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Are Two Better than One? Comparing Intermolecular and

Intramolecular Indicator Displacement Assays in
Pyrophosphate Sensors

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Manuscript ID
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Liu, Xuejian; The University of Sydney, School of Chemistry
Smith, David; University of Sydney, School of Chemistry
Jolliffe, Katrina A; The University of Sydney, School of Chemistry

Page 1 of 26

ChemComm

Professor Kate Jolliffe PhD UNSW

14/12/2012

Dear Jonathan,

Please find attached a manuscript entitled Are Two Better than One? Comparing
Intermolecular and Intramolecular Indicator Displacement Assays in
Pyrophosphate Sensors for consideration for publication in the thematic issue of
Chemical Communications on Host-guest chemistry.
The manuscript reports the synthesis of three new anion receptors with covalently
attached indicators that were designed to selectively recognise pyrophosphate (PPi) ions
with high affinity in aqueous buffer. The sensitivity and selectivity of these molecules is
compared to analogous receptors that employ indicator displacement assays as a
sensing mechanism. This comparison enabled us to determine that improved
discrimination between PPi and ATP can be obtained through covalent indicator
attachment. This is important as many receptors are unable to discriminate between
these two anions.
The results reported in this manuscript will therefore be of interest to a wide cross section
of the chemical community, in particular to supramolecular chemists, and we consider
this special issue of Chem.Commun. to be an ideal forum for their rapid dissemination.

Yours sincerely,

Prof Katrina (Kate) A. Jolliffe

School of Chemistry
Faculty of Science
Rm No 515, Chemistry Building F11
The University of Sydney
NSW 2006 Australia

T +61 2 9351 2297

F +61 2 9351 3329
E kate.jolliffe@sydney.edu.au
sydney.edu.au/science/chemistry/research/
jolliffe

ABN 15 211 513 464

CRICOS 00026A

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Received 00th January 20xx,

Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x

Are Two Better than One? Comparing Intermolecular and

Intramolecular Indicator Displacement Assays in Pyrophosphate
Sensors
Xuejian Liu, David G. Smith and Katrina A. Jolliffe*

www.rsc.org/

Peptide receptors with Zn(II)-DPA units and a covalently bound

fluorescent coumarin indicator on an oxazole-containing scaffold
are shown to function as more selective pyrophosphate sensors
than the analogous chemosensing ensembles in indicator
displacement assays.

of these molecules to bind selectively to pyrophosphate with

that of the analogous receptors in intermolecular IDAs with a
comparable coumarin indicator.

Anions play crucial roles in biological processes.

4-
Pyrophosphate (P2O7 , PPi) for example, plays an important
role in bioenergetics and metabolic processes. As such, the
selective recognition and sensing of biologically relevant
anions in water has numerous potential applications. For
example, a sensor able to selectively detect pyrophosphate
(PPi) in the presence of other phosphate species, especially
nucleoside triphosphates, in aqueous solution would find use
110
in bioanalytical applications.
A number of recent studies have shown that receptors
containing Zn(II)-dipicolylamino (DPA) units exhibit high
selectivity and affinity towards phosphate oxoanions in water,
and in some cases are able to discriminate between these
1,1114
species.
We have previously reported oxazole-based
cyclic peptide receptors with Zn(II)-DPA binding sites that show
15
high levels of discrimination for PPi, as monitored through
16
17,18
either
colourimetric
or
fluorescent

indicator
displacement assays (IDAs). IDAs have been used successfully
as a means of sensing anions in numerous chemosensing
9,1924
ensembles.
However, for some applications it would be
preferable to use sensors in which the indicator is covalently
attached to the anion recognition moiety.
Intramolecular IDAs, in which the indicator is covalently
attached to the receptor, provide a novel means by which to
25
create molecular sensors. We report here a small family of
oxazole-based peptides (1-3Zn2), with Zn(II)-DPA anion
binding sites, to which a fluorescent coumarin indicator is
covalently bound through a flexible linker, so that it can act as
an intramolecular fluorescent IDA. We also compare the ability

Figure 1: Structures of peptide-based receptors 1-3Zn2, and the previously

reported 4-5Zn2

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Figure 2: Oxazole building blocks 7 and 8, and coumarin derivatives 6 and 9.

Based on our previous studies, in which we found that

chemosensing ensembles comprising receptors 4Zn2 or 5Zn2
with a variety of indicators including coumarin sulfonate 6,
showed good selectivity for pyrophosphate over other
9,1618
phosphate oxoanions in aqueous solution,
we designed
three receptors, 1Zn2, 2Zn2 and 3Zn2 comprising an oxazole-
containing peptide backbone with two side-chain appended
Zn(II)-DPA binding sites and a coumarin fluorophore attached
via a side-chain triazole moiety. 1Zn2 and 2Zn2 differ
structurally in the order of the side chain appendages. In 1Zn2
the Zn(II)-DPA units are adjacent to one another whereas in
2Zn2 the coumarin is located in between them. 3Zn2 is a
linear analogue, designed to ascertain whether the cyclic
peptide structure is required to maintain high selectivity.
Compounds 1-3 were prepared using methods similar to
those previously described (see ESI for full synthetic
8,9,14,16,24,26
schemes).
Briefly, the synthesis of the linear
precursors was achieved using standard solid phase peptide
synthesis techniques with the appropriate oxazole building
blocks (7 and 8) and using Fmoc/HATU chemistry. The linear
precursors to compounds 1 and 2 were prepared staring from
2-chlorotritylchloride resin, while for 3, the linear peptide was
extended from Rink amide resin to provide the C-terminal
amide upon cleavage from the resin. For the synthesis of 1 and
2, cleavage of the linear precursors from the resin was
followed by solution phase cyclisation using the coupling
reagent DMTMMBF4. For all three peptides, the coumarin
indicator was then attached via a copper(I)-catalysed azide-
27,28
alkyne [3+2] cycloaddition (CuAAC) reaction
with azide 9.
The Cbz protecting groups were then removed using a solution
of hydrogen bromide in acetic acid to give the corresponding
diamines after basic workup. These were then subjected to
reductive amination with 2-pyridinecarboxaldehyde using our
previously established conditions, providing the DPA-
functionalised cyclic peptide over two steps. Subsequent
addition of two equivalents of Zn(NO3)2 gave the fully
functionalised anion receptors, 1-3Zn2.
The anion binding behaviour of peptides 1-3Zn2 was then
assessed by titration and compared to that of chemosensing
ensembles comprising either 4Zn2 or 5Zn2 and coumarin
23
indicator 6 under the same conditions. PPi, ADP and ATP
were incrementally added to a solution of 1-3Zn2 in HEPES
buffer (5 mM, 145 mM NaCl, pH 7.4) at 25 C and the
fluorescence emission recorded after each addition. In each
case, addition of the anion resulted in an increase in
fluorescence intensity, suggesting that binding of the anion
resulted in displacement of the coumarin from the Zn(II)-DPA
units with concomitant increase of fluorescence intensity
(Figure 3 and Supplementary Information).

