Development of an in situ culture-free screening test for the rapid detection of Staphylococcus aureus within healthcare environments

Article abstract of DOI:10.1039/c3ob40150b

This article reports the development of a novel fluorometric indicator which shows a rapid response when exposed to coagulase positive Staphylococcus aureus (SA) bacteria (including methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) bacteria). The test is robust and will detect a wide variety of SA strains and there is no significant fluorescence response observed for other species of bacteria commonly found in clinical specimens, including other Staphylococcus bacteria. This research forms the basis of a prototype SA testing kit for the rapid detection of SA within hospital and healthcare environments as an economical prescreen or alternative to the current PCR based testing methodology. Rapid identification of SA carriers will allow hospital infection control teams to be pre-emptive and could significantly reduce the incidence of hospital acquired infections involving this organism.

Full text of DOI:10.1039/c3ob40150b

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
Development of an in situ culture-free screening test
for the rapid detection of Staphylococcus aureus within
healthcare environments†
Cite this: DOI: 10.1039/c3ob40150b
Alex Sinclair,*a Lauren E. Mulcahy,b Lynsey Geldeard,a Samerah Malik,a
Mark D. Fielder*b and Adam Le Gresley*a
This article reports the development of a novel fluorometric indicator which shows a rapid response
when exposed to coagulase positive Staphylococcus aureus (SA) bacteria (including methicillin sensitive
Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) bacteria). The test
is robust and will detect a wide variety of SA strains and there is no significant fluorescence response
observed for other species of bacteria commonly found in clinical specimens, including other Staphylo-
coccus bacteria. This research forms the basis of a prototype SA testing kit for the rapid detection of SA
within hospital and healthcare environments as an economical prescreen or alternative to the current
PCR based testing methodology. Rapid identification of SA carriers will allow hospital infection control
teams to be pre-emptive and could significantly reduce the incidence of hospital acquired infections
involving this organism.
Received 23rd January 2013,
Accepted 18th March 2013
DOI: 10.1039/c3ob40150b
www.rsc.org/obc
bacterial numbers via culturing or amplification of character-
istic DNA via PCR are utilised for the detection of SA.2 Only
Introduction
OStaphylococcus aureus (MRSA and MSSA) infections are a sig- after augmentation of bacteria cell numbers can fluorometric
nificant problem within hospitals worldwide. In Europe alone, molecular probes e.g. Boc-Val-Pro-Arg-amido-methyl-coumarin
around 7% of all patients develop healthcare acquired infec- 2 or visual coagulase tests be used and only after PCR amplifi-
tions and this is estimated to cost up to £11bn annually to diag- cation can the hybrid DNA probes found within the BACLite™
nose and treat. Epidemiology suggests that up to 30% of the UK assay be identified.3,4 These amplification steps are time con-
population are carriers of SA, currently UK hospitals only screen suming (ca. 8 h for culture and 3 h for PCR) and require suit-
surgical patients.1 It has been predicted that for an in-patient able instrumentation and skilled operators to obtain a result
cohort of 70 000, cost savings of ∼£600k could be achieved; for a given sample.3c,5 Couple this time delay to that associated
along with the potential avoidance of 840 hospital acquired with sampling and transport in a clinical setting and this can
infections through the use of a routine pre-screen and treat mean that a result for a sample may not be available until the
strategy.2 When this is considered in the context of the next day for most cases.6,7 These delays may make any infec-
26 million in-patient procedures performed within UK hospitals tion more difficult and more expensive to treat, and increase
in 2010–11 (HESonline), this could lead to a potential national the risk of the SA spreading to other patients before it has
annual saving of £222 million and an annual reduction in the been identified/treated.8 There are two key stages where SA
number of hospital acquired infections of 312 000 cases.
detection can be sped up; these are the transport of a sample
Current clinical detection methods for SA are limited by and the time required for culture or molecular detection prior
sensitivity. In all current detection methods, amplification of to even a presumptive identification. Elimination of both of
these limitations could be accomplished by the development
of a simplified point-of-care assay, however to date no appli-
aSchool of Pharmacy and Chemistry, Faculty of Science, Engineering and Computing,
cations have been developed that are reliable, economical or
Kingston University, Penrhyn Rd, Kingston, Surrey KT1 2EE, UK.
robust enough to satisfy the requirements. It is clear that the
E-mail: alex.sinclair@kingston.ac.uk, a.legresley@kingston.ac.uk;
development of an enhanced molecular probe with sufficient
sensitivity to obviate the need for culturing would be a key step
in shortening the time required for the detection of SA in a
clinical environment.
