 10 mbar), final deprotection and cleavage from
resin was performed with a TFA mixture (TFA/H2O/TIS =
95/2.5/2.5, 1 mL, 3 h). The suspension was filtered and the
resin washed with TFA (2×0.5 mL). The combined TFA so-
lutions were poured into cold ether (10 mL, −30 ◦C), and the
suspension was centrifuged (8000 rpm, 6 min). After decant-
ing the supernatant, the crude product was washed with cold
ether (2×5 mL), dissolved in water, filtered and lyophilised,
yielding a white solid. Analytical Data for C23H23NO3PSAu
Mass spectra were recorded on a Finnigan MAT 8200 in-
strument (EI, 70 eV) or a Finnigan TSQ 700 mass spectrom-
eter (ESI; solvent and detection mode are given in parenthe-
ses). Only characteristic fragments with possible composi-
tion are given in brackets. For fragments containing metals
only the isotopomer with highest intensity was described.
1H and 13C NMR spectra were recorded on Bruker AM 360
(1H at 360.14 MHz, 13C at 90.56 MHz) and Bruker AM 300
spectrometers (1H at 300.16 MHz, 13C at 75.47 MHz). 1H
and 13C NMR spectra were referenced to residual signals of
the deuterated solvents as internal standards (CDCl3 = 7.24
(1H) and 77.0 (13C); DMSO = 2.49 (1H) and 39.5 (13C);
MeOH = 3.31 (1H) and 49.0 (13C)). High-performance
liquid chromatography (HPLC) was performed on a cus-
tomized VarianProStar system on reverse-phase Dynamax
˚
Microsorb 60-8 C18 columns (analytical: C18 60 A, diameter
˚
4.5 mm, 250 mm; preparative: C18 microsorb 60 A, diameter
21.4 mm, 250 mm) with water and acetonitrile, both contain-
ing 0.1 % TFA, as eluent using the linear gradient 5 to 95 %
acetonitrile for 35 min.
Preparation of (triphenylphosphine)gold-Boc-cysteine-
thiolate 3
To Boc-cysteine (37 mg, 0.167 mmol) (2) in ethanol (621.44). – MS (FAB, pos): m/z = 721 [Ph3P–Au–PPh3]+,
(1 mL) was added (triphenylphosphine)gold chloride (83 mg, 645 [M + H – Na]+, 459 [Au–PPh3]+. – 1H NMR (CDCl3,
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J. Caddy et al. · Introduction of Phosphine-Gold(I) Precursors into a Cys-modified Enkephalin Neuropeptide
360.1 MHz): δ = 7.60 – 7.25 (m, 15H, PPh3), 4.43 – 4.37 169.3 (C=OAc), 168.9 (C=OGly), 168.3 (C=OGly), 155.6
(m, 1H, CH), 2.74 (unres. dd, 1H, CHaCHb), 2.61 (unres. dd, (p-CTyr), 137.6 (i-CPhe), 129.9/129.2/127.9/128.0 (o- and m-
1H, CHaCHb), 1.67 (s, 3H, Cα−AcH). – 31P NMR (CDCl3, CPhe and CTyr ), 126.2 (p-CPhe), 114.8 (o-CTyr), 54.4 (Cα-
101.26 MHz): δ = 37.5.
Phe), 53.4 (Cα-Cys), 52.9 (Cα -Tyr), 42.0 (C(CH3)3), 41.9
(CH2-Gly), 41.6 (CH2-Gly), 37.5 (Cβ -Phe), 36.4 (Cβ -Tyr),
30.44 (C(CH3)3), 29.3 (Cβ -Cys), 22.4 (Cα-Ac).
Preparation of tert-butyl-Cys-enkephalin 7
∗
Common fragmentation pattern observed with this pen-
tapeptide.
The synthesis was performed manually in a syringe
equipped with a porous filter (10 mL, MultiSynTech) using
Fmoc-protected Cys(tert-butyl)-Wang resin (200 mg, load-
ing 0.77 mmol/g, Iris Biotech GmbH) (6), Fmoc-protected
amino acid monomers, base, and amine free DMF solvent.
The tyrosine side chain protecting group was tert-butyl. Syn-
thesis cycle: (a) deprotection using two times about 3 mL of
a 20 % piperidine solution in DMF, first at 5 min and then at
10 min, without washing in between; washing 5 times with
about 6 mL of DMF; (b) coupling using a solution of acti-
vated amino acid, for 30 min; and washing 5 times with 3 mL
DMF. The coupling mixture contained the protected amino
acid monomer (5-fold excess), TBTU (242 mg, 4.9-fold ex-
cess), and HOBt (118 mg, 5-fold excess) dissolved in DMF
(2.5 mL); then DIPEA (199 mg, 10-fold excess, activation
period 2 min) was added. After repeating the synthesis cy-
cle four times all the desired couplings had been carried out.