Fig 3. Changes in the fluorescence emission intensity of 1 (top left), 2 (top right), 3
(middle), 4 (bottom left) and 5 (bottom right) at 480 nm upon addition of PPi (), ATP (
), and ADP () in HEPES buffer (5 mM, 145 mM NaCl, pH 7.4) at 25 C.

For receptor 1Zn2, addition of PPi resulted in around a 6.5-fold

fluorescent enhancement, accompanied by a 10 nm
bathochromic shift (Figure 4). Fitting of the titration data to a
1:1 binding model gave a log K = 5.2 as determined by a
nonlinear least squares curve fitting. In contrast, addition of
ADP or ATP induced only a minor fluorescence increase when
added to 1Zn2 (Figure 3 and Supplementary information),
indicating that 1Zn2 exhibits a selective turn-on fluorescence
response in the presence of PPi.

Fig 4 Fluorescence emission spectra of 1Zn2 (5 M) in response to PPI (0-300

M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5 nm/5
nm.

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The response of receptor 2Zn2 to PPi shows a similar 10

nm bathochromic shift, although the increase in emission
intensity is much smaller compared to that observed for 1Zn2,
indicating 2Zn2 is less sensitive towards PPi than 1Zn2 (Figure
3). In addition, similar fluorescent enhancements are observed
for addition of ATP and ADP to 2Zn2 as are observed for PPi
addition, indicating that 2Zn2 is less selective towards PPi over
ADP and ATP than 1Zn2.
These results suggest that the positioning of the two Zn(II)-
DPA binding sites relative to the coumarin indicator on the
cyclic peptide scaffold has a significant influence on anion
binding capability. Positioning the indicator between the two
Zn(II)-DPA units appears to make intramolecular indicator
displacement more difficult, presumably as a result of stronger
interactions between the coumarin and the Zn(II)-DPA units.
This is attributed to the ability of the indicator to bridge both
Zn(II)-DPA binding sites in this configuration. Positioning the
two Zn(II)-DPA binding sites adjacent to each other on the
peptide scaffold is required to achieve selective PPi binding in
the intramolecular IDA, and may be attributed to indicator-
metal interactions with only one of the Zn(II)-DPA units making
the indicator easier to displace in 1Zn2 than in 2Zn2.
This is supported by the behaviour observed for 3Zn2, in
which the Zn(II)-DPA units are also adjacent to one another,
and the coumarin occupies a terminal position. This shows a
very similar overall profile with respect to the three anions as
observed for 1Zn2 (Figure 4 and Supplementary Information).
There is a large fluorescent enhancement in the presence of
PPi, but minimal changes in emission upon the addition of
either ADP or ATP.
As previously observed for a 1:1 mixture of 5Zn2 and
17
coumarin indicator 6, addition of PPi, ATP and ADP to
chemosensing ensembles of either 4Zn2 or 5Zn2 and 6 all
resulted in indicator displacement with an observed increase
in fluorescence (Figure 3 and Supplementary Information).
Little difference was observed between 4Zn2 and 5Zn2 with
similar binding affinities for all three anions observed for both
chemosensing ensembles (apparent log Ka for 5Zn2 for PPi =
17
6.8; ATP = 6.3 and ADP = 6.0), and both 4Zn2 and 5Zn2
showed a slight preference for binding to PPi over ATP and
ADP.
A comparison of the results for 1Zn2 and 2Zn2, with
covalently attached indicators, to those obtained for 4Zn2 and
5Zn2, which use intermolecular indicator displacement assays
to observe binding, suggests that by covalently attaching the
coumarin derived indicator to the receptor a significant
improvement in selectivity for PPi can be obtained, albeit with
lower sensitivity. 1Zn2 shows significantly higher selectivity for
PPi compared to the analogous 4Zn2, although the log Ka for
PPi is an order of magnitude lower for 1Zn2 compared to that
observed for 4Zn2. However, the improved selectivity requires
the indicator to be attached at the correct position on the
scaffold, with 2Zn2 not displaying an improvement in either
affinity or selectivity over the analagous indicator
displacement approach with 5Zn2.
One potential application of fluorescent PPi sensors is to
monitor enzymatic processes. Pyrophosphatase is an enzyme

which catalyses the hydrolysis of PPi to two phosphate ions in

the presence of magnesium. To verify the general capability of
our peptide-derived PPi sensors for real time monitoring of
enzymatic processes, a fluorescence assay was used to
monitor pyrophospahtase activity.

Fig. 5 Real-time assay of PPi hydrolysis catalysed by different amounts of
pyrophosphatase ( = 0 units, = 0.25 units, = 0.5 units, = 1.0 units) in
the presence of 3 (10 M) in Tris buffer (10 mM, 10 mM MgCl2, pH 7.5) at
30 C, ex = 390 nm, slits = 10 nm & 5 nm.

Pyrophosphatase was added to a solution containing either

1Zn2 or 3Zn2, and excess PPi in Tris buffer at 30 C. Differing
quantities of PPi were used with each sensor, based upon the
differing sensitivities exhibited by the two sensors. The
fluorescence response was then measured at regular time
intervals. Addition of pyrophosphatase resulted in a decrease
in fluorescence intensity (emission was monitored at 485 nm),
with a more rapid decrease observed in the presence of larger
quantities of enzyme (Figure 5). This is consistent with a
reduction in PPi concentration as the enzyme binds to and
breaks down the PPi. The coumarin can then bind to the zinc
centres of 1Zn2 or 3Zn2, resulting in quenching of the
fluorescence emission. These enzyme assay results suggest the
general applicability of peptide PPi sensors for monitoring the
enzymatic process of inorganic pyrophosphatase.
In summary, we have developed oxazole-based cyclic
peptide receptors which incorporate a covalently linked
fluorescent coumarin reporter. This allows the receptor to
function as an intramolecular indicator displacement assay,
and to selectivity detect PPi. Compared to an intermolecular
analogue, we observed greatly enhanced selectivity for PPi
over competing phosphate species using systems with
covalently attached indicators. We have demonstrated an
application of these receptors to measure pyrophosphatase
enzyme activity in real time.
We thank the ARC (DP110100682 and DP140100227) for
financial support.

