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Journal ArticleDOI

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

01 Dec 2017-Molecular and Cellular Probes (Academic Press)-Vol. 36, pp 1-9

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.

AbstractThis paper reports on the LGX fluorometric test for enzymatic MRSA/MSSA detection. It highlights the reasons rhodamines have been overlooked and also strategies to improve the synthesis of rhodamine-peptide conjugates. Evaluation of the LGX test for detection of MRSA/MSSA on surfaces is undertaken in the presence of potentially confounding E. coli and S. epidermidis for the first time.

Summary (2 min read)

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.

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Accepted Manuscript
Synthetic scale-up of a novel fluorescent probe and its biological evaluation for
surface detection of Staphylococcus aureus
Luke Bywaters, Lauren Mulcahy-Ryan, Mark Fielder, Alex Sinclair, Adam Le Gresley
PII: S0890-8508(17)30066-X
DOI: 10.1016/j.mcp.2017.06.006
Reference: YMCPR 1301
To appear in:
Molecular and Cellular Probes
Received Date: 28 March 2017
Revised Date: 1 June 2017
Accepted Date: 25 June 2017
Please cite this article as: Bywaters L, Mulcahy-Ryan L, Fielder M, Sinclair A, Le Gresley A,
Synthetic scale-up of a novel fluorescent probe and its biological evaluation for surface detection of
Staphylococcus aureus, Molecular and Cellular Probes (2017), doi: 10.1016/j.mcp.2017.06.006.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
1
Title: Synthetic scale-up of a novel fluorescent probe and its
biological evaluation for surface detection of Staphylococcus aureus
Luke Bywaters
1
, Lauren Mulcahy-Ryan
2
, Mark Fielder
2
, Alex Sinclair
1
and Adam Le
Gresley
1
*
1
Chemistry and Pharmaceutical Sciences, SEC Faculty, Kingston University, Kingston-upon-Thames,
KT1 2EE, United Kingdom.
2
Applied and Human Sciences, SEC Faculty, Kingston University, Kingston-upon-Thames, KT1 2EE, United
Kingdom
* Corresponding author
Abstract:
This paper reports on the LGX fluorometric test for enzymatic MRSA/MSSA detection. It
highlights the reasons rhodamines have been overlooked and also strategies to improve the synthesis of
rhodamine-peptide conjugates. Evaluation of the LGX test for detection of MRSA/MSSA on surfaces is
undertaken in the presence of potentially confounding E. coli and S. epidermidis for the first time.
Keywords: Rhodamine, Synthesis, Peptide, Fluorescence, Conjugate, Protease, Pathogen
Introduction
The growth in the number of infections caused by antibiotic resistant pathogens has prompted healthcare
agencies around the world to generate strategic plans.
1
These plans incorporate the restriction of antibiotic use to
slow the emergence of resistant pathogens, as well as the development of new antibiotics, to which resistance
will inevitably emerge. The former part of this strategy requires rapid, affordable diagnostics to determine the
best clinical course of action.
2
The principle drawbacks of PCR are the laboratory requirements and the expense of each test. This drawback is
demonstrated in the UK insofar that PCR is not routinely used to detect problem pathogens such as MRSA.
Bacterial culture is time-consuming (48-72hrs) and as reported in the NHS NOW report specifically concerning
MRSA, it is often the case that patients are discharged before the results of the test are known.
3
PCR techniques rely upon the enzymatic amplification of a gene until sufficient quantities can be detected.
Bacterial culture relies upon the growth rate of a bacteria, augmented by the best media. An alternative approach
involves the targeting of enzymes expressed by bacteria, which cleave between specific amino acid sequences.
4
To observe the actions of bacterially expressed enzymes and in common with other detection methods, a
chromogenic response is arguably the most useful. This is evidenced by the recently reported rhodamine based

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2
fluorogenic probe (1). This probe, (incorporated into a test subsequently referred to as LGX) has shown good
selectivity and sensitivity for both Methicillin Resistant Staphylococcu aureus (MRSA) and Methicillin
Sensitive Staphylococcus aureus (MSSA).
5
Figure 1. Fluorogenic Probe in LGX test (1)
Rhodamine 110 (2) represents one of the most active fluorophores known, with a high extinction coefficient and
a fluorescence quantum yield which is near unity. Rhodamine has been used as a marker in a wide variety of
enyzmic activity studies (serine protease
6
, esterase
7
, caspase
8
, DT diaphorase
9
). Essentially the NH
2
groups of
rhodamine 110 are conjugated to a polypeptide, which mimics the natural substrate of the enzyme in question.
Upon cleavage of the polypeptide mimic, the rhodamine is released and in aqueous solution undergoes a
conversion to a highly fluorescent zwitterionic form. The change in rhodamine's fluorescence quantum yield
from its unconjugated (lactone form), to conjugated (zwitterionic form) is considerable (Rho 110 φ
lactone
= 0.006
φ
zwitterion
= 0.98). This sharp increase in fluorescence is easily detectable by even handheld instruments and often
just the naked eye.
Figure 2. Rhodamine 110 (2)
This paper considers the previous synthetic literature for rhodamine 110 derivatives and reports on new
exploratory approaches to the tailoring of this important fluorophore. Furthermore it addresses why rhodamine
conjugates have not been more widely applied to pathogen detection via bacterial enzymatic action.
The most recent review of rhodamine 110 details the methodology used for the synthesis of rhodamine
derivatives, which is essentially unchanged since the 1980’s.
Rhodamine 110 is reacted with a protected amino acid, which is first activated with a coupling agent. Following
deprotection and subsequent activation, further amino acids are added in a routine fashion, however the yields
are often poor and the costs of the initial rhodamine are high (£100/g). In addition, there is no mention of
racemisation in any of the seminal work, on which that review is based.
10
Recent work by the authors, reports the synthesis of a novel (Boc-Val-Pro-Arg)
2
–Rhodamine (1), however the
authors report considerable difficulty with which Arginine could be attached to rhodamine, which is at odds
with the comparative ease of this achievement by Mangel et al, almost 33 years ago.
11
In reference to the