Staphylocoagulase is an enzyme expressed externally by
∼95% of all known SA bacteria. This enzyme normally
Tel: +44 (0)20 8417 2465
bSchool of Life Sciences, Faculty of Science, Engineering and Computing, Kingston
University, Penrhyn Rd, Kingston, Surrey KT1 2EE, UK.
E-mail: m.fielder@kingston.ac.uk; Tel: +44 (0)20 8416 2843
†Electronic supplementary information (ESI) available: Full NMR of all com-
pounds and HRMS of final compound along with additional biological data not
included within paper. See DOI: 10.1039/c3ob40150b
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
peptide substrate to a chromophore/fluorophore,12,14 however,
there is no literature evidence for the successful coupling of
the staphylocoagulase tripeptide substrate Val-Pro-Arg to rhod-
amine and we hypothesised that we could utilise this prop-
erty to generate a more effective colour change indicator. The
proposed rhodamine based probe 3 would function in a very
similar manor to the commercially available AMC probe 2,
however, in the case of probe 2, the difference in UV properties
between the peptide bound and peptide unbound form are
small, making this compound very difficult to observe at low
concentrations, requiring lengthy cell culturing before it can
be employed. Replacement of the coumarin with rhodamine
would generate a colourless compound 3 that would become
visibly coloured on exposure to MRSA/MSSA.
Due to the extinction co-efficient of rhodamine being many
times that of coumarin, (68 800 vs. 4500 M−1 cm−1), we pre-
dicted that detection could occur at a lower concentration of
either dye or bacteria and/or obviate the need for the lengthy
cell culture when compared to use of the coumarin dye system.
The synthetic route started with us first synthesising the tri-
peptide Boc-N-Val-Pro-Arg(NO2)-OH through conventional
liquid phase peptide coupling using EDCI and PFP ester
methodology.15 Before attempting to couple the tripeptide to
the rhodamine, a model coupling of rhodamine to Boc-N-Arg-
(NO2)-OH using EDCI was carried out, however this was found
to give a complex mixture of products with no obvious sign of
the desired product after purification. A second model study
was then carried out, coupling Boc-NH-Val-OH with rhod-
amine and after screening a range of coupling reagents, such
as EDCI, EDCI/HOBT, EDCI/oxyma and COMU in a range of
different solvents, times and temperatures, we obtained a
moderate yield of the desired product using 4 equivalents of
each of EDCI, Boc-N-Val-OH and NEt3 in anhydrous DMF
under microwave irradiation at 80 °C for 24 hours. Column
chromatography of this mixture was hindered by the presence
of both unreacted rhodamine and the mono-coupled Boc-Val-
rhodamine, both of which exhibit two interconverting spots
corresponding to the closed lactone and the open zwitterionic
form, analogous to 1a and 1b. A 55% yield of purified (Boc-
Val)2-rhodamine was obtained after chromatography.
Fig. 1 Transition to coloured zwitterionic form of rhodamine 1. The commer-
cially available AMC probe 2 and our improved probe LGX 3.
complexes with a second enzyme prothrombin to produce a
staphylothrombin complex which naturally metabolises fibri-
nogen into fibrin by cleavage of a beta loop containing the
amino acids X-Val-Pro-Arg-X.9 The tripeptide Val-Pro-Arg has
been shown to be an effective substrate mimic of fibrinogen,
and the existing commercial fluorescent probe, Boc-Val-Pro-
Arg-amido-methyl-coumarin (AMC) 2 utilises this enzymatic
cleavage to release the fluorophore coumarin, which can be
detected though changes in the fluorescence emission spec-
tra.3a,d However, the sensitivity of the coumarin system is
limited due to a low extinction co-efficient of ca. 4500 M−1
cm−1, a relatively narrow bathochromic shift on loss of the tri-
peptide and a low overall molar fluorescence coefficient 4.15 ×
109 RFU M−1 (λex. = 380 nm, λem. = 460 nm), requiring a signifi-
cant period of cell culture before bacterial cell count is signifi-
cant enough for this test to be utilised.10
Application of these conditions for direct coupling rhod-
amine and the Boc-Val-Pro-Arg(NO2)-OH tripeptide were
attempted but it was found impossible to isolate an analyti-
cally pure sample of our target compound from this complex
crude mixture. These coupling conditions were then replicated
using a variety of different sidechain-protected arginines, such
as NO2, di-NO2, Cbz, Pfp, di-Pfp and di-Cbz Boc-arginine.
