Bis [ zinc ( II ) dipicolylamino ]-functionalised peptides as high affinity receptors for pyrophosphate ions in water

A library of bis[zinc(II)dipicolylamine]-functionalised linear peptides was prepared using an efficient solid phase peptide synthesis approach and their use as chemosensors for anions in water was investigated using indicator displacement assays. High affinity and selectivity for pyrophosphate (PPi) over adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were observed and additional aromatic side chains provided enhanced discrimination between PPi and ATP.

Commercial materials were used as received unless otherwise noted.Amino acids, coupling reagents and resins were obtained from Novabiochem or GL Biochem.Anhydrous CH 2 Cl 2 was obtained by distillation over CaH 2 prior to use.Anhydrous DMF was purified by passage through neutral alumina using an Innovative Technology, Inc., PureSolv™ solvent purification system.HPLC grade DMF used for solid phase peptide synthesis was obtained from LabScan or Merck.

Synthesis
As the syntheses for all the compounds were similar, general outlines of the procedures used are given below.

Solid-Phase Peptide Synthesis
Solid-phase peptide synthesis was conducted in Torviq polypropylene fritted-syringes.

Amino acid coupling:
A preactivated solution of protected amino acid (2 eq. or 4 eq.relative to resin capacity for Lys or other amino acids respectively), HBTU (1.1 eq.relative to peptide) and i Pr 2 NEt (2 eq.relative to peptide) in dry DMF (0.1 M) was added to the resin and agitated at rt for 2 h.The resin was then washed with DMF (× 5), CH 2 Cl 2 (× 5) and DMF (× 5).
Reductive amination: All DPA functionalised peptides were prepared following a modification of the method described by Quinti et al. 2 The resin was swollen in dry DMF at rt for 30 min before being treated with 2-pyridinecarboxaldehyde (20 eq.) with 1% acetic acid in DMF and agitated at rt overnight.Sodium triacetoxyborohydride (25 eq.) was then added to the suspension and agitated at rt overnight.The resin was washed with DMF (× 5), CH 2 Cl 2 (× 5) and DMF (× 5) and subjected to another cycle of reductive amination procedure to ensure completion of the reaction.

Cleavage:
The resin was treated with a solution of trifluoroacetic acid/H 2 O/triisopropylsilane (95:2.5:2.5 v/v/v) for 1 h.The resin was drained and then washed with trifluoroacetic acid (× 4).The cleavage solution and acid washes were combined and concentrated in vacuo.

Ac-Lys(DPA)-Lys(DPA)-NH 2 , 1
Dipeptide 1 was synthesised on Rink amide resin (0.422 g, 0.261 mmol, resin capacity 0.62 mmol g -1 ), utilising the general methods for Fmoc-SPPS, Alloc deprotection and reductive amination.Cleavage from the resin was then achieved through treatment with a solution of TFA, triisopropylsilane and water (95:2.5:2.5 v/v/v) for 1 h.The crude peptide was then purified by preparative RP-HPLC (0 to 50% B over 30 min).The appropriate fractions were lyophilised, affording the linear dipeptide 1 as a TFA salt, which was treated with basic anion exchange resin to give the desired dipeptide 1 (t R = 17.3 min) as a yellow oil.Yield: 28.2 mg, (16%); [α] D -17 (c 0.6 in MeOH); 1  Tripeptide 3 was synthesised on Rink amide resin (0.401 g, 0.251 mmol, resin capacity 0.62 mmol g -1 ), utilising the general methods for Fmoc-SPPS, Alloc deprotection and reductive amination.Cleavage from the resin was then achieved through treatment with a solution of TFA, triisopropylsilane and water (95:2.5:2.5 v/v/v) for 1 h.The crude peptide was then purified by preparative RP-HPLC (0 to 50% B over 30 min).The appropriate fractions were lyophilised, affording the linear tripeptide 3 as a TFA salt, which was treated with basic anion exchange resin to give the desired tripeptide 3 (t R = 17.0 min) as a yellow oil.