Notes and references

1 S. Lee, K. K. Y. Yuen, K. A. Jolliffe and J. Yoon, Chem. Soc.
Rev., 2015, 44, 17491762.
2 J. F. Zhang, S. Kim, J. H. Han, S. J. Lee, T. Pradhan, Q. Y. Cao,
S. J. Lee, C. Kang and J. S. Kim, Org. Lett., 2011, 13, 5294
5297.

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3 W. H. Chen, Y. Xing and Y. Pang, Org. Lett., 2011, 13, 1362

1365.
4 N. L. Han, K. M. K. Swamy, K. K. Sook, J. Y. Kwon, Y. Kim, S. J.
Kim, J. Y. Yeo and J. Yoon, Org. Lett., 2007, 9, 243246.
5 D. H. Lee, S. Y. Kim and J. I. Hong, Angew. Chemie - Int. Ed.,
2004, 43, 47774780.
6 J. H. Lee, A. R. Jeong, J. H. Jung, C. M. Park and J. I. Hong, J.
Org. Chem., 2011, 76, 417423.
7 H. N. Lee, Z. Xu, S. K. Kim, K. M. K. Swamy, Y. Kim, S.-J. Kim
and J. Yoon, J. Am. Chem. Soc., 2007, 129 , 38283829.
8 K. K. Y. Yuen and K. A. Jolliffe, Chem. Commun., 2013, 49,
48246.
9 S. J. Butler and K. A. Jolliffe, Org. Biomol. Chem., 2011, 9,
34713483.
10 S. K. Kim, D. H. Lee, J.-I. Hong and J. Yoon, Acc. Chem. Res.,
2009, 42, 2331.
11 H. T. Ngo, X. Liu and K. A. Jolliffe, Chem. Soc. Rev., 2012, 41,
492865.
12 A. Ojida, Y. Mito-oka, K. Sada and I. Hamachi, J. Am. Chem.
Soc., 2004, 126, 24542463.
13 T. Sakamoto, A. Ojida and I. Hamachi, Chem. Commun., 2009,
141152.
14 S. J. Butler, K. A. Jolliffe, W. Y. G. Lee, M. J. McDonough and
A. J. Reynolds, Tetrahedron, 2011, 67, 10191029.
15 R. B. P. Elmes and K. A. Jolliffe, Chem. Commun., 2015, 51,
495168.
16 X. Liu, H. T. Ngo, Z. Ge, S. J. Butler and K. A. Jolliffe, Chem.
Sci., 2013, 4, 16801686.
17 S. J. Butler and K. A. Jolliffe, Chem. - An Asian J., 2012, 7,
26212628.
18 M. J. McDonough, A. J. Reynolds, W. Y. G. Lee and K. A.
Jolliffe, Chem. Commun., 2006, 29712973.
19 S. L. Wiskur, P. N. Floriano, E. V. Anslyn and J. T. McDevitt,
Angew. Chemie - Int. Ed., 2003, 42, 20702072.
20 B. P. Morgan, S. He and R. C. Smith, Inorg. Chem., 2007, 46,
92629266.
21 M. K. Coggins, A. M. Parker, A. Mangalum, G. A. Galdamez
and R. C. Smith, European J. Org. Chem., 2009, 343348.
22 X. Sun, K. Lacina, E. C. Ramsamy, S. E. Flower, J. S. Fossey, X.
Qian, E. V Anslyn, S. D. Bull and T. D. James, Chem. Sci., 2015,
6, 29632967.
23 R. G. Hanshaw, S. M. Hilkert, H. Jiang and B. D. Smith,
Tetrahedron Lett., 2004, 45, 87218724.
24 V. E. Zwicker, X. Liu, K. K. Y. Yuen and K. A. Jolliffe, Supramol.
Chem., 2016, 28, 192200.
25 T. Minami, Y. Liu, A. Akdeniz, P. Koutnik, N. A. Esipenko, R.
Nishiyabu, Y. Kubo and P. Anzenbacher, J. Am. Chem. Soc.,
2014, 136, 1139611401.
26 V. E. Zwicker, B. M. Long and K. A. Jolliffe, Org. Biomol.
Chem., 2015, 13, 78227829.
27 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
Angew. Chemie - Int. Ed., 2002, 41, 25962599.
28 C. W. Torne, C. Christensen and M. Meldal, J. Org. Chem.,
2002, 67, 30573064.

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Covalently Attached Indicator

Page 6 of 26

Indicator Displacement Assay

OH
OH

O
O

N
Zn2+

-O

Zn2+

N
N

O
O

O
N
HN
N

N
H

NH
N
O

H
N

O OH
N
H
N

O
O

Better PPi selectivity

-O

P
O

O-

H
N

O
O

O
N
HN
N

NH
O

-O

vs.

N
Zn2+

Zn2+ N
OH

N
O

3S

O-

Higher PPi affinity

Pyrophosphate recognition using indicator displacement assays is compared to that of

analogous receptors with covalently attached indicators.

