It was now required to find conditions under which these various carboxylic acids 1a-6a could be reliably coupled to cysteine in order to give a library of potential mimics. Initially, conditions similar to those used for the synthesis of di(O-ethyl-L-cysteinyl)-5-bromoisophthalamide using EDCI as a coupling reagent were used. While in some cases the desired products were detected by mass spectrometry, invariably these reactions did not proceed cleanly or fully to completion giving mixtures of singly and doubly functionalised fluorophores. Reactions with L-cysteine ethyl ester were also attempted via the formation of acid chlorides from the respective acids. In reactions where a significant amount of product was obtained, purification of these products proved problematic. Due to the ease of oxidation of the free thiols in these products, attempts to purify these compounds by recrystallisation or column chromatography resulted in mixtures equally or more complex than the initial reaction products. Evidence from mass spectrometry suggested that oxidative disulfide formation could be responsible for some of this observed instability. In the case of the singly functionalised compounds this was necessarily intermolecular disulfide formation while the doubly functionalised mimics showed more complex mixtures of possible intermolecular and intramolecular disulfides.
It was decided to attempt the couplings with a range of S-protected cysteines that would give products stable enough to be isolated and purified which could then be deprotected when required. Various methods of amide formation were attempted with several different protected cysteines which were either obtained from commercial sources or synthesised as shown in Scheme 4.20.
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Scheme 4.20 Preparation of non-commercially available protected cysteines.
The results of the various couplings are summarised in Table 4.1. For reactions using carbodiimide coupling reagents the carboxylic acids were treated with the carbodiimide at 0 C before addition of the cysteine derivative. Where attempts were made using either acid chlorides or N-hydrosuccinimide (NHS) active esters as intermediates, the starting acids were reacted to form the intermediate and the crude product redissolved after work-up and treated with the cysteine derivative. The majority of reactions were tested on the acids 2a and 3a as more of these two materials were available at the time.
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Cysteine Acids used Conditions* Result
CO2Et
H2N
SH
2a EDCI Incomplete
2a EDCI, HOBt Incomplete
2a EDCI, Heat No Product
2a SOCl2 No Product
2a, 3a, 4a (COCl)2 No Product. Evidence of SM for 3a CO2Et H2N S NHEt O 2a (COCl)2 No product
2a, 3a EDCI Some impure product. Failure in deprotection
CO2H
H2N
S NHEt O
3a EDCI Acetyl transfer giving
EDU† adduct
2a EDCI, HATU, DMAP No Product
CO2H
H2N
S S
2a EDCI, HATU, DMAP No Product
1a, 2a, 3a NHS, DCC No Product. Some starting materials recovered 2a NHS, EDCI No Product CO2Me H2N S S
1a-6a EDCI Successful coupling. Products stable in column
chromatography except N^C^N complexes
Table 4.1 Peptide coupling reactions.
There was some success in the coupling of S-(N-ethylaminocarbonyl)-cysteine ethyl ester using EDCI, where the correct products were obtained and purification was feasible using column chromatography, although the products tended to separate poorly from some impurities. Attempts to remove the carboxamide protecting groups using NaHCO3 (aq), a pH 10.6 NaHCO3/NaOH
buffer or 0.1M NaOH were unsuccessful, with only unreacted material recovered from the reaction. However, when the deprotection was attempted on the singly functionalised iridium complex, 3a, using 1M NaOH, the product
*
Reactions were either carried out in dry THF for the organic fluorophores or a mixture of dry THF and either DCM or dry acetonitrile for iridium complexes where solubility in pure THF was poor.
†
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isolated was not the free thiol as expected but the complex shown in Figure 4.7, identified by HRMS. It was theorised that instead of attack of hydroxide as a nucleophile at the carboxamide functional group, the hydroxide acted as a base, removing the ester -hydrogen leading to loss of sulfur as shown in Scheme 4.21. This is unusual since the original report of the use of this protecting group found no evidence of dethiolation.234
Figure 4.7 Unexpected product of deprotection.
Scheme 4.21 Mechanisms for the desired removal of carboxamide protecting group (above)
and the undesired dethiolation reaction (below).
It was therefore decided to attempt the coupling with the free acid equivalent, S- (ethylaminocarbonyl)-L-cysteine, since the -hydrogen in this case would not be acidic so this side reaction could be avoided. However, as with all the coupling reactions attempted on cysteines with unprotected acid functional groups, no product was obtained.
In fact, as noted in Table 4.1, when this cysteine was reacted with 3a, there was evidence from mass spectrometry of acetyl transfer after reaction with EDCI giving an N-acylurea species as shown in Scheme 4.22. This tends to occur in
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the use of carbodiimides when reaction with the amine is very slow allowing the competing intramolecular acetyl transfer reaction to take over.
Scheme 4.22 Acetyl transfer giving N-acylurea in reactions with carbodiimides.
In the end, S-(tert-butylmercapto)-L-cysteine methyl ester was found to give reproducible results in couplings using EDCI with compounds 1a-4a giving products stable enough to isolate and purify by column chromatography. As a result of these reactions the four compounds, 1b-4b, shown in Figure 4.8 were available for testing as potential PDI mimics. Given the likelihood of instability with respect to oxidation after deprotection, these compounds were stored in their protected forms until required for assays.
Figure 4.8 S-protected potential luminescent PDI mimics 1b, 2b, 3b and 4b.
Reactions using the two Ir(N^C^N) complexes 5a and 6a, however, were less successful, yielding mixtures of products. The desired compounds were
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possibly isolated in low yield by column chromatography and identified by mass spectrometry, however these products were not sufficiently pure and their identity could not be confirmed by other spectroscopic methods such as NMR. In the case of the singly functionalised complex 5a, one of the major side products isolated and identified (in approx 50% yield) was the acetyl transfer product, indicating that one possible issue with these structures is slow reactivity between the EDC activated acid and the cysteine amine. Unfortunately, despite these complexes with terdentate ligands showing promise, a lack of material and insufficient time to synthesise fresh complexes meant that the synthesis of potential mimics using these particular luminophores could not be completed.