SUPPORTING INFORMATION Antimicrobial activity of chlorinated amino acids and peptides

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1 SUPPORTING INFORMATION Antimicrobial activity of chlorinated amino acids and peptides Melanie S. A. Coker*, Wan-Ping Hu, Senti T Senthilmohan and Anthony J. Kettle Free Radical Research Group, University of Otago, Christchurch, New Zealand. Syft Technologies Ltd, Christchurch, New Zealand. Department of Chemistry, University of Canterbury, Christchurch, New Zealand. The chemical stability of chloramines The concordance between chloramine reactivity and loss in dichloramine absorbance did not always hold. In some instances the decay of dichloramines resulted in spectral changes that masked their loss, while with other dichloramines the rate of decrease in UV absorbance was not matched by the loss in chloramine reactivity. For example, the spectral changes of Ala-Phe dichloramine (Figure S1A) indicate that as the dichloramine decayed it formed products with strong UV absorbance. Since this occurred with only a few of the dichloramines, it is likely that in their case breakdown products underwent subsequent reactions. Gly dichloramine lost its absorbance at 35 nm with a half-life of 2 min (Table 1) and a new maximum appeared at 255 nm (Figure S1B). The half-life of 13 min for its chloramine reactivity was much longer. These results indicate that Gly dichloramine decayed to a monochloramine (λ max 252 nm (14)) that subsequently decayed more slowly than the parent (Scheme 1). The same UV decay pattern was also seen with Glu dichloramine. The other amino acid dichloramines decayed far too rapidly to observe this phenomenon. Asn monochloramine and dichloramine were the only species which exhibited an increase in the maximal absorbance over the detection period. Figure S1C shows the UV spectra of Asn monochloramine over 1 min. Both Asn monochloramine and dichloramine formed a peak with maxima at approx. 273 nm. 1

2 To further identify the products of the decomposition of Asn monochloramine and dichloramine we used LC/MS. The products of the decay of Asn monochloramine and dichloramine were separated on a Luna CN 1A column (particle size 5 µm, 15 x 2. mm) (Phenomenex, Torrance, CA) using a Surveyor HPLC Pump (Thermo Corp., San Jose, CA). The column was maintained at 22 C. The products were eluted at a flow rate of 2 µl/min using an isocratic method: 95% solvent A (1 mm ammonium acetate, ph 6.8) and 5% solvent B (acetonitrile). The injection volume was 2 µl. The HPLC was coupled to an ion-trap mass spectrometer (ThermoFinnigan LCQ Deca XP Plus, Thermo Corp.) equipped with an electrospray ionization source. The mass spectrometer was operated with positive ionization using full scan mode (scan range 1 1 m/z) and selected ion monitoring. The electrospray voltage was set at 5. kv. The capillary temperature was 275 C. Nitrogen, the sheath gas, was 35 in units. Selected ion monitoring was performed with collision energy of 28%. Decomposition of Asn monochloramine led to rapid formation of species with a maximum absorbance of 265 nm. LC/MS analysis of this unknown product revealed that it was consistent with decarboxylation of the monochloramine ([M+H] m/z) to form the imine ([M+H] m/z). Other peaks were also apparent with maximum absorbances at 27 and 273 nm, however the masses of these species could not be readily identified. For Asn dichloramine we detected a peak with a maximum absorbance of 273 nm which increased rapidly over 3 mins and then remained constant for at least two hours. LC/MS analysis of this unknown product revealed that had the expected 3:1 ratio for a chlorine containing compound ([M+H] + at m/z and 123. m/z). Analysis of the fragmentation products of this compound showed that it was likely to be a stable chlorimine derived from the decarboxylation of Asn dichloramine. We found that decomposition of both Asn monochloramine and dichloramine to produce the imine and chlorimine, respectively, are consistent with published literature. Amino acid chloramines have a carboxyl group on the α-carbon which 2

3 promotes the rapid decay of monochloramine to imines and dichloramines to chlorimines via decarboxylation (Scheme 1). 3

4 Figure S1. UV spectra of chloramines with unusual decay patterns. Loss in chloramine absorbance with time at 22 C for A) Ala-Phe dichloramine, B) Gly dichloramine and C) Asn monochloramine. Spectra recorded every min for 1 min. 2.5 A Absorbance Wavelength (nm) 2. B Absorbance Wavelength (nm) 2. C Absorbance Wavelength (nm) 4

5 Figure S2. LC/MS analysis of Asn monochloramine. Asn monochloramine was analysed two min after formation and analysed by LC/MS. A) UV chromatogram at 275 nm. The HPLC peak indicated by the arrow had B) a UV spectra with maximum at 265 nm and C) mass consistent with the imine, the product of decarboxylation of the monochloramine (87. m/z). A B C 5