When Boc-N-Arg(CBz)2-OH was utilised, the number of side
products was minimised, and it was possible to isolate an ana-
lytically pure sample of the (Boc-N-Arg(Cbz)2)2-rhodamine 4 in
an 18% yield (∼90% pure samples could be obtained in a
maximum of 42%).
Rhodamine 1 is a commonly available dye that has been
widely used in a number of biological applications, either as a
cell stain or as a fluorescent tag.11 It has a high extinction co-
efficient of ca. 66 800 M−1 cm−1 and an overall molar fluore-
scence coefficient of 1.90 × 1011 RFU M−1 (λex. = 492 nm, λem.
=
523 nm).12 Rhodamine also exists in equilibrium between
two tautomers, a closed lactone tautomer 1a which is colour-
less and an open zwitterionic tautomer 1b which is strongly
coloured (Fig. 1).13
Synthesis
With the arginine functionalised rhodamine 4 in hand,
The principle of coupling peptides to rhodamine is not in exposure to TFA in MeOH yielded 6 in 94%. The resulting
itself, particularly new; many groups of enzymes e.g. serine glassy solid was then coupled with Boc-Val-Pro-OH using
proteases can be detected and quantified through coupling a EDCI and oxyma in anhydrous DMF. After initial flash
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 2 Left: compound 3 in PBS buffer at 100 μM. Right: compound 3 in PBS
buffer at 100 μM after exposure to MRSA coated inoculation loop and left for
1 h at 20 °C.
Scheme 1 (a) HO–Arg(Cbz)2NHBoc, EDCI, pyridine, DMF, 18%. (b) TFA, DCM,
94%. (c) HO-Pro-Val-NHBoc, COMU, NEt3, DMF, 14%. (d) H2, Pd/C, MeOH, DMF,
77%.
100 to 106 colony forming units per mL (CFU mL−1) were gen-
erated by serial dilution in 1 × PBS.
chromatography, NMR analysis was not conclusive, so further
purification via preparative HPLC resulted in an analytically
pure sample of 5 in an overall yield of 14% (Scheme 1).
This product was then dissolved in a 1 : 1 mixture of an-
hydrous DMF–methanol over 5% Pd/C before exposure to hydro-
gen at atmospheric pressure and temperature for 48 hours to
yield the target (Boc-Val-Pro-Arg)2-rhodamine 3, which, after
workup, was pure by NMR, in a yield of 77%.
UV-Vis spectra for 3 correlated with that for other rhod-
amine amide probes with a νmax at 240 nm and similar extinc-
tion coefficient. There was no observed absorbance for 3
within the region of interest (400–600 nm) and the νmax and
extinction coefficient of rhodamine has been shown to be con-
stant between pH 6 and pH 9.16,17
Solutions of 50 μM and 100 μM LGX were prepared asepti-
cally (containing human prothrombin as detailed in the ESI†).
In order to detect the presence of staphylocoagulase and thus
the efficacy of LGX, varying cell concentrations (100–106) CFU
mL−1 (50 μM LGX) and 100–104 CFU mL−1 (100 μM LGX) were
added to a microtitre plate (Nunclon 96 well plates) in a 1 : 1
ratio with either 50 μM or 100 μM of LGX solution. This pro-
vided a final LGX concentration of 25 μM or 50 μM, respect-
ively. The aspiration is to develop a visual indicator as a point-
of-care test, however in order to determine the full sensitivity
of our system at this early stage, the enhanced sensitivity
afforded by fluorometry vs. colourimetry was utilised. Hand
held fluorometers could also be utilised at the point-of-care in
order to generate an accurate read out. The relative fluore-
scence was then recorded every fifteen minutes over a six
hour time period (λex. = 488 nm and λem. = 525 nm). Positive
Biological testing
Preliminary testing involved simply preparing a 100 μM solution controls used in each experiment were a control strain of
of LGX (3) in 1 M phosphate buffered saline solution (PBS). An MRSA (NCTC 12493), and the negative controls were 10 clinical
inoculation loop was then streaked though a confluent growth isolates of Escherichia coli (E. coli), the mean of which served
of MRSA on an agar plate and the cells were suspended in a as a control. A 1 : 1 ratio of 100μM LGX solution and 1 × PBS
cuvette containing our LGX solution. Fluorescence readings was used, resulting in a final LGX concentration of 50μM
were then taken over time and showed a steady increase in which was used as a control and termed “LGX alone”.