Page 7 of 26

ChemComm

Supporting Information

Are Two Better than One? Comparing Intermolecular and

Intramolecular Indicator Displacement Assays in Pyrophosphate
Sensors
Xuejian Liu, David G. Smith and Katrina A. Jolliffe
School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.
Email: kate.jolliffe@sydney.edu.au; Tel: +61 2 9351 2297
General Procedures for SPPS .............................................................................................................................. 2
General procedures for SPPS on 2-chlorotrityl chloride resin .............................................................................. 2
Loading of Fmoc-protected oxazole-based amino acid onto 2-chlorotrityl chloride resin and capping ............ 2
Loading of amino acid onto Rink amide AM Resin ............................................................................................... 2
Fmoc deprotection .................................................................................................................................................. 2
Solid Phase Peptide coupling .................................................................................................................................. 2
Capping of Rink amide AM Resin ........................................................................................................................... 2
Peptide cleavage from 2-chlorotrityl resin ............................................................................................................. 2
Peptide cleavage from rink resin ............................................................................................................................ 2
Macrolactamisation ................................................................................................................................................ 2
General procedures for functionalization of peptide side chains and synthesis of metal complexes ............... 3
Reaction schemes .............................................................................................................................................. 3
Synthesis ............................................................................................................................................................ 4
Boc-Pra-Ser-OMe, (10) ............................................................................................................................................ 5
Boc-Pra-Oxz(Ser)-OMe, (11) ................................................................................................................................... 5
TFA.H-Pra-Oxz(Ser)-OH, (12) ................................................................................................................................... 5
Fmoc-Pra-Oxz(Ser)-OH, (13) ................................................................................................................................... 5
6,7-Dihydroxy-4-azidomethyl coumarin (9) ........................................................................................................... 6
Cyclo[Ala-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Pra-Oxz(Ser)], (15) .................................................... 6
Cyclo[Ala-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Ala(1-DHCMT)-Oxz(Ser)], (16) ................................. 6
Cyclo[Ala-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-Ala(1-DHCMT)-Oxz(Ser)] (1) ................................... 7
Cyclo[Ala-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Pra-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)], (17) .................................................... 8
Cyclo[Ala-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Ala(1-DHCMT)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)], (18) ................................. 8
Cyclo[Ala-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-Ala(1-DHCMT)-Oxz(Ser)-Orn(DPA)-Oxz(Ser)], (2) .................................. 8
Ac-Ala(1-DHCMT)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-NH2 (19) ...................................................... 9
Ac-Ala(1-DHCMT)-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-NH2, (3) ...................................................... 9
NMR spectra .................................................................................................................................................... 11
Fluorescence titrations of 1-3Zn2 with PPi, ADP and ATP ................................................................................ 17
1Zn2 with PPi ........................................................................................................................................................ 17
2Zn2 with PPi ........................................................................................................................................................ 17
3Zn2 with PPi ........................................................................................................................................................ 17
1Zn2 with ADP ...................................................................................................................................................... 18
2Zn2 with ADP ...................................................................................................................................................... 18
3Zn2 with ADP ...................................................................................................................................................... 19
1Zn2 with ATP ....................................................................................................................................................... 19
2Zn2 with ATP ....................................................................................................................................................... 19
3Zn2 with ATP ....................................................................................................................................................... 20
Procedure for fluorescence pyrophosphatase assay ....................................................................................... 20

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General Procedures for SPPS

General procedures for SPPS on 2-chlorotrityl chloride resin
Coupling of Fmoc-protected oxazole-based amino acids was carried out using O-(7-azabenzotriazol-1-yl)-
N,N,N,N'-tetramethyl-uronium hexafluorophosphate (HATU) as the coupling reagent in the presence of N-
methylmorpholine (NMM). Progress of the couplings was monitored by LCMS.

Loading of Fmoc-protected oxazole-based amino acid onto 2-chlorotrityl chloride resin and capping
-1

2-Chlorotrityl chloride resin (resin capacity 1.3 mmol g ) was swollen in anhydrous CH2Cl2 for 1 h in a sinter-fitted
syringe. After filtering off the resin, a solution of the amino acid (1.7 equiv. relative to resin capacity) and DIPEA (3
equiv. relative to resin capacity) in anhydrous CH2Cl2-DMF (9:1 v/v, 0.2 M) was added. Agitated at room
temperature for 72 h, the resin was drained and treated with a solution of methanol-diisopropylethylamine-
CH2Cl2 (2:1:17 v/v/v, 3 x 5 mL x 10 min). The resin was then washed with DMF (3 x 6 mL), CH2Cl2 (3 x 6 mL) and
diethyl ether (3 x 6 mL) before being dried under reduced pressure overnight. The resin loading was calculated to
-1
be 1.15 mmol g according to gravimetric measurements.

Loading of amino acid onto Rink amide AM Resin
-1

Rink Amide AM resin at a loading level of 0.41 mmol g was placed in a sinter-fitted syringe and allowed to swell
in anhydrous DCM for 1 h. After removal of DCM, the resin was treated with a solution of 20% v/v piperidine in
DMF (3 x 5 mL x 10 min) and the resin was then drained and rinsed with DMF (3 x 5 mL), DCM (3 x 5 mL) and DMF
(3 x 5 mL). Afterwards, a solution of Fmoc-protected amino acid (1.5 equiv.), DIPEA (3 equiv.) and HATU (2.5
equiv.) was added to the reaction syringe. After agitation at room temperature for 2 h, the resin was filtered off
and washed with DMF (3 x 5 mL) and DCM (3 x 5 mL).

Fmoc deprotection
The resin-bound peptide was treated with a solution of 10% v/v piperidine in DMF (3 x 5 mL x 10 min) and
subsequently filtered off and washed with DMF (3 x 5 mL), CH2Cl2 (3 x 5 mL) and DMF (3 x 5 mL) to give the free
amine.

Solid Phase Peptide coupling
A solution of Fmoc-protected oxazole based amino acid (1.2 equiv. relative to loading), NMM (2.0 equiv. relative
to loading), HATU (1.2 equiv. relative to loading) was added to the reaction vessel. The resulting suspension was
shaken at room temperature for 50 min and then the resin was drained and washed with DMF (3 x 5 mL), CH2Cl2
(3 x 5 mL) and DMF (3 x 5 mL).

Capping of Rink amide AM Resin
Capping was accomplished by treatment of the resin with 20% v/v acetic anhydride in pyridine (3 x 5 min) and
subsequent washing with DMF (3 x 6 mL), DCM (3 x 6 mL) and DMF (3 x 6 mL).

Peptide cleavage from 2-chlorotrityl resin
After the coupling, the resin was washed with DMF (5 x 5 mL) and CH2Cl2 (10 x 5 mL). Then the resin was treated
with a solution of hexafluoroisopropanol-CH2Cl2 (1:4 v/v, 4 x 6 mL x 10 min). Afterwards, the resin was washed
with CH2Cl2 (5 x 5 mL). All solutions were combined and evaporated to give the crude linear peptide.

Peptide cleavage from rink resin
After acetylation, the resin was washed with DMF (3 x 5 mL) and DCM (3 x 5 mL).The resin was then treated with a
solution of trifluoroacetic acid/H2O/triisopropylsilane (95:2.5:2.5 v/v, 2 x 1 h) and washed with DCM (2 x 2 mL). All
solution were combined and evaporated to give the desired product.