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3
original literature dealing with biologically relevant rhodamine conjugates, we observe multiple discrepancies
between the compounds reported and the fluorescence data before and after enzymatic cleavage. There is no
reported yield for the cathepsin C activated rhodamine
12
or caspase conjugates
8
, yet these are reportedly known
in various reviews. The synthesis of the serine protease activated rhodamine by Mangel et al is also highly
improbable in its simplicity and purification via centrifuge.
11
The only characterisation undertaken is TLC and
low resolution MS. The elemental analysis deviates from the empirical formula, but water and HCl are
inexplicably added to bring the observed values within tolerance. The absence of HPLC/NMR facilities when
the work was done in 1982 means that it is unlikely the compound was specifically synthesised. The primary
literature reports the (CBz-Arg)
2
-Rhodamine to be a pink powder, yet the Sinclair group reported a white solid
after HPLC purification. The biological testing of the serine protease activated rhodamine as then reported bears
out the fact that whatever was synthesised is impure. The UV-Vis spectra indicates there is still fluorescence of
the rhodamine conjugate at 525nm. If the fluorescence quantum yield alone is three orders of magnitude lower
for a Bis-NH
2
conjugated rhodamine than for the free fluorophore, this fluorescence should not be visible, yet it
is shown to be fluorescent in the original paper from 1982. The conclusion has to be that a mixture of
rhodamine, mono Arg-Rhodamine and bis Arg-Rhodamine was in fact made. Thus the contrast between
conjugated and unconjugated rhodamine is comparatively small and significant amounts of protease are required
to achieve this. As a consequence, any attempt to achieve a high sensitivity e.g. for the detection of pM
concentrations of an enzyme expressed by a pathogen would be met with failure.
As indicated in the previous section rhodamine conjugates have been somewhat overlooked as the fluorescent
component for practical enzymatic tests and there is a need to develop synthetic methodology to tune the
rhodamine structure efficiently.
Previously reported synthesis has proven capricious with initial coupling reactions of amino acids with
rhodamine being of very low yield and this makes them less amenable to scale-up. We discuss the different
approaches and show an improvement in the overall yield of a recently reported rhodamine conjugate (Boc-Val-
Pro-Arg)
2
–Rhodamine (1), which has shown remarkable selectivity and sensitivity for MRSA and MSSA as
reported by the authors.
5
2. Results
2.1 The xanthone approach
2.1.1 Grignard

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4
The main problem with functionalising the NH
2
groups on rhodamine are the intrinsic lack of nucleophilicity of
these, due to resonance and other effects.
13
To overcome this problem the approach was taken to reduce some of
the possible conjugation through the rhodamine via removal of the top lactone aromatic. The aim was to achieve
a better amino acid coupling yield and introduce the lactone ring at a later stage. The method followed that of A.
Young-Hoon et al whom originally developed the procedure to produce library of rosamines for combinatorial
synthesis. 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.
14
Figure 3. Asymmetric xanthone synthesis, as per A. Young-Hoon et al.
The initial step is an Ullmann condensation requiring high temperatures and long reaction times. Work-up in hot
concentrated sulphuric acid induces ring closure, to produce the xanthone core. In our hands, this step resulted
in the expected xanthone 3 in a consistent yield of 30% (Figure 4 (a)). NMR data confirmed this (See SI-1).
Having functionalised the NH
2
of xanthone 3 with an appropriately protected amino acid e.g. N-Boc-
Arginine(Cbz)
2
–OH to give 4, the upper ring system could be introduced using a Grignard type approach as
indicated in Figure 4.
Whilst the test reaction of phenyl magnesium bromide and unfunctionalised xanthone 6 successfully produced
tertiary alcohol 7 (Figure 4 (b)), the Grignard reaction using acetal protected benzaldehyde on nitro amino
xanthone 3 was unsuccessful in the production of 8, further elaboration to 9 and 10 were therefore not possible
(Figure 4(c)). Upon examination of the crude mixture, the expected signals were observed neither by NMR nor
by MS, instead we obtained a complex and intractable mixture of deeply purple coloured products which could
not be successfully isolated and characterised.
Figure 4. Proposed incorporation of C ring using Grignard followed by mild oxidation.
The reaction was tested a number of times with varying excesses of Grignard reagent without generating the
product of interest (See Table SI-4). This result warranted further investigation, upon which the Bartoli indole
synthesis was implicated. This synthesis detailed the reaction of vinyl Grignard reagents with aryl nitro
compounds, demonstrating the reactivity of the aryl nitro group towards Grignard reagents.
15
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.

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