A last Fmoc-deprotection with 20 % piperidine was carried
out followed by capping. (c) For our purposes an acetyl cap-
ping was used (acetic anhydride/2,6-lutidine/N-methyl imi-
dazole/THF = 1/1/1/7, 3 mL, 1 h); (d) cleavage: After the
resin was successively rinsed with DMF and DCM and dried
under reduced pressure (1 h, 10 mbar), final deprotection
and cleavage from resin was performed with a TFA mixture
(TFA/H2O/TIS = 95/2.5/2.5, 3 mL, 3 h). The suspension was
filtered and the resin washed with TFA (2 × 1.5 mL). The
combined TFA solutions were poured into cold ether (10 mL,
−30 ◦C), and the suspension was centrifuged (8000 rpm,
6 min). After decanting the supernatant, the crude product
was washed with cold ether (2 × 10 mL), dissolved in wa-
ter, filtered and lyophilised, yielding a white solid. The prod-
uct was purified by preparative HPLC. Yield: 178 mg (71 %).
Analytical Data for C31H41N5O8S (643.76). MS (FAB, pos):
m/z = 666 [M + Na]+, 644 [M + H]+, 467 [∗], 320 [∗]. – MS
(ESI pos., MeOH): m/z = 666 [M + Na]+, 644 [M + H]+. –
1H NMR (CDCl3, 360.1 MHz): δ = 9.16 (br s, 1H, OHTyr),
8.45 (d, 1H, J = 7.9 Hz, NHCys), 8.23 (t, 1H, J = 5.8 Hz,
NHGly), 8.07 (d, 1H, J = 8.3 Hz, NHTyr), 8.03 (d, 1H, J =
8.6 Hz, NHPhe), 7.92 (t, 1H, J = 5.6 Hz, NHGly), 7.24 – 7.14
(m, 5H, ArHPhe), 7.01 (d, 1H, J = 8.6 Hz, ArHTyr), 6.23
(d, 1H, J = 8.6 Hz, ArHTyr), 4.61 – 4.57 (m, 1H, Cα−PheH),
4.41 – 4.31 (m, 2H, Cα−TyrH and Cα−CysH), 3.75 – 3.55
(m, 4H, Gly-CH2 × 2), 3.05 – 2.58 (overlapping m, 6H,
Cβ−PheH, Cβ−Tyr H and Cβ−CysH), 1.76 (s, 3H, Cα−AcH),
Preparation of Cys-enkephalin 8
The synthesis was carried out in analogy to that of
tert-butyl-Cys-Enkephalin (7). Instead of Fmoc-protected
Cys(tert-butyl)-Wang resin, Fmoc-protected Cys(Trt)-Wang
resin (200 mg, loading 0.70 mmol/g, Iris Biotech) (4) was
used. (e) In addition a trityl deprotection step was carried
out after capping, accomplished by 2 min of manual wash-
ing with 5 % TFA, 5 % TIS in DCM, followed by a fur-
ther six 2 min washes on the shaker. The trityl-deprotected
product was then washed with DCM for three wash ses-
sions, dried and subjected to work up conditions. Analytical
Data for C27H33N5O8S (587.65). MS (FAB, pos): m/z = 588
[M + H]+, 467∗, 320a. MS (ESI pos., MeOH): m/z = 610
[M + Na]+, 588 [M + H]+. MS (ESI neg., MeOH): m/z =
587 [M]−, 586 [M – H]−. – 1H NMR (CD3OD, 300.1 MHz):
δ = 7.21 – 7.19 (m, 5H, ArHPhe), 6.97 (d, 1H, J = 8.4 Hz,
ArHTyr), 6.63 (d, 1H, J = 8.4 Hz, ArHTyr ), 4.66 – 4.58 (m,
1H, Cα−PheH), 4.51 – 4.48 (m, 1H, Cα−TyrH), 4.41 – 4.36
(m, 1H, Cα−CysH), 3.80 – 3.60 (m, 4H, Gly-CH2 ×2), 3.19 –
2.66 (overlapping m, 6H, Cβ−PheH, Cβ−Tyr H and Cβ−CysH),
1.87 (s, 3H, Cα−AcH). – 13C NMR: (CD3OD, 75.4 MHz) δ =
174.8 (C=OCys), 173.7 (C=OPhe), 173.4 (C=OTyr), 172.7
(C=OAc), 172.2 (C=OGly), 171.4 (C=OGly), 157.3 (p-CTyr ),
138.4 (i-CPhe), 131.3 (m-CTyr), 130.4 (o-CPhe), 129.9 (m-
CPhe), 128.9 (i-CTyr), 127.8 (p-CPhe), 116.3 (o-CTyr ), 57.1
(Cα-Phe), 56.2 (Cα -Cys), 56.1 (Cα-Tyr), 43.9 (CH2-Gly),
43.4 (CH2-Gly), 38.5 (Cβ -Phe), 37.8 (Cβ -Tyr), 26.6 (Cβ -
Cys), 22.5 (Cα-Ac).
∗
Common fragmentation pattern observed with this pen-
tapeptide.
Preparation of (triphenylphosphine)gold-cysteine-
enkephalin-thiolate 9
The synthesis was carried out in analogy to that of Cys-
enkephalin 8, using Fmoc-Cys(Trt)-Wang resin (300 mg,
loading 0.70 mmol/g, Iris Biotech) (4). After the trityl de-
protection (e above) the resin was dried and transferred into
a schlenk under argon. (f) Gold complexation: To the reac-
tion vessel was added DCM (2 mL) and DIPEA (10 equiv-
alents) followed by the desired gold source (5 equivalents).
1.28 (s, 9H, C(CH3)3). – 13C NMR (CDCl3, 90.6 MHz):
δ = 171.9 (C=OCys), 171.7 (C=OPhe), 171.1 (C=OTyr), The reaction vessel was placed on the shaker where it was
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465
allowed to react overnight. All further steps were carried 4.71 – 4.62 (m, 1H, Cα−PheH), 4.45 – 4.38 (m, 2H, Cα−Tyr
H
out under argon. Filtration and washing with three DCM and Cα−CysH), 3.84 – 3.64 (m, 4H, Gly-CH2 × 2), 2.98 –
washing sessions completes the complexation. (d) Cleav- 2.78 (overlapping m, 6H, Cβ−PheH, Cβ−Tyr H and Cβ−CysH),
age: After complexation the resin was successively rinsed 1.75 (s, 3H, Cα−AcH), 1.29 (s, 9H, C(CH3)3). – 13C NMR
with DMF and DCM and dried under reduced pressure (1 h, (CD3OD, 90.6 MHz): δ = 138.4 (i-CPhe), 135.3 (d, J =
10 mbar), cleavage from resin was performed with a TFA 13.9 Hz, ortho), 133.4 (d, J = 2.4 Hz, para), 130.9 (d, J =
mixture (5 % TIS, 10 % phenol and 85 % TFA, 3 mL, 3 h). 79.0 Hz, ipso), 130.6 (d, J = 11.4 Hz, meta), 129.9 (m-CTyr),
The suspension was filtered and the resin washed with TFA 129.5 (o-CPhe), 129.0 (m-CPhe and i-CTyr), 127.7 (p-CPhe),
(2×1.5 mL). The combined TFA solutions were poured into 116.3 (o-CTyr ), 57.0 (Cα -Phe), 56.4 (Cα-Cys), 56.2 (Cα-
◦
cold ether (10 mL, −30 C), and the suspension was cen- Tyr), 44.0 (C(CH3)3), 43.9 (CH2-Gly), 43.6 (CH2-Gly), 38.2
trifuged (8000 rpm, 6 min). After decanting the supernatant, (Cβ -Phe), 37.8 (Cβ -Tyr), 30.7 (C(CH3)3), 34.5 (Cβ -Cys),
the crude product was washed with cold ether (2 × 10 mL), 22.6 (Cα-Ac). – 31P NMR (CD3OD, 101.26 MHz): δ = 37.8.
dissolved in water, filtered and lyophilised, yielding a peach
Acknowledgements
solid. Unfortunately subjection to HPLC resulted in decom-
position of the synthesized product and all further charac-
The authors would like to thank Project AuTEK (Mintek
terization was carried out without HPLC purification. Yield: and Harmony Gold) for permission to publish this paper and
(73 %). Analytical Data for C45H47N5O8PSAu (1045.90). – for financial support. The authors are grateful to H. Rudy
MS (FAB, pos): m/z = 1046 [M]+, 721 [Ph3P–Au–PPh3]+, (MS), A. Seith (MS), T. Timmermann (NMR) and T. Coelho
588 [M + H – AuPPh3]+, 459 [Au–PPh3]+). – MS (ESI (NMR) for technical assistance. Further thanks go to X. de
pos., MeOH): m/z = 1046 [M]+, 721 [Ph3P–Au–PPh3]+, 610 Hatten for helpful discussions with regard to cysteine chem-
[M – AuPPh3 + Na]+, 459 [AuPPh3]+. – 1H NMR (CD3OD, istry. NMN likes to thank Prof. H. Raubenheimer for an
360.1 MHz): δ = 7.52 – 7.35 (m, 15H, PPh3), 7.26 – 7.19 (m, invitation to Stellenbosch and the possibility to finish this
5H, ArHPhe), 7.02 (dd, 1H, HTyr,ar), 6.68 (dd, 1H, HTyr,ar), manuscript in his own office.
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