Macrolactamisation
A solution of the crude linear peptide in DMF (0.05 M) was treated with DMTMM.BF4 (1.3 equiv.) and DIPEA (3
equiv.) and the resulting mixture was stirred at room temperature for 16 h. The solution was concentrated and
the resultant residue was partitioned between water (15 mL) and chloroform-isopropanol (3:1 v/v, 50 mL). The
aqueous phase was further extracted with chloroform-isopropanol (3:1 v/v, 7 x 50 mL). The combined organic
fractions were dried over MgSO4 and evaporated to give the crude cyclic peptide which was then purified by

S2

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preparative RP-HPLC [gradient of 10% to 60% acetonitrile (0.1% TFA) in water (0.1% TFA) over 60 min] to afford
the desired product as a colourless solid. Yields: 46%-53%.

General procedures for functionalization of peptide side chains and synthesis of metal complexes
Step 1. Cbz deprotection: A solution of hydrogen bromide in acetic acid (33% v/v, 3.0 mL) was added to the Cbz-
protected cyclic peptide (0.07 mmol) and stirred at room temperature for 2h. The reaction mixture was treated
with diethyl ether (80 mL) to give a precipitate which was collected by centrifugation. The obtained
dihydrobromide salt was then partitioned between chloroform-isopropanol (3:1 v/v, 20 mL) and aqueous NaOH
(0.2 M). The aqueous phase was further extracted with chloroform-isopropanol (3:1 v/v, 9 x 20 mL). The
combined organic layers were dried (MgSO4) and the solvent was removed under reduced pressure to give the
free diamine.

Step 2. Reductive amination: To a stirred solution of the free diamine in anhydrous DMF (5 mL) was added 2-
pyridinecarboxaldehyde (20 equiv.) and sodium triacetoxyborohydride (20 equiv.). The resulting mixture was
saturated with nitrogen and stirred at room temperature for 16 h. The mixture was then concentrated and
treated with aq. NaOH (0.1 M, pH 8). The aqueous phase was further extracted with chloroform-isopropanol (3:1
v/v, 10 x 20 mL). The combined organic fractions were dried over MgSO4 and the solvent was evaporated to give
the crude functionalized peptide which was then purified by preparative RP-HPLC [gradient of 0 to 40%
acetonitrile (0.05% ammonia) in water (0.05% ammonia) over 50 min] to afford the desired product.

Step 3. Metal complex preparation: To a solution of the peptide in methanol (3 mL) was added an aqueous
solution of zinc nitrate (2.0 equiv.). The mixture was stirred at room temperature for 5 min and subsequent
evaporation of the solvent afforded the Zn(II) complex in quantitative yield.

Reaction schemes
Scheme S1
O
O
O

N
H

OH
O

HCl.H 2N

OH

N
H

ii
iii

H
N

O
O

N
H

OH

11

10

iv, v

O
O

N
H

N
O

O
OH

vi
N

TFA.H 2N
O

13

12

(i) HBTU, HOBt, DIPEA, CH3CN, 0 C, (ii) CH 2Cl 2,XtalFluor-E, -78 C, (iii) BrCCl 3, DBU, CH 2Cl 2, 0 C, (iv) NaOH, MeOH, H 2O, (v) TFA, CH 2Cl 2,
(vi) Fmoc-succinimide, 1,4-dioxane.

S3

O
OH

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Scheme S2
NHCbz
NHCbz
O
O
SPSS

Cl

NH

R'

R' =
R' =

R'' =

NHCbz

complex formation

NHCbz
O

N
O

NH

R'

N
H

i) Cbz deprotection
ii) reductive amination

HN

R'

H
N

O
O

R''

O
NH

H
N

NHCbz R'' =

N
H

HN

R' =

NHCbz

O
NH

R''

O
O

17

R'' =

copper(I)-catalysed azide-alkyne
[3+2] cycloaddition (CuAAC) reaction

N
H

R' =

H
N
R''

R'
N

R'' =

NHCbz

15

HN

N
Zn2+

NH

21

N
O

HN

FmocHN

20

N
H

i) cleavage from resin

ii) macrolactamisation

R''

N
H

HN

R'
N

H
N

R''
HO

HO
OH

N
OH
R'' =

R' =
N

16

HO

R' =
N

OH

R'' =

NHCbz

N
N

18

R'' =

R'' =

R' =
NHCbz

N
O

Scheme S3

Synthesis

OH

O
R' =

HO

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ChemComm

Boc-Pra-Ser-OMe, (10)

To a solution containing Boc-Pra-OH (10.0 g, 46.9 mmol) and the hydrochloride salt of H-Ser-OMe (7.30 g, 46.9
mmol) in CH3CN (300 mL) was added HBTU (21.4 g, 56.4 mmol) and HOBt (7.60 g, 56.2 mmol). The resulting
mixture was cooled to 0 C and DIPEA (24.7 mL, 140 mmol) was added slowly. The reaction mixture was stirred at
rt for 16 h. The solvent was removed under reduced pressure and the residue dissolved in chloroform (800 mL),
washed with aqueous sodium hydrogen carbonate solution (3 x 60 mL), dried (MgSO4) and concentrated to give a
yellow oil, which was purified by flash column chromatography (silica gel; EtOAc/hexane, from 1:2 to 1:1, v/v) to
20
1
afford the desired dipeptide 10 as a colourless oil (14.5 g, 82%). []D = -4.3 (c 0.65 MeOH); H NMR (300 MHz,
CD3OD): 4.58 4.47 (m, 1H), 4.36 4.22 (m, 1H), 3.91 (dd, J = 11.5, 4.0, 1H), 3.79 (dd, J = 11.5, 4.0, 1H), 3.74 (s, 3H), 2.75
13
2.64 (m, 2H), 2.63 2.51 (m, 2H), 1.46 (s, 9H); C NMR (126 MHz, CDCl3): 170.7, 170.6, 155.7, 80.6, 79.4, 71.7, 62.7,
+
55.0, 53.1, 52.7, 28.3, 22.6; HRMS (ESI) calcd. for C14H22N2O6Na [M + Na] 337.1376, found 337.1370.

Boc-Pra-Oxz(Ser)-OMe, (11)

Step 1: A solution of dipeptide 10 (8.50 g, 27.0 mmol) in anhydrous CH2Cl2 (300 mL) was cooled to -78 C, then
XtalFluor-E (9.29 g, 40.6 mmol) was added portionwise. The reaction mixture was stirred at -78 C for 3 h and then
warmed to 0 C. Aqueous saturated sodium hydrogen carbonate (300 mL) was added to quench the reaction and
the organic phase was separated. The aqueous layer was subsequently warmed to room temperature and further
extracted with chloroform (3 x 300 mL). The combined organic fractions were dried over MgSO4 and concentrated
under reduced pressure to yield the crude oxazoline as a yellow oil.

Step 2: BrCCl3 (3.2 mL, 32.5 mmol) and DBU (4.8 mL, 32.1 mmol) was added to a solution containing oxazoline in
anhydrous CH2Cl2 (300 mL) at 0 C. The resulting mixture was stirred at room temperature for 3 h and then
quenched by the addition of saturated ammonium chloride solution (400 mL). The aqueous layer was further
extracted with chloroform (4 x 300 mL). The combined organic fractions were dried (MgSO4) and evaporated
under reduced pressure to give a yellow oil which was purified by flash chromatography (silica gel, EtOAc/hexane,
20
1
1:4 v/v) to afford 11 as a colourless solid (4.80 g, 60%). mp 83-84 C; []D = -41.6 (c 0.68 MeOH); H NMR (500
MHz, CDCl3): 8.19 (s, 1H), 5.54 (d, J = 7.6, 1H), 5.10 (m, 1H), 3.87 (s, 3H), 2.87 - 2.80 (m, 2H), 1.96 (s, 1H), 1.41 (s,
13
9H); C NMR (125 MHz, CDCl3): 163.6, 161.5, 154.9, 144.5, 133.4, 80.6, 78.2, 72.1, 52.3, 47.9, 28.3, 24.5; HRMS
+
(ESI) calcd. for C14H18N2O5Na [M + Na] 317.1113, found 317.1108.

TFA.H-Pra-Oxz(Ser)-OH, (12)

Step 1: A solution of 11 (2.94 g, 10.0 mmol) in methanol (50 mL) was treated with a solution of NaOH (4.00 g, 100
mmol) in water (15 mL), and the mixture was stirred at room temperature for 16 h. The reaction mixture was then
acidified with hydrochloric acid (1.0 M) until pH 6 and evaporated to give a solid which was suspended in
chloroform/isopropanol (3:1 v/v, 800 mL). The suspension was subsequently filtered and the filtrate was
concentrated to give a colourless foam (2.80 g).
Step 2: The colourless foam obtained in step 1 (2.80 g) was dissolved in CH2Cl2 (40 mL) and treated with
trifluoroacetic acid (40 mL) at 0 C. This was stirred at room temperature for 1.5 h, the reaction mixture was then
concentrated to afford the product 12 as a yellow oil (2.79 g, quant. over two steps).

Fmoc-Pra-Oxz(Ser)-OH, (13)

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Compound 12 (2.79 g, 10.0 mmol) was treated with aqueous saturated sodium carbonate solution until pH 9
followed by addition of a solution of Fmoc-succinimide (3.20 g, 9.5 mmol) in 1,4-dioxane (20 mL). The mixture was
diluted with 1,4-dioxane/water (1:1 v/v, 50 mL) and stirred at room temperature for 16 h. After removal of the
solvents under reduced pressure, the residue was diluted with water (200 mL) and adjusted to pH 5 with
hydrochloric acid (1.0 M, 125 mL). The solution was filtered to give the crude product which was triturated with
20
hexane/Et2O (5:1 v/v, 2 x 60 mL) to give 13 as a colourless solid (3.33 g, 83%). m.p. 136-138 C; []D = -39.1 (c 0.45
1
MeOH); H NMR (300 MHz, CD3OD): 8.46 8.41 (s, 1H), 7.79 (d, J = 7.5, 2H), 7.67 (d, J = 7.5, 2H), 7.39 (t, J = 7.5,
2H), 7.30 (t, J = 7.5, 2H), 5.04 (t, J = 6.8, 1H), 4.39 (t, J = 6.4, 2H), 4.23 (t, J = 6.8, 1H), 2.98 2.78 (m, 2H), 2.37
13
2.34 (s, 1H). ; C NMR (75 MHz, CDCl3): 163.2, 162.6, 156.1, 144.3, 143.5, 141.1, 133.6, 127.5, 126.9, 124.9, 119.7,
+
78.0, 77.4, 71.5, 67.0, 46.9, 23.5; HRMS (ESI) calcd. for C23H18N2O5Na [M + Na] 425.1113, found 425.1104.

6,7-Dihydroxy-4-azidomethyl coumarin (9)

To a solution of 4-chloromethyl-6,7-dihydroxy coumarin (0.45 g, 2.0 mmol) in acetone (5 mL) was added a solution
of sodium azide (0.25 g, 4.0 mmol) in water (0.5 mL). The mixture was then stirred at room temperature for 16 h
and ice water (10 mL) was added, the resulting precipitate was isolated and dried to give the desired product 9 as
-1
a brown solid (0.33 g, 71%). IR max/cm 3485, 3427, 3124, 3088, 3045, 2190, 1728, 1624, 1468, 1412, 1362, 1283,
1
1238, 1215, 1156, 1047, 874; H NMR (300 MHz, DMSO): 6.98 (s, 1H), 6.76 (s, 1H), 6.24 (s, 1H), 4.71 (s, 2H);
+
HRMS (ESI) calcd. For C10H7N3O4Na [M + Na] 256.0334, found 256.0331.

Cyclo[Ala-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Pra-Oxz(Ser)], (15)

Cyclic peptide 15 was synthesized according to general procedures for SPPS on 2-chlorotrityl chloride resin
followed by macrolactamisation). The crude product was used directly in the next step without purification.







Cyclo[Ala-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Ala(1-DHCMT)-Oxz(Ser)], (16)

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Compound 16 was synthesized in 41% yield following the procedure described above; H NMR (500 MHz, CD3OD):
8.40 8.32 (m, 3H), 8.29 (s, 1H), 8.07 (s, 1H), 7.36 7.21 (m, 10H), 6.99 (s, 1H), 6.75 (s, 1H), 5.77 (s, 2H), 5.54 (s,
1H), 5.50 5.34 (m, 2H), 5.03 (d, J = 7.6, 4H), 3.24 3.05 (m, 4H), 2.20 1.90 (m, 4H), 1.74 1.45 (m, 4H), 1.65 (d,
J = 6.6, 3H).

Cyclo[Ala-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-Ala(1-DHCMT)-Oxz(Ser)] (1)

Cyclic peptide 15 was prepared as a yellow solid (32 mg, 55%) following the procedure described above for
20
-1
synthesis of 13. []D = -107.8 (c 0.18 MeOH); IR max/cm 3352 (br), 3112, 3036, 1743, 1718, 1663, 1592, 1524,
1
1346, 1212; H NMR (500 MHz, MeOD) = 8.39 (m, 4H), 8.32 (s, 1H), 8.31 (s, 1H), 8.30 (s, 1H), 8.28 (s, 1H), 7.91
(s, 1H), 7.73 (m4H), 7.57 (t, J = 8.6, 4H), 7.25 7.20 (m, 4H), 6.98 (s, 1H), 6.73 (s, 1H), 5.76 (m, 3H), 5.49 (s, 1H),
5.46 (q, J = 7.0, 1H), 5.29 (m, 2H), 3.76 (m, 8H), 3.52 (m, 2H), 2.58 (m, 4H), 2.14 2.02 (m, 2H), 2.02 1.95 (m, 2H),
13
1.92 1.83 (m, 2H), 1.65 (d, J = 7.1, 3H), 1.67 - 1.55 (m, 4H). C NMR (125 MHz, DMSO-d6): 165.0, 164.6, 163.6,
161.0, 160.0, 159.7, 151.1, 151.0, 149.1, 143.9, 143.3, 142.8, 136.9, 135.9, 135.8, 135.6, 124.8, 123.1, 122.5,
+
108.1, 102.9, 60.1, 53.3, 49.7, 46.7, 46.4, 42.6, 30.5, 29.2, 23.4, 19.0; HRMS (ESI) calcd. for C64H62N17O12 [M + H]
1260.4758, found 1260.4760.

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Cyclo[Ala-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Pra-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)], (17)

Cyclic peptide 17 was synthesized according to the general procedures for SPPS on 2-chlorotrityl chloride resin
followed by macrolactamisation. The crude product was used directly in the next step without purification.

Cyclo[Ala-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Ala(1-DHCMT)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)], (18)

To a solution of the crude cyclic peptide 18 (0.17 g, 0.2 mmol) in DMF (5 mL) was added azide 10 (0.04 g, 0.2
mmol), CuSO4.5H2O (0.05 g, 0.2 mmol), and sodium ascorbate (0.04 g, 0.2 mmol). The suspension was then
allowed to stir at rt for 16 h. The suspension was concentrated and the residue partitioned between aqueous
EDTA solution (20 mL) and chloroform/isopropanol (3:1 v/v, 200 mL). The aqueous phase was further extracted
with chloroform/isopropanol (3:1 v/v, 7 x 30 mL) and the combined organic fractions were evaporated to give the
crude product which was then purified by preparative RP-HPLC [gradient of 10% to 60% acetonitrile (0.1% TFA) in
1
water (0.1% TFA) over 60 min] to afford the desired product as a brown solid (91 mg, 39%). H NMR (300 MHz,
CD3OD): 8.40 - 8.33 (m, 3H), 8.31 (s, 1H), 7.92 (s, 1H), 7.40 - 7.21 (m, 10H), 6.99 (s, 1H), 6.76 (s, 1H), 5.82 5.71
(m, 3H), 5.51 (s, 1H), 5.44 5.35 (m, 3H), 5.10 4.98 (m, 4H), 3.65 3.39 (m, 2H), 3.25 3.07 (m, 4H), 2.23 1.86
(m, 4H), 1.74 1.44 (m, 4H), 1.66 (s, 3H).







Cyclo[Ala-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-Ala(1-DHCMT)-Oxz(Ser)-Orn(DPA)-Oxz(Ser)], (2)

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Step 1: Cyclic peptide 13 (0.05 g, 0.043 mmol) was treated with a solution of hydrogen bromide in acetic acid (33%
v/v, 2.5 mL) and the mixture was stirred at room temperature for 30 min. The resulting solution was then added
to diethyl ether (60 mL) dropwise. The precipitate was collected by centrifuge and dissolved in MeOH-water (4:1

v/v), the solution was then treated with basic ion exchange resin (Ambersep 900 hydroxide) to give the free
diamine in quantitative yield.

Step 2: Synthesis of 2 was achieved according to the general procedure for reductive amination. Subsequent
preparative RP-HPLC purification [gradient of 0 to 30% acetonitrile (0.05% ammonia) in water (0.05% ammonia)
20
over 50 min] afforded the desired product as a yellow solid (30 mg, 55%). []D = -100.3 (c 0.22 MeOH); IR
-1
1
max/cm 3365 (br), 3142, 3025, 1752, 1745, 1678, 1601, 1516, 1455, 1362, 1184, 1118; H NMR (400 MHz,
CD3OD) 8.43 - 8.36 (m, 4H), 8.36 - 8.33 (m, 3H), 8.31 (s, 1H), 7.93 (s, 1H), 7.75 (t, J = 7.9, 4H), 7.58 (d, J = 8.0, 4H),
7.27 - 7.18 (m, 4H), 6.98 (s, 1H), 6.70 (s, 1H), 5.80 - 5.72 (m, 3H), 5.53 5.44 (m, 1H), 5.39 (s, 1H), 5.30 - 5.23 (m,
2H), 3.76 (s, 8H), 3.58 3.43 (m, 2H), 2.61 - 2.54 (m, 4H), 2.13 2.00 (m, 2H), 2.00 1.83 (m, 2H), 1.66 (d, J = 6.5,
13
3H), 1.65 1.52 (m, 2H), 1.29 (s, 2H), C NMR: (125 MHz, DMSO-d6): 164.5, 164.3, 164.3, 163.2, 160.4, 159.6,
159.5, 159.5, 159.3, 159.3, 150.7, 148.7, 148.4, 143.5, 142.8, 142.3, 142.3, 142.18, 136.4, 135.5, 135.3, 135.2,
124.3, 122.6, 122.6, 122.0, 108.0, 107.5, 102.5, 59.7, 52.9, 52.8, 49.2, 46.4, 42.2, 31.3, 30.0, 29.4, 29.0, 29.0, 29.0,
+
28.7, 28.7, 26.2, 22.9, 22.1, 18.6, 13.9, 13.6; HRMS (ESI) calcd. for C64H62N17O12 [M + H] 1260.4764, found
1260.4774.

Ac-Ala(1-DHCMT)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-NH2 (19)

Preparation of 19 was achieved according to the general procedures for SPPS on Rink Amide AM resin. After
acetylation, the click reaction was carried out using azide 9 (1.5 equiv.), CuSO4.5H2O (0.5 equiv.) and sodium
ascorbate (0.5 equiv.).
Ac-Ala(1-DHCMT)-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-Orn(DPA)-Oxz(Ser)-NH2, (3)

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Step 1: A solution of hydrogen bromide in acetic acid (33% v/v, 6 mL) was added to the tripeptide 19 (0.13 g, 0.12
mmol) and the suspension was stirred at room temperature for 50 min. The resulting mixture was then added to
diethyl ether (80 mL) dropwise. The yellow precipitate was collected by centrifuge and dissolved in MeOH/water
(4:1 v/v), the solution was then treated with basic ion exchange resin (Ambersep 900 hydroxide) to give the free
diamine (0.10 g, quant.).

Step 2: Synthesis of 3 was achieved according to the general procedure for reductive amination. Preparative RP-
HPLC purification [gradient of 0 to 22% acetonitrile (0.05% ammonia) in water (0.05% ammonia) over 50 min]
20
-1
gave the desired product 3 as a yellow solid (68 mg, 48%). []D = -30.2 (c 0.21 MeOH); IR max/cm 3126, 3063,
1
1746, 1377, 1214; H NMR (400 MHz, CD3OD) 8.42 8.35 (m, 4H), 8.32 (s, 1H), 8.30 (s, 1H), 8.28 (s, 1H), 7.90 (s,
1H), 7.74 (t, J = 7.5, 4H), 7.58 (d, J = 7.5, 4H), 7.26 7.19 (m, 4H), 7.01 (s, 1H), 6.73 (s, 1H), 5.76 (s, 2H), 5.47 (s,
1H), 5.49 5.40 (m, 1H), 5.23 5.13 (m, 2H), 3.76 (s, 8H), 3.45 (dd, J = 15.2, 7.5, 1H), 3.37 3.32 (m, 1H), 2.58 (t, J
13
= 6.1, 4H), 2.18 2.03 (m, 2H), 2.02 1.90 (m, 2H), 1.93 (s, 3H), 1.70 1.47 (m, 4H); C NMR (75 MHz, DMSO)
169.4, 163.9, 163.7, 163.4, 161.9, 160.6, 160.0, 160.0, 159.4, 151.3, 150.7, 148.8, 148.4, 143.2, 143.1, 142.6,
142.4, 142.0, 136.6, 136.1, 135.4, 124.4, 122.8, 122.2, 108.7, 102.8, 59.8, 53.1, 46.9, 46.6, 29.6, 28.9, 23.1, 22.3.
+
HRMS (ESI) calcd. for C60H61N16O11 [M + H] 1181.4706, found 1181.4710.

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NMR spectra

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Fluorescence titrations of 1-3Zn2 with PPi, ADP and ATP

Stock solutions of receptors (5 M of 1-3Zn2, and of the anion (2 mM) were prepared respectively. An aliquot of
receptor solution (2.5 mL) was placed in a 1 cm quartz glass cuvette and incubated at 25 C for 5 min, the
fluorescence emission spectrum from 400-700 nm or 360-600 nm was recorded (ex = 390 nm for 1-3Zn2).
Aliquots of a solution containing the anion were then added to the sample cuvette, followed by stirring for 20
seconds, the fluorescence emission spectrum was collected.

To assess the apparent association constant for the receptor-anion, the fluorescence data over the range 440-580

nm or 380-520 nm was fitted with nonlinear least squares curve fitting program HypSpec (Hyperquad package,
119
global analysis) based on 1:1 binding model.

1Zn2 with PPi

(left) Fluorescence emission spectra of 1Zn2 (5 M) in response to PPi (0-300 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5 nm/5
nm. (right) Binding isotherm () obtained by fitting the observed fluorescence intensities at 480 nm () to a 1:1 binding model using non-linear regression.

2Zn2 with PPi

(left) Fluorescence emission spectra of 2Zn2 (5 M) in response to PPi (0-500 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5 nm/5
nm. (right) Observed fluorescence intensities at 480 nm ().

3Zn2 with PPi

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(left) Fluorescence emission spectra of 3Zn2 (5 M) in response to PPi (0-10 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5 nm/5
nm. (right) Binding isotherm () obtained by fitting the observed fluorescence intensities at 480 nm () to a 1:1 binding model using non-linear regression.

1Zn2 with ADP

(left) Fluorescence emission spectra of 1Zn2 (5 M) in response to ADP (0-300 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5
nm/5 nm. (right) Observed fluorescence intensities at 480 nm ().

2Zn2 with ADP

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(left) Fluorescence emission spectra of 2Zn2 (5 M) in response to ADP (0-500 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5
nm/5 nm. (right) Observed fluorescence intensities at 480 nm ().

3Zn2 with ADP

(left) Fluorescence emission spectra of 3Zn2 (5 M) in response to ADP (0-100 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5
nm/5 nm. (right) Observed fluorescence intensities at 480 nm ().

1Zn2 with ATP

(left) Fluorescence emission spectra of 1Zn2 (5 M) in response to ATP (0-300 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5
nm/5 nm. (right) Observed fluorescence intensities at 480 nm ().

2Zn2 with ATP

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(left) Fluorescence emission spectra of 2Zn2 (5 M) in response to ATP (0-500 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5
nm/5 nm. (right) Observed fluorescence intensities at 480 nm ().

3Zn2 with ATP

(left) Fluorescence emission spectra of 3Zn2 (5 M) in response to ATP (0-500 M) in HEPES (5 mM, 145 mM NaCl, pH 7.4) at 25 C. ex = 390 nm, slits = 5
nm/5 nm. (right) Observed fluorescence intensities at 480 nm ().

Procedure for fluorescence pyrophosphatase assay

A stock solution containing receptor and PPi (4-10 equiv. relative to the receptor concentration) in 10 mM Tris
buffer (pH 7.5, 10 mM MgCl2) was prepared. An aliquot of 3.0 mL of the solution was placed in a 1 cm quartz glass
cuvette. After incubation at 30 C for 8 min, inorganic pyrophosphatase (0.25, 0.5, 1.0, 2.0 units) was added to the
sample cuvette. The resulting solution was stirred for 6 seconds and the fluorescence spectra were periodically
collected.

Real-time assay using 1Zn2 to monitor PPi hydrolysis catalysed by different amounts of pyrophosphatase ( = 0 units, = 0.25 units, = 0.5 units,
= 1.0 units) in the presence of 3 (10 M) in Tris buffer (10 mM, 10 mM MgCl2, pH 7.5) at 30 C, ex = 390 nm, slits = 10 nm & 5 nm.

S20