Synthetic scale-up of a novel fluorescent probe and its biological evaluation for surface detection of Staphylococcus aureus.

TL;DR: The LGX fluorometric test for enzymatic MRSA/MSSA detection is reported on, highlighting the reasons rhodamines have been overlooked and also strategies to improve the synthesis of rhodamine-peptide conjugates.

About: This article is published in Molecular and Cellular Probes.The article was published on 2017-12-01 and is currently open access. It has received 2 citations till now.

Summary

Introduction

• M AN US CR AC CE PT ED 1 highlights the reasons rhodamines have been overlook d and also strategies to improve the synthesis of rhodamine-peptide conjugates.
• This drawback is demonstrated in the UK insofar that PCR is not routinely used to detect problem pathogens such as MRSA.
• The change in rhodamine's fluorescence quantum yield from its unconjugated (lactone form), to conjugated (zwitterionic form) is considerable (Rho 110 φlactone = 0.006 φzwitterion = 0.98).
• The elemental analysis deviates from the empirical formula, but water and HCl are inexplicably added to bring the observed values within olerance.
• The biological testing of the serine protease activated rhodamine as then reported bears out the fact that whatever was synthesised is impure.

2.1 The xanthone approach

• The aim was to achieve a better amino acid coupling yield and introduce the lactone ring at a later stage.
• These conditions were adapted to produce the initial required nitro amino xanthone (3) for further modification towards a number of novel unsymmetrical rhodamines for further study.
• The initial step is an Ullmann condensation requiring high temperatures and long reaction times.
• It is possible that attack on the deactivated ketone group of the xanthone was slower than on the nitro group demonstrating the incompatibility of nitro arenes with Grignard reagents, aiding explanation of the observed results.

2.1.3 Directed metalation

• As one of the better known routes to aryl containing structures, directed metalation was investigated in the context of rhodamine conjugate synthesis.
• The Wuts group explored the use of dialkyl hydrazides as directed lithiating agents and this showed promise.
• After confirmation, the same reaction conditions were applied to the simplest unfunctionalised xanthone core structure (6) with success and supplementary information for data SI – 9).
• Acidification with concentrated HCl resulted in the slow formation of the spirophthalide which crystallised over night to form needle like white crystals which were washed with cold ether and isolated.
• Upon examinatio by 1H NMR these appeared to be the expected product, M AN US CR IP T AC CE PT ED 7 the previously unreported rhodamine core structure 21.

2.2.1 Optimising coupling agents and conditions

• The ability to scale-up the synthesis of rhodamine conjugates for affordable, real-world application had, at that point not been achieved, and the authors turned their attention back to the original coupling approaches to the rhodamine 110 core structure (2).
• The problems associated with the coupling of arginine to rhodamine are a useful exemplar reaction to the coupling of any amino acid to rhodamine.
• 21,22 Equally as important, the yield was consistent throughout several runs and represents a sound method for scaling the initial coupling.

2.2.2 Scale-up problems with deprotection of Cbz groups

• The previously reported synthesis of (Boc-Val-Pro-Arg)2–Rhodamine involves the direct attachment of an exhaustively CBz protected Boc-arginine with a particularly poor yield (17%), with subsequent amino acids being added prior to deprotection via hydrogenation.
• This approach is not amenabl to scale-up and larger amounts of (Boc-Val-Pro-Arg(CBz)2)2–Rhodamine cannot be successfully deprotected either by using standard or non-standard hydrogenation techniques.
• Since all hydrogenation steps failed to selectively r move the CBz groups and leave the central lactone intact when scaled up, an alternative approach was adopted.
• The authors were delighted to observe the reappearance of the lactone functionality through disappearance of the signal for the benzylic proton in question.
• Details of the synthetic chemistry, including general experimental spectroscopic data are available in supplementary information.

2.3.1 Preparation of bacterial samples

• Clinical isolates of MRSA, MSSA, S. epidermidis and E. coli, were revived from - 80°C and cultured overnight on Brain Heart Infusion (BHI) agar at 37°C, followed by two subcultures on nutrient agar.
• These were placed in an incubator overnight to ensure thorough drying of each plate.
• Swabbing was performed as described above in the experimental section.
• This process was repeated 3 times and the results shown in Figure 9 and 10 are averages with standard deviation error bars.

2.3.2 Preparation of LGX

• The appropriate amount of human prothrombin in 1 x PBS was then added to produce final prothrombin concentrations of 83.6 nM and 41.8 nM, respectively.
• The concentrations of LGX, subsequently discussed, refer to the concentration of the active fluorophore 1.
• The presence of coagulase positive bacteria was determined by addition of 100 µM LGX solution followed by5µL prothrombin.
• The fluorescence was then recorded (excitation 488 nm and emission 525 nm) at 15 minute intervals over a 2.5 hour time period using a benchtop fluorimeter (BMG FLUOstar OPTIMA).

3. Discussion

• As can be observed, the majority of samples test poitive for MRSA (denoted by a fluorescence reading exceeding 20,000 RFU).
• Figure 7 demonstrates the effects of co-culturing E. coli and S. epidermidis on the efficacy of detecting MRSA using the LGX system.
• It is noted that the presence of FBS in the experimental system may interfere with the fluorescence potential of LGX, and this requires further investigation.

Figures

Figure 9. Fluorescence response of LGX for the different co-cultures of MRSA (20% of 103 CFU/ml) over 2.5 hours. Results also include LGX (100uM), Negative Control (E. coli 103 CFU/ml) and Postive Control (MRSA 103 CFU/ml).