Master’s Degree programme
in Sustainable Chemistry and TechnologiesSecond Cycle (D.M. 270/2004) Final Thesis
Synthesis and Characterization of NHC*s
Gold(I) Complexes with Biological
Molecules in the Coordination Sphere
Supervisor
Ch. Prof. Marco Bortoluzzi
Assistant supervisors
Ch. Prof. Marina Rubini and Ch. Prof. Matthias Tacke
Graduand
Francesca Adami
Matriculation Number 843047
Academic Year
Index
1. INTRODUCTION 1 1.1. Uses of gold in medicine, 1
1.1.1. Gold nanoparticles, 1
1.1.2. Luminescence of Au compounds, 2 1.1.3. Anti-parasitic, 3
1.1.4. Antitumoral action, 4
1.1.4.1. Possible mechanisms of action, 4 1.1.4.2. Gold(III) complexes, 5
1.1.4.3. Gold(I) complexes, 7
1.1.4.3.1. Phosphole complexes, 7 1.1.4.3.2. Auranofin, 8
1.1.4.3.3. Gold(I) N-Heterocyclic carbenes, 8
2. SCOPE 13
3 EXPERIMENTAL 14 3.1. Instruments, 14
3.2. General conditions, 14
3.3. Synthesis of 1,3 dibenzyl - 4,5 diphenyl imidazolium bromide, 14
3.4. Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) Chloride, 15 3.5. Synthesis of N-acetyl-L-cysteine methyl ester, 15
3.6. Synthesis of N-acetyl-L-cysteine ethyl ester, 16 3.7. Syntheses of the dipeptides, 17
3.7.1. Cys-Tyr, 19 3.7.2. Ala-Cys, 19 3.7.3. Cys-Ala, 19
3.8. Synthesis of the Cysteine Acetylated-Alanine Amidated Dipeptide, 20
3.8.1. Reaction number 1, 20
3.8.2. Reaction number 2, 20
3.9.1. Reaction number 1, 21 3.9.2. Reaction number 2, 22
3.10. Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (N-Acetyl-L-Cysteine), 22
3.10.1. Reaction number 1, 22 3.10.2. Reaction number 2, 23 3.10.3. Reaction number 3, 23
3.11. Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (N-Acetyl-L-Cysteine-Methyl Ester), 24
3.11.1. Reaction number 1, 24 3.11.2. Reaction number 2, 24 3.11.3. Reaction number 3, 25 3.11.4. Reaction number 4, 25
3.12. Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (N-Acetyl-L-Cysteine Ethyl Ester), 26
3.13. Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (Alanine-Amidated Cysteine dipeptide), 26
3.13.1. Reaction number 1, 26 3.13.2. Reaction number 2, 27
3.14. Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (Cysteine Acetylated-Alanine Amidated), 27
4. RISULTS E DISCUSSION 28
4.1. Synthesis of the imidazolium salt, 29 4.2. Synthesis of Gold(I) NHC Chloride, 30 4.3. Esterification reactions, 31
4.4. Dipeptide syntheses, 34
4.4.1. Acetylation of the dipeptides, 42
4.5. Coupling reactions, 46
ACKNOWLEDGEMENTS
I would like to express a special thanks to my relator, Professor Marco Bortoluzzi, for giving me the opportunity to do my research work abroad and for his help throughout the whole period.
I want to really thank Professor Marina Rubini and Professor Matthias Tacke for accepting me in their laboratories and for the encouragement they gave me during the experience.
I also want to thank all the people in the laboratory for welcoming me so kindly: Danielle, Cillian, Cian, Dhiyaa, Daniel, Nada, Hessa, Kirti, Jenny, Fahad and Sondos.
INTRODUCTION
Gold is a late transition metal located in the third row of the last group of the transition metals. Its atomic number is 79 and the electronic configuration of the outer shell of the element is 5d106s1. The main oxidation states of the element are Au(0), Au(I) and Au(III), while Au(II) tends to disproportionate to Au(I) and Au(III). Due to its position in the periodic table the relativistic effects are high on the gold, stabilizing the 6s and destabilizing the 5d orbitals; this is also the reason for the tendency of the element to give linear compounds in the monovalent state, since the orbitals 6s and 5dz2 gets closer in energy[1].
Uses of gold in medicine
The metal has been used in medicine since ancient times, there are reports of gold therapeutic application as early as 2500 BC, having its origin in the ancient imperial Chinese culture. It was then used to treat smallpox, skin ulcers and measles and people thought that gold had immortal properties, leading to its association with longevity. The features attributed to the metal pushed the alchemists to search for ways to make drinkable gold elixirs, overshadowing the toxic side effects that were also observed.
However, the modern use of gold-based drugs started in 1890 with the German physician and bacteriologist Robert Koch, who discovered the anti-tubercular activity of potassium dicyanoaurate in vitro, which unfortunately does not persist in vivo. A more successful attempt was done in 1929 by the French physician Jacques Forestier, who used sodium aurothiopropanol sulfonate to successfully treat rheumatoid arthritis. These findings led to the development of Auranofin which was introduced into the clinic in 1985, after being approved by the FDA (Food and Drug Administration) in 1976, and is used to treat certain cases of arthritis[2].
A brief summary of the application of gold in the pharmaceutical field is reported.
Gold nanoparticles
Nanoparticles are structures with a diameter between 1 and 10 nanometers and have interesting properties that differ from the ones that the element expresses either in bulk or in a molecule. Depending on the size, shape, degree of aggregation and the nature of the protecting organic shell on their surface, they display electronic structures that reflect the electronic band structure of the nanoparticles, having to submit to quantum-mechanical rules.
There are two main phenomena related to this[3]. First, the quantum size effect that is observed when the size of the particle is small enough to become comparable to the wavelength of the electron, which leads the particles to behave electronically as zero-dimensional quantum dots (or boxes). The electrons are therefore trapped in these boxes and show a characteristic collective oscillation frequency of the plasma resonance, giving rise to the so-called plasmon resonance band (PRB) observed near 530 nm in the 5-20 nm diameter range. An example of medicinal application of this is described by Taglietti et al.[4]; they reported the synthesis of monolayers of gold nanostars (GNS) grafted on mercaptopropyl-trimethoxysilane-coated glass slides. In the formed monolayers, the localized surface plasmon resonance of GNS can be tuned and, upon irradiation in the near-IR range (NIR, 750–1100 nm), it undergoes thermal relaxation, inducing local hyperthermia and efficient killing of Staphylococcus aureus biofilms.
Then, since in nanoparticles, unlike in bulk metals, there is a gap between the valence band and the conduction band, standing electron waves with discrete energy levels are formed. This leads to the observation of single-electron transitions which occur between a tip and a nanoparticle, when the temperatures are low enough to allow the electrostatic energy (given by e2/2C) to be greater than the thermal one (given by kBT).
The optical properties and the great biocompatibility of gold nanoparticles (AuNPs) are really interesting in the biological and pharmaceutical fields. The resonance frequency lies in the visible region of the electromagnetic spectrum but any change to the environment of these particles (such as surface modification or aggregation) causes colorimetric changes of the dispersions.
AuNPs have therefore been widely explored and have important applications as probes. They are used for chemical sensing (heavy metal cations, DNAs and proteins detection), for studies of carbohydrate-protein interactions and enzymatic activities and for imaging applications[5].
Luminescence of Au compounds
Luminescence is a spontaneous emission of light by a substance not resulting from heat and is thus a form of cold-body radiation. The luminescence properties[6,7] of phosphinegold(I) compounds have been extensively investigated and a wide variety of transitions are considered responsible for these properties (intra-ligand transitions, metal-centered transitions and ligand-to-metal charge transfer). The presence of aurophilic interactions, caused by the relativistic effect on the metal, is very important in the structural chemistry of gold and creates additional interest in these systems. These interactions can provide a stabilization energy to crystal packing similar to the hydrogen bonding interactions. They influence the luminescence properties exhibited in certain
phosphinegold(I) compounds enhancing the transition probability by lowering the energy gap between the emitting and receiving energy levels.