6 Figure S3. Real-time response of volatile ammonia monochloramine and dichloramine as detected by SIFT-MS. Detection of volatile gases of A) ammonia monochloramine and B) ammonia dichloramine in the headspace above 1 mm solutions. Insets show standard curve of volatile ammonia monochloramine and ammonia dichloramine as detected by SIFT-MS determined from the maximum response. NH 3, 17 m/z ( ); NH 2 Cl, 51 m/z ( ); NHCl 2, 85 m/z ( ). Isotopes of these species were present at the expected ratios of 3:1 for NH 2 Cl (51 and 53 m/z), and 9:6:1 for NHCl 2 (85, 87 and 89 m/z). Concentration (ppb) A Concentration (ppm) [NH2Cl] (mm) Concentration (ppb) B Concentration (ppm) [NHCl2] (mm)

7 Analysis of products formed from the decomposition of dichloramines Upon analysis of Glu-Val-Phe monochloramine by LC/MS, three major peaks were detected in the total ion chromatogram (Figure S4B). Peak i was unreacted Glu-Val- Phe. No products could be identified in peak ii, which did not change during the incubation. The major ions in peak iii were attributed to Glu-Val-Phe oxopeptide, and the Glu-Val-Phe aminol (Fig 5B inset). Glu-Val-Phe monochloramine was not detected by LC/MS. The abundance, of both the Glu-Val-Phe oxopeptide and the Glu-Val-Phe aminol increased with time after initial formation of the peptide monochloramine (Figure S4C). The abundance of unreacted Glu-Val-Phe remained constant over 9 min (data not shown). Based on these results we conclude that the monochloramine of Glu-Val-Phe decays slowly with time to produce an oxopeptide and an aminol as well as other undetected species. 7

8 Figure S4. Identification of the products formed during decay of Glu-Val-Phe monochloramine. (A) Glu-Val-Phe monochloramine was prepared by reacting 1.25 mm HOCl with 5 mm peptide in 1 mm phosphate buffer ph 7.4, diluted 1 fold into H 2 O and infused into the mass spectrometer. (B) Thirty min after preparing Glu- Val-Phe monochloramine the solution was separated and analysed by LC/MS. The inset shows the MS spectrum of peak iii. (C) The relative abundance of Glu-Val-Phe oxopeptide ( ) and the Glu-Val-Phe aminol ( ) were followed as the parent monochloramine decayed. Peak areas were determined from the extracted masses. Results are representative of at least three experiments. 8

9 1 A 394. Relative Abundance m/z Relative Abundance B i m/z ii ii Relative Abundance (1^9) C

10 Asp-Phe methyl ester dichloramine was analysed by an isocratic HPLC method. The area of peak i, the monochloramine, did not change over the course of the experiment. Other masses in both peaks ii and iii were consistent with likely in-source decomposition products of the Asp-Phe methyl ester chlorimine species i.e. decarboxylation of the chlorimine (m/z 283. and 285.); decarboxylation and hydrolysation of the chlorimine (m/z 25.); decarboxylation and chlorine exchange of the chlorimine (m/z 249.1) and fragmentation at the peptide bond to the Phe amino acid (m/z 18.) (Figures 6B&C). It appeared that there were many pathways for the decomposition of Asp-Phe methyl ester dichloramine as supported by the appearance of numerous peaks separated by HPLC (Figure S5). One peak showed no changes over 2 min, but a considerable 5 to 1-fold increase in relative peak area over 24 hours. This species was consistent with the decay of the chlorimine, via hydrolysis, to Phe (data not shown). This indicates that the chlorimine is relatively stable but does slowly hydrolyse over long periods. HPLC analysis revealed four major kinetic profiles. The first profile, exhibited by peaks labelled 1 and 4, was characteristic of transient species that were in highest abundance after 3 min of incubation of the Asp-Phe methyl ester dichloramine at 22 C. It is likely that these were reactive products that underwent subsequent reactions. Peak 1 had an absorbance maximum at 31 nm indicating it was a dichloramine species. Peak 4 had an absorbance spectrum characteristic of phenylalanine (λ max nm). The second kinetic profile was displayed by peak 2. This also reached a maximum after 3 min but slowly declined after this time. The third kinetic profile was positive and linear over 2 min, as displayed by peaks 5, 7 and 8. Peak 5 absorbed very strongly at 36.4 nm. This suggested formation of a less polar dichloramine species. A fourth kinetic profile, very similar to the chlorimine, was evident with peaks 3, 6 and 9, which showed an exponential increase to a maximum over 15 min. All of these species have absorbance maxima close to 25 nm indicating loss of one chlorine atom from the dichloramine. 1

11 Figure S5. Time course analysis of products from the decay of Asp-Phe methyl ester dichloramine. Representative profiles of unknown products formed from the decay of Asp-Phe methyl ester dichloramine..16 i ii iii x x x1 3 Relative Peak Area 5x1 3 3x1 3 2x x1 3 6x1 3 4x x1 3 2x