absorbance at 525 nm over time. In addition to this, on remov-
As can be observed in Fig. 3, the results of all concentrations
ing the sample from the spectrophotometer, a visible colour of MRSA (mean values for 15 bacterial strains at a given concen-
change was observed, raising the potential for a visual colouri- tration) are plotted against the mean value of the negative
metric readout to be obtained in the future clinical point-of- control E. coli strains, at a concentration of 102 CFU mL−1 for
care test, significantly simplifying the testing process, see Fig. 2. both LGX concentrations (for demonstrative purposes). The
Screening was then carried out involving 15 clinical isolates mean value for LGX solution alone was used as a principal
of MRSA (as detailed in ESI†). These were cultured on nutrient control for all SA strains, and a significantly greater increase in
agar overnight at 37 °C. Discrete colonies of each isolate were fluorescence intensity in MRSA samples was observed across
tested for coagulase activity using a staphylase test kit (Oxoid), the 6 hour time period when compared to the controls. Taking
following standard procedures, and each MRSA isolate was a lower threshold limit of 20 000 relative fluorescence units
shown to be coagulase positive. Following this, an inoculum of (RFU) as a positive result, it can be seen that at a 50 μM concen-
each respective sample was cultured overnight in nutrient tration of LGX, and at all bacterial concentrations of MRSA,
broth under aerobic, shaking conditions at 37 °C. Each sample there is a clear positive result (please see ESI† for 25 μM data).
was washed in sterile 1 × phosphate buffered saline (PBS) and It is also noteworthy that there is an observed ‘instantaneous’
varying concentrations of bacterial suspension, ranging from positive reaction, and this can be explained by the slight lag
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 3 The efficacy of 50 μM LGX in detecting MRSA concentrations as low as
102 CFU mL−1. Average of fifteen bacterial strains (n = 3). Error bars represent
standard deviation.
Fig. 4 The efficacy of 50 μM LGX in demonstrating both selectivity and sensi-
tivity in detecting coagulase positive MRSA and MSSA, compared to coagulase
negative S. epidermidis and E. coli at concentrations of 103 CFU mL−1. Average
of ten to fifteen bacterial strains (n = 3). Error bars represent standard deviation.
time between mixing, placing the sample in the fluorometer
and the first reading, demonstrating the impressive rate of the bacterium juxtaposed with the results demonstrated in this
initial reaction. This graph also highlights the stability of LGX– experiment (staphylase test data available on request). There is a
prothrombin mixture over the experimental time, which shows significant increase in fluorescence for coagulase positive MRSA
no obvious increase in fluorescence in the absence of bacteria, and MSSA when compared to coagulase negative E. coli and
yet the presence of a staphylocoagulase containing microorgan- S. epidermidis (please see ESI† for 25 μM data). It is important to
ism rapidly stimulates a fluorescent response. The speed of the note that this method of detection does not require the over-
reaction is ascribed to the enzyme complex formed between night culturing on agar which is necessary in performing the
staphylocoagulase and prothrombin, which is the active peptidase staphylase test, thus our method of determining the presence of
which cleaves the substrate mimic, at a rate relative to the bac- SA, may be considered to be a culture independent method of
terial concentration for each specific strain, demonstrating that bacterial detection, and is superior in terms of speed of results.
the compound can rapidly detect SA concentrations of bacteria as
low as 102 CFU mL−1
To further determine the efficacy of LGX it was necessary to
compare it to the existing AMC probe 2. A 500 μM or 100 μM of
.