There are two main classes of phosphinegold(I) luminescence compounds: halides and thiolates. For the latter, even though the phosphine ligand often contains a chromophore, the luminescence properties are determined mainly by the nature of the thiolate ligand, since the process starts with the excitation of the sulfur that later transfers the charge to the gold. Quantum mechanical calculations on the electronic structures and transitions are however usually employed to determine the nature of the absorbing and emitting states.
Luminescence has been used to explore photochemical and photophysical processes in various media. A particularly notable application is the use of luminescent phosphinegold(I) thiolates as alkali metal ion sensors; another important feature is the dependence of the luminescence properties of the compound on the pH of the aqueous solution as well as on the solvent polarity.
Anti-parasitic
Gold complexes have been used to treat parasitic diseases[8] such as malaria. It was after malaria parasites became resistant to chloroquine, Fig. 1, one of the most successful drugs ever used to treat an infectious disease, that the research started to focus on creating functionalized gold phosphines compounds.
Fig. 1 – Chloroquine and chloroquine gold derivative structures
The first attempts revolved around attaching ligands that were already known to be active against the parasite such as chloroquine, primaquine, etc.. In vitro and in vivo testing showed that the action
N Cl HN N N Cl HN N Au P PF6 Chloroquine [Au(PPh3)(CQ)][PF6]
These findings encouraged the development of new gold antimalarial agents with a variation in the structure of the complex, changing the phosphine ligand (with the purpose of inducing changes in the electronic and steric properties), the counteranion, the gold oxidation state (both Au (I) and Au (III)) and also the ligand itself (with sugar derivatives for example).
Antitumoral action
Possible mechanisms of action
Gold drugs are prodrugs[9], this means that the molecules undergo transformations which lead to their active forms. In particular gold(I) complexes react in vivo, with molecules or sites of proteins and enzymes, giving rapid ligand displacement reactions. Even though little is known about the real mechanism of action, the experimental data show that gold(I) is transported in the body by attachment to sulfur residues in proteins and that metabolic elimination of gold(I) as [Au(CN)2] -occurs, with some evidence of gold(III) formation in oxidative bursts.
Gold drugs action as antirheumatic[10] seems to be related to the metal ability to target various enzymes, such as cyclooxygenases (COX), and deactivate their active sites. There are similar reports of auranofin inhibiting selenium-gluthathione peroxidase, at micromolar concentrations. Furthermore, gold(I) species with labile ligands and several gold(III) complexes interact readily with isolated DNA and therefore this biomolecule cannot be excluded as a possible target for gold drugs.
The most important enzyme targeted is however thioredoxin reductase[11], TrxR; auranofin selectivity towards this molecule is approximately 1000-fold higher in comparison to other related enzymes. Even though a unique mode of action has not been reported, there are several studies that underline the key role of TrxR inhibition for gold metallodrug pharmacology, considering both gold(I) and gold(III) compounds.
Thioredoxin reductase is a homodimeric protein belonging to the family of glutathione reductase like enzymes, it catalyzes the NADPH dependent reduction of thioredoxin (Trx) disulfide and many other oxidized cell constituents, through its active site which contains the Gly-Cys-Sec-Gly motif [where Gly stands for glycine, Cys for cysteine and Sec for selenocysteine]. The enzyme shows a broad substrate specificity and is involved in numerous metabolic pathways and pathophysiological conditions (tumors, infectious diseases, rheumatoid arthritis, etc.). A particularly interesting property is its action as an antioxidant, which prevents cells from oxidative stress, a key factor for
DNA damage. Overexpression of TrxR has been observed in numerous tumor cell lines and potent cytostatics, like cisplatin, are effective inhibitors of TrxR.
The fact that thioredoxin reductase represents an effective druggable cancer target has received significant experimental support, especially considering that some established antitumor agents used
in clinic several years ago were retrospectively found to act as potent inhibitors of thioredoxin reductase. The enzyme can be efficiently inhibited by various compounds ranging from natural to synthetic organic products (such as quinines), from simple metal ions (like Ca(II), Zn(II), Mn(II), Cd(II) and Tl(I)) to structurally elaborated metal coordination compounds (for example Au, Pt and Ru derivatives).
The inhibitory properties of gold complexes against thioredoxin reductase were characterized in more detail by Gromer et al. and in their paper it was reported that the enzyme, in its physiological, NADPH-reduced form, is strongly inhibited by auranofin, with IC50 values as low as 4 nM. In contrast, the gold(III) compound tetrachloroaurate turned out to be a far weaker inhibitor.
It seems likely that gold(I) complexes, being soft electrophilic centers, interact with the enzyme on its nucleophilic sulfur and selenium residues, with a preferential binding to the last one.
Both gold(I) and gold(III) complexes were, on the other hand, active against mitochondrial thioredoxin reductase, heavily influencing mitochondrial functions, such as membrane permeability properties, leading to swelling and loss of membrane potential.
It has been also shown by Mohr et al.[12] that, for a variety of gold complexes, the anti-tumour activity is mediated by an enhanced production of reactive oxygen species (ROS). For this reason, a mechanism involved in the induction of apoptosis is oxidative stress, caused by the formation of ROS due to deregulated mitochondrial activity.
In conclusion, several gold compounds have been evaluated as potential inhibitors of thioredoxin reductase, most of which caused profound enzyme inhibition with IC50 values typically ranging from nanomolar to micromolar. An important factor is the soft character of the gold complex, that supports the view that the selenol group is very likely the primary binding site for gold compounds. This is strictly correlated to the necessity for the compounds to possess at least one labile ligand in order to allow a direct gold–selenium bond creation. Gold(III) compounds, less soft in nature, display a lower inhibitory potency but still appreciable. These complexes are more likely to act through a different mechanism, probably causing for instance oxidative damage to the enzyme.
Gold(III) complexes
Based on their structural and electronic similarity to cisplatin, one of the most important compounds used to treat a wide range of cancers, gold(III) antitumor drugs represent a promising class of
potential anticancer agents[10]. The main setback for the development of gold(III) complexes as therapeutic drugs is their low stability under physiological conditions, which remains a critical parameter in the drug development of these species. Gold(III) complexes with various ligands (such as Au–N, Au–S or Au–C) have been prepared and biologically investigated.
AuCl3(Hpm) and AuCl2(pm), Fig. 2, two gold chloride species with pyridine ligands, showed good cytotoxic activity, whose action was confirmed to be binding to the DNA. Both complexes were relatively stable in organic solvents but underwent hydrolysis of the chloride ligand in aqueous buffer media. On the other hand, there are examples of bi- and tri- pyridine complexes, such as [Au(bipy)(OH)2][PF6] and [Au(phen)Cl2]Cl, Fig. aa., stable and active as cytotoxic agents in physiological buffers at 37°C.
Of particular interest are gold(III) dithiocarbamate complexes, Fig. 2. Fregona et al.[13] reported the synthesis of several gold(III) dithiocarbamato derivatives, of the type [AuX2(L)] (X=Br, Cl; L=various dithiocarbamates), that are promising anticancer agents with fine-tuned stability, good antiproliferative activity, and encouraging selectivity. Remarkably, beside the higher activity in comparison to cisplatin, they also showed lower toxicity towards normal tissues. The main intracellular targets of these complexes were detected in proteasome, the thioredoxin system and mitochondria.
In 2018 they also reported the synthesis of five new Au(III)-peptidodithiocarbamato complexes[14] that differ with regard to the amino acid sequence and/or the chiral amino acid configurationability. The gold(III)-based moiety was functionalized to exploit the targeting properties of the peptidomimetic ligand toward two peptide transporters (namely PEPT1 and PEPT2), which are upregulated in several tumor cells. All the compounds had GI50 values in the low micromolar range.
Fig. 2 – Examples of gold(III) complexes
N Au Cl Cl Cl N Au Cl O Cl OH N N Au HO OH PF6 Au N N N Cl 2 2 Cl N S Au S Cl Cl
7
Gold(I) complexes Phosphole complexes
An interesting class of gold compounds are the gold phosphole complexes[10]. These gold agents are characterized by a phosphacyclopentadiene ligand attached to the central metal and are potent inhibitors of thioredoxin reductase and the related glutathione reductase. The complex GoPI, Fig. 3, shows EC50 (half maximal effective concentration) values for enzyme inhibition in the low nanomolar range and is probably the most potent inhibitor of glutathione reductase reported so far.