Ac-Lys(DPA)-Leu-Lys(DPA)-NH 2 , 4
Tripeptide 4 was synthesised on Rink amide resin (0.505 g, 0.313 mmol, resin capacity 0.62 mmol g -1 ), utilising the general methods for Fmoc-SPPS, Alloc deprotection and reductive amination.Cleavage from the resin was then achieved through treatment with a solution of TFA, triisopropylsilane and water (95:2.5:2.5 v/v/v) for 1 h.The crude peptide was then purified by preparative RP-HPLC (0 to 70% B over 60 min).The appropriate fractions were lyophilised, affording the linear tripeptide 4 as a TFA salt, which was treated with basic anion exchange resin to give the desired tripeptide

Ac-Lys(DPA)-Pro-Lys(DPA)-NH 2 , 5
Tripeptide 5 was synthesised on Rink amide resin (0.502 g, 0.311 mmol, resin capacity 0.62 mmol g -1 ), utilising the general methods for Fmoc-SPPS, Alloc deprotection and reductive amination.Cleavage from the resin was then achieved through treatment with a solution of TFA, triisopropylsilane and water (95:2.5:2.5 v/v/v) for 1 h.The crude peptide was then purified by preparative RP-HPLC (0 to 70% B over 60 min).The appropriate fractions were lyophilised, affording the linear tripeptide 5 as a TFA salt, which was treated with basic anion exchange resin to give the desired tripeptide 5 (t R = 24.7 min) as a colourless oil.

Ac-Lys(DPA)-Trp-Lys(DPA)-Gly-NH 2 , 9
Tetrapeptide 9 was synthesised on Rink amide resin (0.609 g, 0.250 mmol, resin capacity 0.41 mmol g -1 ), utilising the general methods for Fmoc-SPPS, Alloc deprotection and reductive amination.Cleavage from the resin was then achieved through treatment with a solution of TFA, triisopropylsilane and water (95:2.5:2.5 v/v/v) for 1 h.The crude peptide was then purified by preparative RP-HPLC (0 to 70% B over 60 min).The appropriate fractions were lyophilised, affording the linear tetrapeptide 9 as a TFA salt, which was treated with basic anion exchange resin to give the desired tetrapeptide 9 (t R = 27.7 min) as a colourless oil.

Formation of bis[zinc(II)] complexes
A solution of the appropriate peptide in MeOH (1 mL) was added to an aqueous solution of Zn(NO 3 ) 2 (2 eq.relative to peptide) and the mixture was stirred at rt for 30 min.The mixture was then concentrated under reduced pressure and the residue lyophilised, affording the bis[Zn(II)] complex as a nitrate salt.

Zinc Complexation -Representative Spectra of Compound 1
Partial 1 H NMR spectra in CD 3 OD of compound 1 (a) before and (b) after addition of two equiv. of Zn(NO 3 ) 2 to yield the bis[Zn(II)] complex.Benzylic protons denoted by black filled circle.
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Anion Binding Studies under Mimicked Physiological Conditions
All titrations were performed in aqueous solutions buffered at pH 7.4 HEPES (5 mM) in the presence of 145 mM sodium chloride at 25 °C.

UV-Vis Titrations of Indicator with Receptor
Stock solutions of indicator 11 (20 µM) and of each 1-10•Zn 2 receptor was prepared (1000 µM).2.5 mL of the HEPES buffer solution was transferred to one of a matched pair of 1 cm quartz glass cuvettes as a reference.The other cuvette was filled with 2.5 mL of indicator 11 solution and the UV absorbance (λ abs = 250-750 nm) was recorded.Aliquots of the receptor solution were then added to both cuvettes.After each addition, the resulting solution was stirred with a magnetic stirrer and the UV absorbance was recorded.Typically, up to 10 equivalents of the receptor were added.
To determine the association constants for the indicator-receptor complexes, the UV absorbance (λ abs = 250-750 nm) was plotted as a function of receptor concentration and the raw titration data was analysed using nonlinear least squares curve procedure based on the equilibria for 1:1 binding, 4 utilising the commercially available software programme HypSpec ® (Hyperquad ® package). 3

UV-Vis Titrations of Indicator-Receptor Ensembles with Anions
A stock solution of receptor (20 µM) was prepared.A stock solution of 1:1 indicator 11receptor ensemble solution (20 µM) was also prepared.2.5 mL of the receptor solution was transferred to one of a matched pair of 1 cm quartz glass cuvettes as a reference.The other cuvette was filled with 2.5 mL of the indicator 11-receptor solution and the UV absorbance was recorded for 250-750 nm.Aliquots of the anion solution (2000 µM) were then added to both cuvettes.After each addition, the resulting solution was stirred with a magnetic stirrer and the UV absorbance was recorded.Typically, up to 10 equivalents of the anion were added.
To determine the association constants for the anion-receptor complexes, the UV absorbance (λ abs = 250-750 nm) was plotted as a function of anion concentration and the raw titration data was analysed using nonlinear least squares curve fitting procedure based on the equilibria for indicator displacement, 4 utilising the commercially available software programme HypSpec ® (Hyperquad ® package). 3ectronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013

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Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013

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Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 S 32 Job Plot for the indicator-receptor binding of 11 and 1, 8-10•Zn 2 (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013

Zn 2 Receptor 7•Zn 2 Receptor 8•Zn 2
Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013