In order to determine whether this is a species-specific 2 (coumarin) solution was prepared with 1 × PBS as described
phenomenon and indeed whether the compound exhibits selec- for compound 3 (method adapted from Ford et al., 1999),18 and
tivity between bacterial strains (as detailed in the ESI†), 15 clini- the complete solution was assayed as before with cell concen-
cal isolates of each of MRSA, MSSA and E. coli and 10 strains of trations of bacteria (106, 105, 104, 103, 102 and 100 CFU mL−1) in
coagulase negative staphylococcal (CNS) species (S. epidermidis, a microtitre plate. The coumarin probe 2 has previously been
S. warneri, M. luteus and S. hominus), were cultured on nutrient shown to be effective in the detection of MRSA at higher bac-
agar overnight at 37 °C. A sample of each type of bacteria was terial concentrations (∼108 CFU mL−1),3d and when mixed with
again maintained and tested for coagulase using a staphylase other bacteria.3e Staphylocoagulase activity was determined by
test kit (Oxoid), and each MRSA and MSSA was shown to be coa- fluorescence spectrophotometry using literature precedent (λex
=
gulase positive, and each E. coli and S. epidermidis was shown to 355 nm, λem. = 460 nm) at 15 minute intervals over a 6 hour time
be coagulase negative. Samples of 103 CFU mL−1 concentrations period (n = 3 samples were assayed in each case).3d Strains of
of each respective bacterial species were assayed under the MRSA and MSSA were assayed for fluorescence intensity using
same parameters as previously described.
As can be observed in Fig. 4, mean values for 15 bacterial using 50 μM and 25 μM of LGX on identical bacterial samples.
strains at concentrations of 103 CFU mL−1 of bacteria amply
Fig. 5 (for demonstrative purposes showing only concen-
100 μM and 500 μM coumarin. This was compared to screens
demonstrate the efficacy of LGX as both a selective and sensitive trations of 103 CFU mL−1 and concentrations of 50 μM LGX
means of determining the presence of coagulase positive bac- and 500 μM coumarin – see ESI† for 25 μM LGX data) illus-
teria. As previously described, all bacterial strains were assessed trates that the fluorescence produced by LGX, when compared
for coagulase activity, and the coagulase status of each to the coumarin system, is significantly greater, to the extent
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Experimental section
Rhodamine-(Arg-(CBz)2-NHBoc)2 (4)
To a solution of HO–Arg(Cbz)2NHBoc (4.61 mmol, 2.5 g, 6 eq.),
EDCI (4.61 mmol, 883 mg, 6 eq.), oxyma (4.60 mmol, 654 mg, 6
eq.) in anhydrous DMF (5 mL) was added freshly distilled anhy-
drous pyridine (5 mL) followed by rhodamine 110 (0.763 mmol,
280 mg, 1 eq.) and the solution was stirred at room temperature
under a nitrogen atmosphere for 10 days. The reaction was then
diluted with EtOAc (20 mL), washed with saturated aqueous
NaHCO3 (2 × 10 mL), 1 M aqueous LiCl (2 × 10 mL) and 10%
aqueous CuSO4 (3 × 10 mL), dried over MgSO4 and concentrated to
give a green oily solid. This crude product was then purified by
column chromatography over silica gel, eluting with 25% EtOAc in
CHCl3 to yield the product as a white amorphous solid (0.14 mmol,
200 mg, 18%): δH (400 MHz, CDCl3), 9.45 (2H, s, broad, NH-23),
9.29 (2H, s, broad, NH-21), 9.01 (2H, s, broad, NH-15), 8.02 (1H, dd,
Fig. 5 MRSA and MSSA detection using LGX, compared to 500 μM coumarin,
using bacterial concentrations of 103 CFU mL−1. Average of ten bacterial strains
(n = 3). Error bars represent standard deviation.
where one may consider the results incomparable. Even at
500 μM of coumarin, fluorescence fails to reach the lower
threshold limit (20 000 RFU) for a positive result according to
our LGX test. When comparing the efficacy of the LGX com-
pound with the studies performed by Holliday et al.,3d whereby
a bacterial cell concentration of 108 CFU mL−1 was necessary
to produce a positive result with the coumarin system, our
LGX system has proved to be far superior in terms of speed,
selectivity and sensitivity, with it able to detect cell concen-
trations of >102 CFU mL−1 within 30 minutes.