Fig. 3 – GoPI complex structure
A broad range of compounds with multiple phosphine ligands attached to the gold(I) central atom has been investigated. The lead compound for this class is [Au(dppe)2]+, Fig. 4, a tetrahedral bischelated gold complex, which showed in vitro and in vivo antitumor activity and induced DNA protein cross-links and DNA strand breaks in cells. The complex is kinetically stable in the presence of thiols and its action appears in general to be different from that of auranofin. Structure– activity relationship studies suggested that the optimal length of the bridge connecting the two phosphor atoms is 2–3 carbon atoms.
Fig. 4 – [Au(dppe)2]+ complex structure
P Au N N Cl GoPI Au PR2 PR2 R2P R2P Cl [Au(dppe)2]Cl R = Ph N N R2 AuCl R1 Y Y N N R2 Au R1 Y Y S N N NHC*-Au-Cl NHC*-Au-(2-mercapto-pyrimidine) N N AuCl N N Au NHC*-Au-Cl NHC*-Au-S-R S O AcO OAc OAc OAc P Au N N Cl GoPI Au PR2 PR2 R2P R2P Cl [Au(dppe)2]Cl R = Ph N N R2 AuCl R1 Y Y N N R2 Au R1 Y Y S N N NHC*-Au-Cl NHC*-Au-(2-mercapto-pyrimidine) N N AuCl N N Au S O AcO OAc OAc OAc
Auranofin
The most common medical use for gold compounds is the treatment of rheumatoid arthritis, an autoimmune inflammatory disease with the characteristic progressive erosion of the joints leading to their deformities, immobility and pain. A lot of gold drugs are still used to treat this illness mainly focusing on alleviating the symptoms and preventing the progressive destructive processes. The application of these drugs is fundamental because the damages are generally irreversible and cannot be influenced by anti-inflammatory agents and analgesics.
The discovery of auranofin[10], triethylphosphino gold(I) 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-1-thiolate, Fig. 5, was of particular interest since it can be administered orally, while the other gold salts are usually given by injection.
Fig. 5 – Structure of Auranofin
Auranofin is a neutral, linear two coordinate gold phosphine complex containing a carbohydrate ligand. Further studies revealed that auranofin displayed also promising cell growth inhibiting action in vitro but limited efficacy in experimental in vivo models. An early structure–activity relationship study on 63 auranofin analogues had demonstrated the importance of the phosphine ligand, as derivatives lacking this moiety were significantly less active. This study demonstrated that in vitro inactive compounds also were not active in vivo, but that activity in vitro did not necessarily translate into activity in vivo. For this reason, it is so far unclear if auranofin itself might be a suitable antitumor drug candidate; however, an interest in the research on gold complexes for cancer chemotherapy has definitely been triggered.
Gold(I) N-Heterocyclic carbenes
N-Heterocyclic carbenes[15] (NHCs) are an important class of ligands, usually used instead of phosphines for their similar properties as s donors. There is a tendency to substitute phosphines because they are toxics, pretty expensive and generally not stable enough in the reaction conditions. NHCs, on the other hand, are easy to prepare and exhibit versatile ligand properties, forming stronger bonds to metal centers than phosphines. The complexes are therefore more stable toward
O O O O O O O O O S Au P
moisture, air, and heat, playing important roles in catalysis and biomedical applications as well as other fields such as luminescent and functional materials.
N-heterocyclic carbenes[16] are molecules that have the carbene carbon linked to two nitrogen atoms through covalent bonds, leaving it with two unbounded valence electrons.
Carbenes linked to late transition metals are also called Fisher carbenes, and the bond that is formed is a dative one, with the two electrons coupled in the lower energy orbital. In this case, unlike for the phosphines, there is little p back-bonding, because there is low interaction between the metal and the p orbital that is not hybridized, since it is already conjugated with the orbitals of the other atoms of the cycle, Fig. 6.
Fig. 6 – NHCs* resonance structures
NHCs metal complexes[15] are as versatile as their applications, ranging from zinc over rhodium to platinum, the latter of particular interest since the discovery of cisplatin as an effective anticancer drug. In relation to their biological application gold(I/III) NHCs complexes have definitely gained importance after auranofin exhibited a moderate antitumor activity.
Ott et al. synthesized a series of benzimidazol-2-ylidene-gold(I) complexes and found strong antiproliferative effects against both cancerous and noncancerous cells as well as thioredoxin reductase (TrxR) inhibition for the NHC*-Au-PPh3 complexes.
In 2012, Schuh et al.[17] reported the synthesis and characterization of gold(I) complexes with 1,3-substituted imidazole-2-ylidene and benzimidazole-2-ylidene ligands of the type NHC*-Au-L (where L was either Cl or 2-mercapto-pyrimidine), Fig. 7. These complexes were tested for their antiproliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), as well in the nontumorigenic human embryonic kidney cell line (HEK-293T), showing in some cases important cytotoxic effects. They also showed promising inhibition of TrxR and glutathione reductase (GR) at low micromolar range.
7
Schema 9
Nonostante che originariamente entrambi i tipi di leganti fossero stati chiamati con lo stesso nome, abbastanza presto ne sono state riconosciute le peculiarità. In particolare, i carbeni di Schrock (alchilideni) danno con gli early transition metals autentici legami doppi, mentre nel caso dei carbeni di Fisher, più adatti ai late transition metals, il legame che si forma è sostanzialmente un legame dativo singolo. Questo è dovuto alla scarsa disponibilità ad accettare elettroni dell’orbitale p non coinvolto nell’ibridazione poiché spesso coniugato con gli orbitali degli altri atomi costituenti lo scheletro del carbene.
I carbeni N-eterociclici (NHC) in particolare, si devono considerare carbeni di Fisher con i due elettroni accoppiati nell’orbitale a più bassa energia. La stabilità di questo tipo di carbeni va ricercata nelle formule limite di risonanza rappresentate in Schema
10. Schema 10 M R' R singoletto Fischer M R' R tripletto Schrock N C N R' R N C N R' R N C N R' R N C N R' R
Fig. 7 – Generals structures of NHC*-Au-L compounds synthesized by Schuh’s research group
Previous reports by Scilliano et al.[15] on bis-NHC* silver and gold complexes showed increased anticancer activity when Ag was replaced with Au in lung cancer cell lines.
Other important classes of gold(I) compounds are the pseudohalides and thiolates. The firsts have on their coordination sphere an easily replaceable ligand, so the metal can bond with anions such as C-, SCN-, N3- or NCO- due to their stronger connection to the gold. The possibility to coordinate azides has gained interest for cycloaddition reactions with terminal alkynes, so they can not only be used as potential anticancer drugs but also as organometallic synthons for further synthetic procedures.
The thiolates, on the other hand, being soft bases, bond strongly to soft metals like gold(I). Phosphine gold(I) thiolates have therefore been explored for their anticancer potential, coordinating to the metal substrates that were already known to be biologically active such as 6-mercaptopurinate, an immunosuppressant used in the treatment of acute lymphocytic leukemia. The results were encouraging, since enhanced cytotoxicity was observed in comparison to the free thiol on several human cancer cell lines.
Matthias Tacke’s research group has been working on several gold NHCs complexes[18]. Some lead compounds are 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) chloride, NHC*−Au−Cl, Fig. 8, and its 2′,3′,4′,6′‐tetra‐O‐acetyl‐β‐d‐glucopyranosyl‐1′‐thiolate derivative (NHC*−Au−SR, Fig. 8). These exhibit average GI50 (concentration for 50% of maximal inhibition of cell proliferation) values of 1.78 and 1.95 μm on the NCI (National Cancer Institute) 60 cancer cell panel and induce apoptosis through thioredoxin reductase (TrxR) inhibition with IC50 (half minimal inhibitory concentration of a substance) values of 1.5 μm for and 3.1 μm, being therefore promising anticancer drug candidates for clinical testing in the nearby future.
P Au N N Cl GoPI Au PR2 PR2 R2P R2P Cl [Au(dppe)2]Cl R = Ph N N R2 AuCl R1 Y Y N N R2 Au R1 Y Y S N N NHC*-Au-Cl NHC*-Au-(2-mercapto-pyrimidine) N N AuCl N N Au NHC*-Au-Cl NHC*-Au-S-R S O AcO OAc OAc OAc
Fig. 8 – Structures of NHC*−Au−Cl and NHC*−Au−SR compounds synthesized by Tacke’s research group
NHC-AuCl exhibited its best growth inhibition activity against the leukemia cell lines SR and K-562, with GI50 values of 319 nM and 608 nM. Particularly good results were also obtained for NHC-AuCl and NHC-AuSR both against the prostate cancer cell line PC3 with GI50 values of 1.84 μM and 1.83 μM, the breast cancer line MDA-MB-468, for which GI50 values of 1.39 μM and 1.54 μM and the renal cell line CAKI-1 with GI50 values of 1.74 μM and 2.00 μM. Both compounds showed a significant effect as TrxR inhibitors killing cancer cells through enhanced oxidative stress. Computational methods were used to determine that NHC-AuSR is a potential ligand for a glucose transporter, which is overexpressed in many tumors.
In the same research group, Dada et al. then synthesized and examined three novel NHC*-Au-pseudohalides and three novel NHC*-Au-thiolate[19]. The most promising results were found for the cyclohexyl thiolate, the thiocyanate and the azide derivatives, Fig. 9, after being tested on the 60 cell lines tested in the NCI. The GI50 values against the several human cancer cell lines indicate low micromolar activity. NHC*-Au-SCN gave the best results with an average GI50 value of 0.47 µM against MCF7 (breast cancer line), while for the other compounds the average GI50 was similar to the previously synthesized NHC*-Au-chloride and NHC*-Au-thioglucoside.
Fig. 9 – Examples of structures of NHC*−Au pseudohalides and thiolate synthesized by Dada P Au N N Cl GoPI Au PR2 PR2 R2P R2P Cl [Au(dppe)2]Cl R = Ph N N R2 AuCl R1 Y Y N N R2 Au R1 Y Y S N N NHC*-Au-Cl NHC*-Au-(2-mercapto-pyrimidine) N N AuCl N N Au NHC*-Au-Cl NHC*-Au-S-R S O AcO OAc OAc OAc N N Au NHC*-Au-SCN N N Au NHC*-Au-(Azide) N3 SCN N N Au S NHC*-Au-(Cycolhexyl thiolate)
Furthermore, interesting results were obtained in collaboration with Dr. Bernardes[20], who was able to bioconjugate human serum albumin (HSA, the most abundant protein in the blood that display an important role as transporter) to the gold NHC complex. The conjugation was carried out through a selective transmetallation reaction between the chloride gold derivative and the free cysteine moiety on the protein. The IC50 values of the gold precursor and the product for the selected cancer cell lines were comparable and in the micromolar range.
These findings suggest that conjugating NHC gold compounds to biological molecules is a promising field of research.
SCOPE
The thesis project is based on the synthesis of Au(I) complexes for antitumoral purposes. The idea is to complex the metal with two ligands, one is an N-heterocyclic carbene and the second is a biological molecule. The work can be divided in two parts, the first one involves the synthesis of the precursors, the NHC*-Au-Cl complex and the dipeptides, and the second part focuses on finding the best conditions for the coupling reaction between the gold and the sulfur of the biological moiety.
EXPERIMENTAL Instruments
1H and 13C NMR spectra were recorded on a 300 or 400 MHz Varian spectrometer at room temperature using TMS as an internal standard and deuterated solvents, CDCl3, and D2O as stated for the specific synthesis. 1H NMR chemical shift were referenced to TMS, DDS or residual chloroform in CDCl3 (7.26 ppm). 13C NMR chemical shift were referenced to chloroform in CDCl3 (77.3 ppm). Chemical shifts are reported as d values in parts per million (ppm) and coupling constants (J) were recorded in hertz (Hz).
Mass spectra were recorded on a quadrupole tandem mass spectrometer (Quattro Micro, Micromass/Waters Corp., USA), using solutions in 100% water or 100% acetonitrile. Mass spectra were obtained using the electron spray positive ionization technique for all compounds.
Infrared spectra were recorded on a Bruker ALPHA FT-IR spectrometer.
General conditions
Unless stated, all chemical reagents were used as supplied. All reactions were carried out in oven-dried glassware. The solvents used were of analytical grade and were used without further purification. All solvents were evaporated under reduced pressure with a bath temperature of 40°C.
Synthesis of 1,3 dibenzyl - 4,5 diphenyl imidazolium bromide
4.5 diphenyl imidazole, 0.220 g [≈ 1 mmol], and potassium carbonate, 0.207 g [≈ 1.5 mmol], were
stirred in acetonitrile, 30 mL, for 15 minutes. Then benzyl bromide, 0.240 mL [≈ 2 mmol], was added and the mixture was left stirring for 3 days.
The solution was filtered and the solid was washed with acetonitrile, 10 mL, and the transparent solution was concentrated down to 2-3 ml at the rotavapor. The product was precipitated with
diethyl ether, 50 mL, filtered and dried in vacuum for 2 hours.
The mother liquors of 1,3 dibenzyl - 4,5 diphenyl imidazolium salt were first dried and then dissolved in acetonitrile and precipitated again with diethyl ether in order to get an higher yield.
The product was obtained with a yield up to 80%.
1H NMR (300 MHz, CDCl3, d ppm): 11.25 (s, 1H, CHimidazolium), 7.46 - 7.27 (m, 11H, CHbenzyl + CHphenyl), 7.14 – 7.08 (m, 9H, CHbenzyl + CHphenyl), 5.51 (s, 4H, CH2 benzyl).
Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) Chloride
1,3 dibenzyl - 4,5 diphenyl imidazolium salt, 0.2382 g [≈ 0.49 mmol], was put in a flask with
approximately 12 mL of dichloromethane. The flask was covered with aluminium foil, because the reaction is light sensitive, and silver oxide, 0.0646 g [≈ 0.28 mmol], was added and the reaction begun.
After 4 hours, chloro dimethyl sulphide gold(I), 0.1464 g [≈ 0.497 mmol], was added for the transmetalation step; this reaction is light sensitive as well. The reaction was then stopped after 6 hours and, in order to eliminate the unreacted silver oxide, the solution was filtered on a silica column (approximately 3 cm).
The transparent solution was concentrated down to 2-3 mL at the rotavapor and the product was precipitated with pentane.
The liquors of the gold complex were first dried and then dissolved in dichloromethane and precipitated again with diethyl ether in order to get a higher yield.
The product was obtained with a yield up to 76%.
1H NMR (300 MHz, CDCl3, d ppm): 7.33 - 7.31 (m, 2H, CHbenzyl + CHphenyl), 7.25 – 7.20 (m, 12H, CHbenzyl + CHphenyl), 7.04 – 6.96 (m, 7H, CHbenzyl + CHphenyl), 5.44 (s, 4H, CH2 benzyl).
Synthesis of N-acetyl-L-cysteine methyl ester
N-acetyl-L-cysteine, 0.3000 g [≈ 2 mmol] was put in a flask with methanol, 15 ml, and one drop of
concentrated H2SO4, in an oil bath at 90°C, at reflux.
The reaction was monitored with TLC plates, taken in a solution 1:1 of cyclohexane/ethyl acetate. There is the clear spot for the aromatic groups and there is a yellow spot after using the ninhydrin stain.
After 24 hours the reaction was stopped, and the solvent was removed connecting the flask first at the rotavapor and then to the vacuum Schlenk line. An oily product was obtained.
The product started to slowly crystallize from the CDCl3 used to do the NMR spectrum while being dried at the Schlenk line.
The product was obtained with a yield of 89%.
1H NMR (400 MHz, CDCl3, d ppm): 6.36 (s, 1H, NH), 4.91-4.87 (dt, J = 7.4, 4.1 Hz 1H, CH), 3.79 (s, 3H, CH3 ester), 3.03-2.99 (ddd, J = 9.0, 4.2, 1.5 Hz, 2H, CH2), 2.07 (s, 3H, CH3 acetyl), 1.35 – 1.31 (t, J = 9.0 Hz, 1H, SH).