4
3JHH = 6.02 Hz, JHH = 2.01 Hz, CH-12), 7.56–7.69 (2H, m, CH-10
and CH-11), 7.43, (2H, s, CH-2), 7.38–7.20 (20H, m, CH-27, CH-28,
3
CH-29, CH-33, CH-34 and CH-35), 7.05 (1H, dd, JHH = 6.02 Hz,
4JHH = 2.01 Hz, CH-9), 6.77 (2H, d, 3JHH = 6.02 Hz, CH-4), 6.69 (2H,
3
dd, JHH = 6.02 Hz, CH-5), 5.70, (2H, s, broad, NH-36), 5.30–5.02
(8H, m, CH2-25 and CH2-31), 4.39, (2H, m, CH-17) 4.07–3.87 (4H,
m, CH2-20), 1.86–1.62 (8H, m, CH2-18 and CH2-19), 1.41 (18H, s,
CH3-39); δC (100 MHz) 171.1, 171.0, 169.5, 163.6, 160.8, 156.2,
155.7, 153.1, 139.6, 136.4, 135.1, 134.4, 134.4, 129.7, 128.9, 128.8,
128.5, 128.4, 128.3, 128.0, 128.0, 126.2, 124.9, 124.1, 115.4, 114.2,
107.9, 82.4, 80.4, 69.1, 67.2, 54.8, 44.0, 28.5, 28.3, 25.0.
Conclusions
We have developed a novel, selective fluorogenic assay, which
obviates the need for time consuming culturing or genetic
amplification, currently core to most hospital SA tests. Our test
has proved itself to be rapid, presently giving a preliminary posi-
tive/negative result within 30 minutes for bacterial concen-
trations commonly found on skin (>102 CFU mL−1). The testing
methodology described within this paper is currently under-
going further biological screening within our labs and work is
ongoing towards the development of a rapid point-of-care test
kit.19 This test kit would allow routine SA testing to become sim-
plified, freeing up hospital microbiology from large scale SA
screening and providing rapid preliminary results. This would
allow hospital infection control procedures to become pre-
Rhodamine-(Arg-(CBz)2-NH2)2 (6)
emptive rather that responsive, reducing the costs, both monet- To a solution of Rho-(Arg-(Cbz)2-NHBoc)2 (0.14 mmol, 200 mg,
ary and impact upon infection, associated with treating hospital 1 eq.) in DCM (2 mL) was added TFA (0.3 mL) and the reaction
acquired SA infections. We are currently conducting research to was stirred under nitrogen atmosphere for 6 hours. The reaction
further increase the sensitivity of this detection technique, was then quenched with 1 M aqueous NaOH (1 mL) and
alongside application towards a point-of-care diagnostic kit for extracted. The organic layer was then washed with saturated
use as an economical routine screening tool.
aqueous NaCl (3 × 1 mL), dried over MgSO4 and concentrated to
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
yield product as an amorphous solid which used immediately (0.5 mL) was added 5% Pd/C (1 mg) and the mixture was stirred
(0.13 mmol, 156 mg, 94%).
under a hydrogen atmosphere for 48 hours. The reaction
mixture was then concentrated, dissolved in methanol and fil-
tered through celite. Concentration yielded the product as an
amorphous white solid (0.0140 mmol, 22 mg, 77%): δH
(600 MHz; CD3OD) 10.11 (2H, d, 3JHH = 7.7, NH-24), 8.48 (2H, d,
Rhodamine-(Arg-(Cbz)2-Pro-Val-NHBoc)2 (5)
3
3JHH = 7.0, NH-25), 8.17 (2H, s, NH-15), 8.05 (1H, d, JHH = 7.6,
CH-12), 7.89 (1H, s, CH-2b), 7.82 (1H, s, CH-2), 7.79 (1H, dd,
3JHH = 8.5, 4JHH = 6.9, CH-11) 7.73 (1H, dd, 3JHH = 8.5, 4JHH = 6.9,
CH-10), 7.41 (2H, s, NH-21), 7.22 (1H, d, 3JHH = 7.8, CH-4), 7.21
3
3
(1H, d, JHH = 7.8, CH-9), 7.19 (1H, d, JHH = 7.8, CH-4b), 6.75
3
3
(2H, d, JHH = 7.8, CH-5), 6.75 (2H, d, JHH = 7.8, CH-5b), 6.65
3
(2H, JHH = 7.8, NH-35), 4.52–4.96 (4H, m, CH-17 and CH-27),
4.20 (2H, m, CH-32), 3.93 (2H, m, CHH-30), 3.70 (2H, m,
CHH-30) 3.24 (4H, m, CH2-20), 2.28 (4H, m, CH2-18), 2.12 (2H,
m, CHH-28), 2.04–1.96 (6H, m, CHH-28, CHH-29 and CH-33),
1.87 (2H, m, CHH-29) 1.45 (18H, s, CH3-38) 1.01 (6H, m, CH3-
34) 0.96 (6H, m, CH3-34); δC (125 MHz; CD3OD) 173.2, 172.2,
171.0, 169.8, 163.8, 157.4, 156.7, 151.5, 140.7, 135.4, 130.0,
128.0, 126.2, 124.6, 123.7, 115.6, 114.1, 107.4, 82.7, 79.2, 73.3,
60.4, 58.0, 53.7, 48.6, 48.3, 44.1, 40.7, 35.6, 30.2, 29.4, 29.2, 28.9,
27.3, 24.9, 24.7, 22.3, 18.4, 17.2. (3 additional peaks for C-2b,
C-4b and C-5b); m/z (CI) 618.3317 [M + 2H]; HRMS: Found
618.3322 (z = 2) [M + 2H], C62H86N14O13 [M] requires 1235.4322
(z = 1), 617.7161 (z = 2).
A solution of Rho-(Arg-(Cbz)2-NH2)2 (0.132 mmol, 156 mg, 1 eq.),
HO-Pro-Val-NHBoc (0.264 mmol, 92 mg,
2 eq.) and NEt3
(0.396 mmol, 40 mg, 55 μL, 3 eq.) in anhydrous DMF (1 mL) was
cooled to 0 °C before the addition of COMU (0.264 mmol, 114 mg,
2 eq.). The reaction was stirred under a nitrogen atmosphere for
1 hour at 0 °C before warming to room temperature over a further
3 hours. The reaction was then diluted with EtOAc (2 mL), washed
with saturated aqueous NaHCO3 (2 × 1 mL), 1 M aqueous LiCl (3 ×
1 mL) then dried over NaSO4 and concentrated to give crude
product. Product was purified by preparative HPLC to yield the pure
product as a colourless glassy solid (0.0182 mmol, 32 mg, 14%): δH
(600 MHz; CDCl3) 9.42 (2H, m, NH-23), 8.83, (2H, s, NH-21), 8.05
3
(1H, d, JHH = 7.6, CH-12), 7.77 (1H, s, CH-2b), 7.70–7.61 (3H, m,
Acknowledgements
CH-2, CH-10 and CH-11), 7.57 (1H, s, NH-36), 7.51 (1H, s, NH-36b),
7.41–7.36 (10H, m, CH-27, CH-28 and CH-29), 7.34–7.30 (4H, m,
The authors would like to thank Kingston University, Enter-
prise and PARC for the funding to support this project, as well
as the EPSRC mass spectrometry service, Swansea, UK, for pro-
viding high resolution mass spectra.
3
CH-33), 7.26–7.20 (6H, m, CH-34 and CH-35), 7.10 (1H, d, JHH
=
3
3
6.4, CH-9), 7.09 (1H, d, JHH = 8.4, CH-4), 7.03 (1H, d, JHH = 8.4,
3
3
CH-4b), 6.69 (1H, d, JHH = 8.4, CH-5), 6.68 (1H, d, JHH = 8.4,
CH-5b), 5.27 (4H, s, CH2-31), 5.20 (2H, d, 3JHH = 12.0, CH2-25), 5.16
3
(2H, d, 3JHH = 8.6, NH-47), 5.06 (2H, d, JHH = 12.0, CH2-25b), 4.53
(2H, m, CH-17), 4.34 (2H, m, CH-38), 4.25 (2H, m, CH-44), 4.07 (4H,
m, CH2-20), 3.73 (2H, m, CHH-41), 3.58 (2H, m, CHH-41), 2.07–1.66
(18H, m, CH2-18, CH2-19, CH2-39, CH2-40, CH-45), 1.47 (18H, s,
CH3-50), 0.97–0.84 (12H, m, CH3-46); δC (125 MHz; CDCl3) 172.0,
170.0, 163.5, 161.1, 155.9, 151.6, 140.0, 136.2, 135.0, 134.5, 129.7,
128.9, 128.9, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 126.3, 125.1,
123.9, 114.2, 108.2, 82.6, 79.7, 69.2, 67.2, 60.5, 60.3, 57.3, 57.2, 54.0,
47.7, 44.0, 31.3, 29.7, 28.4, 25.2, 25.0, 19.4, 17.5, 17.5, 14.2.