13C NMR (100 MHz, CDCl3, d ppm): 170.6, 169.8 (C=O), 53.5, 52.8 (OCH3), 26.8 (CH2), 23.1 (CH3).
IR (ATR, cm-1): 3298 (N-H stretching), 2565 (S-H stretching), 1731 (C=O ester stretching), 1642 (C=O amide stretching).
Synthesis of N-acetyl-L-cysteine ethyl ester
N-acetyl-L-cysteine, 0.3000 g [≈ 2 mmol] was put in a flask with ethanol, 15 mL, and one drop of
concentrated H2SO4, in an oil bath at 90°C, at reflux.
The reaction was monitored with TLC plates, taken in a solution 1:1 of cyclohexane/ethyl acetate. There is the clear spot for the aromatic groups and there is a yellow spot after using the vanillin stain.
After 24 hours the reaction was stopped, and the solvent was removed connecting the flask first at the rotavapor and then to the Schlenk system. An oily product was obtained.
In order to purify the product and obtain it as a solid compound, several attempts have been done, but without success. First a silica column was started with sand on top and a solution of
ethanol/cyclohexane, 1:4, as solvent. Then, the collected fractions were dissolved it in a small
amount of ethanol, approximately 5-6 mL, and precipitated with diethyl ether. The flask was left in the freezer overnight in order to let it precipitate properly. A second precipitation was attempted from a CH2Cl2 solution, approximately 3 mL, first with pentane and then, after the precipitation of
an agglomerate of an off-white solid, also some petroleum ether. The flask was left again in the freezer overnight. The cold solution had crystals but as soon as it warmed up, they became an oil.
The product was obtained with a yield of 37%.
1H NMR (300 MHz, CDCl3, d ppm): 6.40 (s, 1H, NH), 4.89-4.84 (dt, J = 7.4, 4.0 Hz 1H, CH), 4.31-4.20 (m, 2H, CH2 ester), 3.04-3.00 (m, 2H, CH2 thiol), 2.07 (s, 3H, CH3 acetyl), 1.33 – 1.28 (t, J = 7.1 Hz, 1H, SH), 1.31 (s, 3H, CH3 ester).
Syntheses of the dipeptides
All the syntheses of the dipeptides follow the same steps. In the laboratory three dipeptides have been synthesized: Cysteine-Amidated Tyrosine (Cys-Tyr), Alanine-Amidated Cysteine (Ala-Cys) and Cysteine-Amidated Alanine (Cys-Ala).
The procedure described subsequently is the general optimized method.
The Rink Amide AM resin, 0.3000 g [≈ 0.22 mmol active sites] was put in the reactor and the first step, removing the F-moc (fluorenil metilossicarbonil ester) moiety from the resin, begun. 3-4 mL of a solution of 40% in volume piperidine in DMF (dimethylformamide) were added in the reactor and it could be readily noticed that the resin swelled. The reactor was stirred with a mechanical machine for approximately 5 minutes.
To be sure that all the side products and unreacted materials were removed, after each step of the reaction the same washing procedure was used: addition of approximately 3 mL of DMF, manual shaking for one minute and removal of the solvent; this was repeated three times.
The reaction was started again by adding 3-4 mL of a solution of 20% in volume piperidine in
DMF. The reactor was stirred with a mechanical machine for approximately 15 minutes; then the
solvent and the by-products were removed, and the reactor washed.
The first coupling reaction was started by adding 0.4621 g [≈ 0.89 mmol, 4eq] of PyBOP (benzotriazol-tri-pirrolidin-phosphonium hexafluorophosphate), 0.3093 mL [≈ 1.78 mmol, 8eq] of
DIPEA (diisopropyl-ethyl-amine) and 4 equivalents of the first protected amino acid [≈ 0.89 mmol]
initially dissolved in the minimum quantity of DMF. The protections on the amino acid are the F-moc on the amine moiety, the trityl group on the thiol of the cysteine and the tertbutyl group on the phenol of the tyrosine.
The reactor was then put on the mechanical stirring machine for one hour and after that time the solvent was removed, and the resin washed.
1.78 mmol, 8eq] of DIC (diisopropyl carbodiimide) and 4 equivalents of the first protected amino acid [≈ 0.89 mmol], initially dissolved in the minimum quantity of DMF, were added.
The reactor was then put on the mechanical stirring machine and left there overnight. The solvent was then removed, and the resin washed.
In order to remove the F-moc moiety from the first amino acid, 3-4 mL of a solution of 40% in volume piperidine in DMF were added in the reactor, which was stirred with a mechanical machine for approximately 5 minutes, and then the reactor was washed.
The reaction was started again by adding 3-4 mL of a solution of 20% in volume piperidine in
DMF.
The reactor was stirred with a mechanical machine for approximately 15 minutes; then the solvent and the by-products were removed, and the reactor washed.
The first coupling reaction with the second amino acid was started by adding 0.4621 g [≈ 0.89 mmol, 4eq] of PyBOP (benzotriazol-tri-pirrolidin-phosphonium hexafluorophosphate), 0.3093 mL [≈ 1.78 mmol, 8eq] of DIPEA (diisopropyl-ethyl-amine) and 4 equivalents of the second protected amino acid [≈ 0.89 mmol], initially dissolved in the minimum quantity of DMF.
The reactor was then put on the mechanical stirring machine for one hour and after that time the solvent was removed, and the resin washed.
In order to obtain a high yield a second coupling reaction with the same amino acid was done. So, 0.1262 g [≈ 0.89 mmol, 4eq] of OxymaPure (ethyl-2-cyano-hydroxyimino-acetate), 0.278 mL [≈ 1.78 mmol, 8eq] of DIC (diisopropyl carbodiimide) and 4 equivalents of the second protected amino acid [≈ 0.89 mmol], initially dissolved in the minimum quantity of DMF, were added.
The reactor was then put on the mechanical stirring machine for one hour and a half and after that time the solvent was removed, and the resin washed.
In order to remove the F-moc moiety from the second amino acid, 3-4 mL of a solution of 40% in volume piperidine in DMF were added. The reactor was stirred with a mechanical machine for approximately 5 minutes; then the solvent and the by-products were removed, and the reactor washed.
The reaction was started again by adding 3-4 mL of a solution of 20% in volume piperidine in DMF and the reactor was stirred with a mechanical machine for approximately 15 minutes; then the solvent and the by-products were removed, and the reactor washed.
The liquid phase was removed and the solid was washed 3 times with approximately 4 mL of
methanol; in order to dry the resin, 5 mL of diethyl ether were added, and the resin was dried in
vacuum.
The next step was the detachment of the pedtide from the resin, for this reason 3 mL solution of 95% trifluoroacetic acid (TFA) 2.5% water and 2.5% triethyl silane were added and it was
immediately clear that the reaction had started: the transparent solution became yellow first and red shortly after, as well as the resin. The reactor was left on the stirring machine for 4 hours.
The solvent and the products were taken away and the resin was washed with approximately 2 mL of TFA. In order to eliminate some of the solvent recovered, the solution was purged with nitrogen. Then a solution of 9.5 mL of water with 0.5 mL of acetonitrile was added and the mixture became milky opaque.
The solution was lyophilized overnight, and a TLC of the product dissolved in water was taken in a solution 3:1:1 of butanol/acetic acid/water.
Cys-Tyr
The product was obtained with yields up to 74%.
MS (m/z, QMS-MS/MS): 285.0461 [M+ + H]. The MS spectrum was recorded in water.
Ala-Cys
The product was obtained with yields up to 82%.
1H NMR (400 MHz, D2O, d ppm): 4.25-4.20 (q, J = 7.3 Hz, 1H, CHAlanine), 4.13-4.11 (t, J = 5.4 Hz, H, CHCysteine), 3.04-2.92 (ddd, J = 27.6, 15.1, 5.7 Hz, 2H, CH2, Cysteine), 1.31-1.29 (d, J = 7.3 Hz, 3H, CH3, Alanine).
13C NMR (100 MHz, D2O, δ ppm): 177.2 (C=OAlanine), 167.7 (C=OCysteine), 53.9 (CHCysteine), 49.6 (CHAlanine), 25.0 (CH2, Cysteine), 16.5 (CH3, Alanine).
MS (m/z, QMS-MS/MS): 192.0798 [M+ + H]. The MS spectrum was recorded in water.
Cys-Ala
The product was obtained with yields up to 97%.
1H NMR (400 MHz, D2O, d ppm): 4.41-4.38 (t, J = 6.3 Hz, H, CHCysteine), 4.05-4.00
(q, J = 7.2 Hz, 1H, CHAlanine), 2.87-2.75 (ddd, J = 29.0, 14.3, 5.4 Hz, 2H, CH2, Cysteine), 1.44-1.42 (d, J = 7.1 Hz, 3H, CH3, Alanine)
13C NMR (100 MHz, D2O, δ ppm): 170.9 (C=OAlanine), 55.5 (CHCysteine), 48.9 (CHAlanine), 25.01(CH2, Cysteine), 16.4 (CH3, Alanine).
MS (m/z, QMS-MS/MS): 192.0802 [M+ + H]. The MS spectrum was recorded in water.
Synthesis of the Cysteine Acetylated-Alanine Amidated Dipeptide
Reaction number 1
Cysteine-alanine amidated dipeptide (previously synthesized), 0.1300 g [≈ 0.68 mmol], was
dissolved in 6 ml of a solution of 0.1% acetic acid in water (pH ≈ 3 checked with the pH paper indicator) and cooled down in an ice bath.
Then, 8.5 ml of a cold solution 0.5 M of acetic anhydride in acetonitrile was added.
The reaction was monitored for the firsts 10 minutes with TLCs, in a solution 3:1:1 of
butanol/acetic acid/water, stained in ninhydrin.
The product was then dried at the Schlenk line and checked at the NMR.
Something has reacted but not everything, the peaks in the NMR spectra are more than expected, but there are also the ones of the product.
1H NMR (300 MHz, D2O, d ppm): 4.41-4.38 (t, J = 6.3 Hz, H, CHCysteine), 4.06-4.00 (q, J = 7.2 Hz, 1H, CHAlanine), 2.85-2.75 (ddd, J = 21.4, 14.3, 7.3 Hz, 2H, CH2, Cysteine), 1.96 (s, 3H, CH3, Acetyl), 1.44-1.42 (d, J = 7.2 Hz, 3H, CH3, Alanine).
Reaction number 2
The amidated and acetylated dipeptide was synthesized following the same steps reported for the syntheses of the amidated dipeptide, the only difference was the introduction of the acetylation reaction.
The acetylation reaction was done after removing the F-moc protection from the amine group of the cysteine and before detaching the dipeptide from the resin.
prepared and added to the reactor, were the dipeptide was attached to the resin with the free amine moiety. The reactor was left stirring on the stirring machine for 15 minutes.
The liquid phase was removed and the solid was washed 3 times with approximately 4 mL of
methanol, and then 5 mL of diethyl ether, before drying it in vacuum. After that, the acid solution
was added in order to detach the protected dipeptide from the resin.
From the NMR and MS spectra it seems the desired product was obtained, but the IR spectrum did not have the SH band, this means that the dimer was obtained instead.
1H NMR (400 MHz, D2O, d ppm): 4.38-4.35 (t, J = 6.3 Hz, H, CHCysteine), 4.20-4.15 (q, J = 7.2 Hz, 1H, CHAlanine), 2.86-2.75 (ddd, J = 24.7, 14.4, 5.3 Hz, 2H, CH2, Cysteine), 1.90 (s, 3H, CH3, Acetyl), 1.27-1.25 (d, J = 7.2 Hz, 3H, CH3, Alanine).
13C NMR (100 MHz, D2O, δ ppm): 175.5 (C=OAlanine), 174.3 (C=OCysteine), 55.2 (CHCysteine), 49.9 (CHAlanine), 25.1 (CH2, Cysteine), 21.5 (CH3, Acetyl), 16.3 (CH3, Alanine).
MS (m/z, QMS-MS/MS): 256.0735 [M+ + Na] The MS spectrum was recorded in water.
IR (ATR, cm-1): 3388 (N-H stretching), 1624 (C=O amide stretching).
Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (L-Cysteine)
Reaction number 1
L-cysteine, 0.0126 g [≈ 9.90x10-2 mmol], and K
2CO3, 0.0055 g [≈ 4.00x10-2 mmol] were put in a
flask with water, 5 mL, and DMF, 1 mL. The mixture was left stirring for approximately 20 minutes.
In the meantime, NHC-Au-Cl, 0.0622 g [≈ 9.90x10-2 mmol] was dissolved in DMF, 7 mL, and then added to the amino acid solution.
The reaction was left stirring overnight, and it was monitored with TLC plates, taken in a solution 5:3 of cyclohexane/ethyl acetate, stained with ninhydrin.
After 24 hours the solvent was removed, and the product was separated from the potassium
organic phase was removed and the product was dissolved in deuterated chloroform in order to get an NMR spectrum.
There was no sign of the product in the spectrum, only the NHC-Au-Cl peaks were visible.
Reaction number 2
L-cysteine, 0.0106 g [≈ 8.25x10-2 mmol], and K
2CO3, 0.0105 g [≈ 8.25x10-2 mmol] were put in a
flask with water, 3 mL, and DMF, 2 mL. The mixture was left stirring for approximately 15 minutes.
In the meantime, NHC-Au-Cl, 0.0524 g [≈ 8.25x10-2 mmol] was dissolved in DMF, 8 mL, and then added to the amino acid solution.
The reaction was left stirring overnight and it was monitored with TLC plates, taken in a solution 5:3 of cyclohexane/ethyl acetate, stained with ninhydrin.
After 24 hours, the solvent was removed, and the product was separated from the potassium
carbonate adding ethyl acetate and water, 30 mL and 5 mL respectively. The organic phase was
dried, and the powder dissolved in CDCl3 in order to take an NMR spectrum.
There was no sign of the product in the spectrum, only the NHC-Au-Cl peaks were visible.
Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (N-Acetyl-L-Cysteine)
Reaction number 1
N-Acetyl-L-Cysteine, 0.0130 g [≈ 0.07 mmol], was dissolved in EtOAc, approximately 2 mL.
Separately, the gold complex, 0.0500 g [≈ 0.07 mmol], was stirred in EtOAc, 8 mL, the minimum amount of solvent required to dissolve the compound.
After half an hour the solution with the gold complex was added to the protected peptide, washing with 5 mL of solvent, for a total of 15 mL, and the flask was put under a controlled atmosphere of N2.
The reaction was monitored with TLC plates, taken in a solution of cyclohexane/ethyl acetate 1:1, stained with ninhydrin.
After 24 hours the reaction was stopped, all the solvent evaporated at the rotavapor and the flask connected to the Schlenk line.
From the NMR spectrum of the crude seemed that something had reacted but not everything. The reaction was started again, adding a base.
Reaction number 2
The reaction mixture from the first reaction was dissolved again in EtOAc, 15 mL, and K2CO3, 0.0222 g [≈ 0.14 mmol], was added as base.
The reaction was monitored with TLC plates, taken in a solution of ethyl acetate/cyclohexane 1:1, stained with ninhydrin.
After 24 hours the reaction was stopped and a solution 8% HCl, 10 mL was added. In order to neutralize the acid a saturated solution of sodium carbonate was added checking the pH of the resulting solution.
The aqueous phase was washed twice with some millilitres of EtOAc. MgSO4 was then added to the
organic phase in order to remove all the water from the solution. The salt was filtered and the
EtOAc evaporated from the flask.
There was no sign of the product in the spectrum, only the NHC-Au-Cl peaks were visible.
Reaction number 3
N-Acetyl-L-Cysteine, 0.0130 g [≈ 0.07 mmol], and the gold complex, 0.0500 g [≈ 0.07 mmol], were
dissolved in EtOAc, 15 mL. The mixture was stirred for four hours to let the reagents dissolve completely and the reaction start, under a controlled atmosphere of N2. After 4 hours, K2CO3, 0.0050 g [≈ 0.04 mmol], was added to push the reaction towards the product.
The reaction was monitored with TLC plates, taken in a solution of ethyl acetate/cyclohexane 1:1, stained with ninhydrin.
After 24 hours the reaction was stopped, the base was filtered, all the solvent evaporated, and an NMR spectrum was taken.
Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (N-Acetyl-L-Cysteine-Methyl Ester)
Reaction number 1
N-acetyl-L-cysteine methyl ester (previously synthesized), 0.0140 g [≈ 0.07 mmol], and K2CO3,
0.0220 g [≈ 0.07 mmol] were stirred for 15 minutes, under a controlled atmosphere of N2, in approximately 15 mL of acetone. Then, the gold complex, 0.0500 g [≈ 0.07 mmol], was added. The reaction was monitored with TLC plates, taken in a solution 1:1 of cyclohexane/ethyl acetate, stained with ninhydrin.
After 24 hours the reaction was stopped, all the solvent evaporated and a solution 8% HCl, 10 mL, was added. The mixture was stirred for a few minutes before adding CH2Cl2, 10 mL. A solution of saturated sodium bicarbonate and one of saturated sodium chloride were added before separating the two phases and drying the organic one over magnesium sulfate. The dried solution was filtered, and all the solvent was evaporated.
There was no sign of the product in the spectrum, only the gold reagent and by-products.
Reaction number 2
N-acetyl-L-cysteine methyl ester (previously synthesized), 0.0150 g [≈ 0.07 mmol], and K2CO3,
0.0220 g [≈ 0.14 mmol] were stirred for 15 minutes, under a controlled atmosphere of N2, in approximately 15 mL of ethyl acetate. Then, the gold complex, 0.0500 g [≈ 0.07 mmol], was added. The reaction was monitored with TLC plates, taken in a solution 1:1 of cyclohexane/ethyl acetate, stained with ninhydrin.
After 24 hours the reaction was stopped, the base was filtered, all the solvent was evaporated, and an NMR spectrum was taken.
There was no sign of the product in the spectrum, only of the reagents.
Reaction number 3
The reagents mixture from the third reaction was dissolved again in EtOAc, 15 mL, and K2CO3,
0.0440 g [≈ 0.28 mmol], was added in the flask, under a controlled atmosphere of N2. The reaction was left stirring.
The reaction was monitored with TLC plates, taken in a solution 1:1 of cyclohexane/ethyl acetate, stained with ninhydrin.
After 24 hours the reaction was stopped, the base was filtered, and all the solvent was evaporated. From the NMR spectrum of the crude it was not clear if the mixture had reacted completely, so a column was started in a Pasteur pipette (cotton at the bottom and silica), using a solution of
cyclohexane/ethyl acetate 1:1 as eluent. The product was dissolved in CH2Cl2, 4 mL, and the
fractions were collected every 1 mL. A TLC of each fraction was taken in a solution of
cyclohexane/ethyl acetate 1:1.
There was no sign of the product in the spectra, only the gold reagent, the protected amino acid and by-products.
Reaction number 4
N-acetyl-L-cysteine methyl ester (previously synthesized), 0.0130 g [≈ 0.07 mmol], and HK2PO4,
0.0250 g [≈ 0.14 mmol] were stirred for 15 minutes, under a controlled atmosphere of N2, in approximately 15 mL of ethyl acetate. Then, the gold complex, 0.0500 g [≈ 0.07 mmol], was added. The reaction was monitored with TLC plates, taken in a solution 1:1 of cyclohexane/ethyl acetate, stained with ninhydrin.
After 24 hours the reaction was stopped, the base filtered, and all the solvent was evaporated. From the NMR spectrum of the crude it was not clear if the mixture had reacted, so a column was started in a Pasteur pipette (cotton at the bottom and silica), using a solution of cyclohexane/ethyl
acetate 1:1 as eluent. The product was dissolved in CH2Cl2, 4 mL, and the fractions were collected
every 1 mL. A TLC of each fraction was taken in a solution of cyclohexane/ethyl acetate 1:1.
There was no sign of the product in the spectra of the different fractions, only the gold reagent, the protected amino acid and by-products.
Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (N-Acetyl-L-Cysteine Ethyl Ester)
N-acetyl-L-cysteine ethyl ester (previously synthesized), 0.1429 g [≈ 0.75 mmol], K2CO3, 0.1033 g
[≈ 0.75 mmol], and NHC-Au-Cl, 0.2207 g [≈ 0.35 mmol] were added in a flask with ethyl acetate, 50 mL, under nitrogen atmosphere for 24 hours.
The reaction was monitored with TLC plates, taken in a solution 1:1 of cyclohexane/ethyl acetate, stained with ninhydrin.
The reaction was stopped, the base was filtered, the product was dried and analysed at the NMR. The product was purified using a small column in a Pasteur pipette (cotton at the bottom and silica). The product was dissolved in CH2Cl2, 4 mL and then a solution EtOAc/Cyclohexane 1:1 was used
as eluent. The fractions were collected every 1 ml. A TLC of each fraction was taken in
EtOAc/Cyclohexane 1:1.
The product was dried again and analysed at the mass spectrometer and at the NMR.
After the purification, the product was obtained with a yield of 60%.
1H NMR (300 MHz, CDCl3, d ppm): 7.33 - 7.30 (m, 2H, CHbenzyl + CHphenyl), 7.24 – 7.22 (m, 10H, CHbenzyl + CHphenyl), 7.04 – 6.96 (m, 8H, CHbenzyl + CHphenyl), 6.72 (s, H, NH), 5.44 (s, 4H, CH2 benzyl), 4.71-4.68 (dt, J = 7.6, 4.8 Hz 1H, CH), 4.15-4.11 (m, 2H, CH2 ester), 3.38-3.23 (ddd, J = 28.2, 13.1, 5.3, 2H, CH2 thiol), 1.95 (s, 3H, CH3 acetyl), 1.25 – 1.20 (t, J = 7.1 Hz, 3H, CH3 ester).
IR (ATR, cm-1): 1732 (C=O ester stretching), 1661 (C=O amide stretching). MS (m/z, QMS-MS/MS): 788.2221 [M+ + H]
The MS spectrum was recorded in acetonitrile.
Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (Alanine-Amidated Cysteine dipeptide)
Reaction number 1
Alanine-Amidated Cysteine dipeptide (previously synthesized), 0.0077 g [≈ 0.04 mmol], and NHC-Au-Cl, 0.0253 g [≈ 0.04 mmol] were added in a flask, in a biphasic environment, 5 mL of deionized water and 5 mL of ethyl acetate, under nitrogen atmosphere.
The reaction was stopped after 24 h and the two phases were separated. The organic phase was collected, and all the solvent was evaporated, in order to take an NMR spectrum.
There was no sign of the product in the spectrum.
Reaction number 2
The reagents mixture from the first reaction was dissolved again in a biphasic environment, 5 mL of
deionized water and 5 mL of ethyl acetate, under nitrogen atmosphere. K2CO3, 0.0055 g [≈ 0.04
mmol], was added and the reaction was left stirring.
The reaction was monitored with TLC plates, taken in a solution 5:3 of cyclohexane/ethyl acetate, stained with ninhydrin.
After 18 hours the reaction was stopped, and the two phases were separated. The organic phase was collected, and all the solvent was evaporated, in order to take an NMR spectrum.
There was no sign of the product in the spectrum, only of gold reagent.
Synthesis of (1,3 dibenzyl - 4,5 diphenylimidazol - 2 ylidene) Gold(I) (Cysteine Acetylated-Alanine Amidated)
Cysteine acetylated-alanine amidated (previously synthesized), 0.0280 g [≈ 0.06 mmol], K2CO3,
0.0440 g [≈ 0.14 mmol], were stirred for 10 minutes, under a controlled atmosphere of N2, in approximately 15 mL of ethyl acetate. Then, the gold complex, 0.0370 g [≈ 0.06 mmol], was added. The reaction was monitored with TLC plates, taken in a solution 1:1 of cyclohexane/ethyl acetate, stained with ninhydrin.
The reaction was stopped after 24 h, the base was filtered, the product was dried and analysed at the NMR.
RESULTS AND DISCUSSION Synthesis of the imidazolium salt
The first reaction involves the synthesis of the N-heterocyclic carbene ligand, obtained through quaternization of both nitrogen atoms. The reaction is carried out following the strategies already tested by the research group[15], as reported in Scheme 1.
Scheme 1 – Synthesis of the imidazolium salt
The base, potassium carbonate, was necessary to deprotonate the reagent in order to facilitate the nucleophilic attack on the benzyl bromide. The solvent, acetonitrile, was chosen for two reasons: its ability to improve the kinetics of the process through its polarity and its capacity to stabilize the ionic product. The imidazolium salt was obtained with high yields, up to 80%.
The peak at 11.25 ppm, in the 1H NMR spectrum, is the most interesting since it is caused by the presence of the imidazolium proton, the hydrogen linked to the carbon located between the nitrogen atoms. The high chemical shift is related to the elevated acidity of the proton. Between 7.46 and 7.08 ppm there are multiplets for the phenyl hydrogens and at 5.51 ppm there is a singlet that integrates 4 for the two -CH2 of the benzyl moieties, Fig. 10.
Scheme 1 Scheme 2 N N N N H Br + 2 K2CO3 CH3CN RT, 3 d H Br N NH H Br Ag2O DCM RT, 4 h Darkness N N AgBr AuS(CH3)2 DCM RT, 6 h Darkness N N AuCl
Fig. 10 - 1H NMR spectrum of 1,3 dibenzyl - 4,5 diphenyl imidazolium bromide
Synthesis of Gold(I) NHC Chloride
The most direct way to obtain in situ the free carbene ligand from an imidazolium salt requires the use of a strong base, which unfortunately is not compatible with the gold precursor, chloro dimethyl sulphide gold(I)[21]. For this reason, it was necessary to synthesize an intermediate silver carbene complex, that was then transmetallated into the gold compound. The advantage of this method is that it is not necessary to generate the free carbene. Furthermore, the reaction can be performed in air and there are not signs of decreased yield nor decomposition.
The only caution needs to be towards carrying out the reaction under the exclusion of light, since silver salts are light sensitive. After generating the NHC-Ag(I) bromide complexes, using 0.5 equivalents of silver oxide, it follows the addition in situ of the gold(I) precursor, as shown in Scheme 2. 1H NMR 300Hz CDCl3 N N H Br
Scheme 2 – Synthesis of the Gold(I) NHC Chloride
The product is easily separated from the solution, precipitating it with pentane, and it can be stored without specific precautions.
The characterization is done again with NMR spectra. The main feature of the 1H NMR spectrum is the disappearance of the peak at 11.25 ppm, indicating that the carbene carbon is not linked to an hydrogen anymore. On the other hand, there are still the multiplets of the phenyl hydrogens at approximately 7 ppm and the singlet for the two -CH2 of the benzyl moieties around 5.51 ppm. Previous works[15] also reported the 13C NMR spectrum where the characteristic carbene carbon peak appears at 171.45 ppm, Fig. 11.
Fig. 11- 1H NMR spectrum of (1,3 dibenzyl - 4,5 diphenyl imidazol - 2 ylidene) Gold(I) Chloride
N N H Br Ag2O DCM RT, 4 h Darkness N N AgBr AuS(CH3)2 DCM RT, 6 h Darkness N N AuCl N N AuCl 1H NMR 300Hz CDCl3
Esterification reactions
The esters, N-acetyl-L-cysteine-methyl ester and N-acetyl-L-cysteine-ethyl ester, were made via Fisher esterification technique by refluxing the N-acetyl precursors in methanol or ethanol in the presence of catalytic amount of H2SO4, Scheme 3.
Scheme 3 - General synthesis of the methyl and ethyl ester derivatives of N-acetyl-L-cysteine
The methyl ester was obtained as small transparent crystals, slowly formed after being dried form the CDCl3 used to do the NMR spectrum. In the 1H NMR spectrum, reported in Fig. 12, around 6.4 there is the broad peak of the NH, at 4.9 ppm the doublet of triplets of the CH, at 3.0 ppm the characteristic second order multiplet of the diasterotopic hydrogens of the thiol CH2, at 2.7 ppm the singlet of the acetyl CH3 and at 1.3 ppm the triplet of the SH hydrogen, that are the same peaks of the reagent; whereas at 3.8 ppm appears the singlet of the methyl ester hydrogens. The assignment of the peaks was confirmed also by the gCOSY spectrum, Fig. 13.
O OH HS HN O ROH H2SO4 (cat) Reflux, 24 h O OR HS HN O R = -CH3, -CH2CH3
Fig. 12 - 1H NMR spectrum of N-acetyl-L-cysteine methyl ester
Fig. 13 - gCOSY NMR spectrum of N-acetyl-L-cysteine methyl ester
1H NMR 300Hz CDCl3 O O HS HN O gCOSY NMR 300Hz CDCl3 O O HS HN O
In the 13C NMR spectrum, Fig. 14, there are the 6 peaks of the carbons of the molecule, which have been assigned using gHSQCAD, and gHMBCAD, bidimensional techniques.
Fig. 14 – 13C NMR spectrum of N-acetyl-L-cysteine methyl ester
The characterization of the product was done also through the FT-IR spectrum, where, at 2565 cm-1, appears the S-H stretching.
The ethyl ester, on the other hand was obtained as an oil, even after various attempts of purification on silica columns and of precipitation with different solvents. The 1H NMR spectrum, Fig. 15, showed however that the product was obtained. There are, in fact, the same peaks of the reagent: around 6.4 the broad peak of the NH, at 4.9 ppm the doublet of triplets of the CH, at 3.0 ppm the multiplet of the CH2, and at 2.0 ppm the singlet of the acetyl CH3; while at 4.3 ppm there is the multiplet of the CH2 hydrogens of the ester and around 1.3 ppm there is a multiplet that integrates 4, that is probably a combination of the peaks of the CH3 hydrogens of the ester and of the triplet of the SH. 13C NMR 100Hz CDCl3 O O HS HN O
Fig. 15 - 1H NMR spectrum of N-acetyl-L-cysteine ethyl ester
Dipeptide syntheses
The dipeptide synthesis follows the same steps, independently from the amino acid sequence. The selected procedure is a solid phase peptide synthesis that allows the rapid assembly of the dipeptide through successive reactions of amino acid derivatives on an insoluble porous support[22].
The Rink Amide AM Resin from Iris Biotech GMBH was used in all the synthesis, Fig. 16.
Fig. 16 – Structure of the Rink Amide AM Resin
1H NMR 300Hz CDCl3 O O HS HN O N H O O H N H3CO OCH3 O O
The resin is polystyrene based with 1% of divinylbenzene; the certificate states that the resin has 0.74 mmol active sites per gram and the active sites are protected with the F-moc moiety (fluorenyl methyloxicarbonyl ester), so a deprotection step is needed before the reaction with the first amino acid.
The removal of the F-moc moiety is done using piperidine and the reaction follows the mechanism presented in Scheme 4.
Scheme 4 – Removal of the F-moc moiety mechanism
The amidic part of the resin is converted through this reaction into the free amine, which will be able to react with the carboxylic moiety of the amino acid.
The next step is the coupling with the first amino acid. The amino acids used are protected on the amine moiety, with the F-moc, and on the reactive sites on the side chains, if there are any, with the trityl group for the thiol in the Cysteine and with the tButyl group for the alcohol in the L-Tyrosine.
Eight equivalents of each amino acid have been used in total for the reaction, in order to push it towards a 100% yield. Two coupling reagents were chosen, Oxime pure (ethyl-2-cyano-hydroxyimino-acetate) and PyBOP (benzotriazol-tri-pirrolidin-phosphonium hexafluorophosphate), and, in the presence of two co-reactants, DIC (diisopropyl carbodiimide) and DIPEA (diisopropyl-ethyl-amine) respectively. The reactions were performed with 4 equivalents of each amino acid, 4 equivalents of the activating agent and 8 equivalents of the corresponding base.
FA06 Resin F-moc deprotection
FA06 Activation of the amino acid with Oxime pure and DIC mechanism
R NH(Fmoc) N C N + O OH R NH(Fmoc) O O N NH + O O NC N HO R NH(Fmoc) O O N CN O O N H NH O + R = -CH3, -CH2S(Trityl), -CH2Ph[pO(tButyl)] NHR O O NH H NHR O O + N H2 + NHR O O + N H2 N H + CO2 + NH2R R = Resin 9-methylene-9H-fluorene