Notes and references
1 A. Voss, Br. Med. J., 2009, 339, b3721.
2 M. Scott, P. Panesar, A. Grosso, A. Urquhart, A. Jeanes and
A. Wilson, J. Infect. Prev., 2011, 12(3), 119.
3 (a) T. Morita, H. Katcv, S. Iwanaga, K. Takada and
T. Kimura, J. Biochem., 1977, 82, 1495; (b) R. Lottenberg,
U. Christensen, C. Jacson and P. Coleman, Methods
Enzymol., 1981, 80, 341; (c) S. Kawabata, T. Morita,
S. Iwanaga and H. Igarashi, J. Biochem., 1985, 98, 1603;
(d) M. Holliday, M. Ford, J. Perry and F. Gould, J. Clin.
Microbiol., 1999, 1190; (e) K. Chilvers, J. Perry, A. James and
R. Reed, J. Appl. Microbiol., 2001, 91, 1118.
Rhodamine-(Arg-Pro-Val-NHBoc)2 (3)
4 M. Struelens, P. Hawkey, G. French, W. Witte and
E. Tacconelli, Clin. Microbiol. Infect., 2009, 15(2), 112.
5 (a) S. Malhotra-Kumar, K. Haccuria, M. Michiels, M. Leven,
C. Poyart, W. Hryniewicz and H. Goossens, J. Clin. Micro-
biol., 2008, 46, 1577; (b) M. John, J. Burden, J. Stuart,
R. Reyes, R. Lannigan, S. Milburn, D. Diagre, B. Wilson and
Z. Hussain, Antimicrob. Chemother., 2009, 63, 493;
To a solution of Rho-(Arg-(Cbz)2-Pro-Val-NHBoc)2 (0.0182 mmol,
32 mg, 1 eq.) in anhydrous DMF (0.5 mL) and anhydrous MeOH
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
(c) D. Baldoni, H. Hermann, R. Frei, A. Trampuz and 14 (a) E. Boonacker, S. Elferink, A. Bardai, B. Fleischer and
A. Steinhuber, J. Clin. Microbiol., 2009, 47, 774.
6 S. Harbarth, C. Fankhauser and J. Schrenzel, J. Am. Med.
Assoc., 2008, 299, 1149.
7 D. Jeyaratnam, C. Whitty, K. Phillips, D. Liu, C. Orezzi,
U. Ajoku and G. French, Br. Med. J., 2008, 336, 927.
8 L. Young and L. Winston, Infect. Control Hosp. Epidemiol.,
2006, 27(12), 1304.
C. Van Noorden, J. Histochem. Cytochem., 2003, 51, 959;
(b) M. J. Blackman, J. Corrie, J. Croney, G. Kelly,
J. Eccleston and D. Jameson, Biochemistry, 2002, 41, 12244;
(c) S. Grant, J. Sklar and R. Cummings, J. Biomol. Screen.,
2002, 7, 531; (d) H. Hug, M. Los, W. Hirt and K. Debatin,
Biochemistry, 1999, 38, 13906; (e) B. Packard, D. Toptygint,
A. Komoriya and L. Brandt, Proc. Natl. Acad. Sci. U. S. A.,
1996, 93, 11640; (f) S. Serim, U. Haedke and S. Verhelst,
ChemMedChem, 2012, 7, 1146.
9 C. Izquierdo and F. Burguillo, Int. J. Biochem., 1989, 21,
579.
10 M. Zimmerman, B. Ashe, E. C. Yurewicz and G. Patel, Anal. 15 S. Han and Y. Kim, Tetrahedron, 2004, 60, 2447.
Biochem., 1977, 78, 47.
16 V. Graziano, W. McGrath, A. DeGruccio, J. Dunn and
11 (a) M. Beija, C. Afonso and J. Martinho, Chem. Soc. Rev.,
W. Mangel, FEBS Lett., 2006, 580, 2577.
2009, 38, 2410; (b) B. Shlyahovsky, V. Pavlov, L. Kaganovsky 17 D. Walker, J. Phys., 1987, 20, 217.
and I. Willner, Angew. Chem., Int. Ed., 2006, 45, 4815.
12 S. Leytus, L. Melhado and W. Mangel, Biochem. J., 1983,
209, 299.
18 M. Ford, J. Perry, I. Robson, S. Morgan, M. Holliday, K. Orr
and F. Gould, J. Hosp. Infect., 1999, 42, 133.
19 A. Sinclair, A. Le Gresley and M. Fielder, UK Pat. 1212853.4,
Kingston University.
13 I. S. Ioffe and V. F. Otten, Zh. Org. Khim., 1965, 1, 336.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem.