SHORT COMMUNICATION Synthesis and isolation of methyl bismuth

Chemical Papers 63 (6) 742–744 (2009) DOI: 10.2478/s11696-009-0071-1

SHORT COMMUNICATION

Synthesis and isolation of methyl bismuth cysteine and its definitive identification by high resolution mass spectrometry a

Joerg Hippler*, a Markus Hollmann, b Heinrich Juerling, a Alfred V. Hirner a Institute

of Environmental Analytical Chemistry, University of Duisburg-Essen, 45141 Essen, Germany b Fraunhofer

Institute for Molecular Biology and Applied Ecology (Fraunhofer IME) Department Environmental & Food Analysis, 57392 Schmallenberg, Germany Received 19 February 2009; Revised 8 May 2009; Accepted 14 May 2009

After the synthesis and isolation of methylated bismuth cysteine, its initial identification by IRspectroscopy was performed, whereas for definitive identification, high resolution mass spectrometry (ESI-TOF-MS and LTQ Orbitrap) was carried out. c 2009 Institute of Chemistry, Slovak Academy of Sciences Keywords: high resolution mass spectrometry, methyl bismuth cysteine, IR, synthesis

Although bismuth compounds have been used for more than 200 years in pharmaceutical applications, mostly as an anti-gastritic and anti-ulcer drug in the form of bismuth subsalicylate and bismuth citrate, relatively little is known about their behavior in vivo. Because of the high affinity of bismuth to sulfurcontaining molecules (Sun et al., 2004), the formation of bismuth cysteine complexes is highly probable and has been already reported in literature (Briand et al., 2004; Burford et al., 2003). For mercury, a heavy element with similar chemical behavior, the formation of cysteine complexes with biological relevance has been recently reported by Krupp et al. (2008). Furthermore, methylation of bismuth in feces and the human body has been reported (Michalke et al., 2002; Boertz et al., 2009). To enable further speciation analysis it is important to have suitable chromatographic techniques as well as stable methylated bismuth standards which are commercially not available. Consequently, we synthesized and isolated methylated bismuth cysteine. All chemicals used were of analytical grade unless stated otherwise. L-Cysteine was obtained from Fluka (> 98 %, Buchs, Schwitzerland). Methyl bismuth dibromide was purchased from VeZerf Laborsynthese GmbH (Idar-Oberstein, Germany). Methyl bismuth cysteine was prepared by adding

384 mg (0.1 mol) of solid methyl bismuth bromide (CH3 BiBr2 ) to a saturated solution of 245 mg (0.2 mol) L-cysteine (Cys) in ultra-pure water in a bismuth to cysteine mole ratio of 1 : 2. The mixture was stirred at room temperature (20 ◦C) under ultra pure argon atmosphere. After 2 h, methanol was added to the yellow reaction mixture until precipitation of a slightly yellow solid occurred. The crystalline product was isolated by filtration through a fiberglass filter and dried in a vacuum desiccator containing silica gel for several days. The product was stored under argon and kept away from light, but it is generally stable when exposed to air. The results of elemental analysis of the product: wi /mass % calculated: C, 14.0; H, 2.4; N, 4.1; S, 9.3; found: C, 12.9; H, 2.6; N, 4.7; S, 10.1 strongly indicate a bismuth to cysteine mole ratio of 1 : 1. Assuming that the center of asymmetry in L-cysteine retained unchanged, the structure of the product ([α]D = 29.0 (c = 4.0 g L−1 , H2 O, 25 ◦C)) is depicted in Fig. 1. IR spectroscopy, high resolution mass spectrometry (ESI-HR-TOF-MS and ESI-Orbitrap) as well as tandem mass spectrometry (ESI-MS/MS) gave the possibility to identify the synthesized compound. The IR spectra (KBr) of bismuth cysteine and methyl bismuth cysteine revealed that in contrast to

*Corresponding author, e-mail: [email protected]

Unauthenticated Download Date | 11/24/15 11:02 AM

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Table 1. Comparison of IR data of L-cysteine and methyl bismuth cysteine L-Cysteine

Methyl bismuth cysteine

Wavenumber (cm−1 )

Assignment

Wavenumber (cm−1 )

Assignment

1588 2551 2964 3170 3420

ν(COO− ) ν(S—H) ν(C—H) ν(N—H) ν(O—H)

1772 – 2925 3116 3462

ν(C— —O) – ν(C—H) ν(N—H) ν(O—H)

NH2

O CH3 BiBr2

HS

OH NH2

S Bi

H3C

O

O

Fig. 1. Reaction scheme of methyl bismuth cysteine preparation.

Fig. 2. Representative ESI-HR-TOF-MS spectrum (positive mode) of methyl bismuth cysteine ([M + H]+ ; m/z = 344.0160) after isolation and subsequent solution in water.

free cysteine, the characteristic IR band position of the thiol group (ν(S—H) = 2550–2600 cm−1 ) was missing in the methyl bismuth cysteine spectra. This observation strongly indicates coordination of a sulfur atom in cysteine to bismuth. The IR spectral data are listed in Table 1. For further characterization of methyl bismuth cysteine, the synthesized compound was dissolved in water and measured first by LTQ OrbitrapTM mass spectrometry (Thermo Fisher Scientific Inc., Waltham, USA). Registered dominant ions at m/z = 344.01706 (R = 36201) [M + H]+ (calculated m/z = 344.01581) strongly indicate the molecular formula of BiC4 H9 NO2 S. For the ESI-HR-TOF-MS analysis, a small amount of methyl bismuth cysteine was dissolved in a mixture of ultra pure water and methanol (ϕr = 1 : 4) which was injected into a BioTOF III system (Bruker, Bremen, Germany) using a flow injection. Data were acquired in an electrospray (ESI)

positive ion mode in the scan range of m/z from 100 to 700 and at the sampling rate of 2 GHz. The mass spectrum of methyl bismuth cysteine in aqueous solution (Fig. 2) shows molecular ions at m/z = 344.0160 [M + H]+ which are consistent with the mass spectra obtained by LTQ OrbitrapTM and the expected molecular ion (structure (1)). The spectrum also contains ions at m/z = 365.9977, some 23 amu higher than the expected molecular ion. These can be identified as the sodium adduct of the molecular ion (calculated m/z = 365.9972); sodium adducts are quite common in electrospray ionization. Additionally, the loss of 45 amu can be observed resulting in a signal at m/z = 298.0097 (calculated m/z = 298.0098) and attributable to the loss of CO2 (structure (2)) during the ionization process as well as to the loss of three hydrogen atoms during the rearrangement of bonds. The fragmentation pathway seems to be similar to the fragmentation of cobalt(II) cysteine complexes as described by Buchmann et al. (2007). A further loss of 15 amu results in a signal at m/z = 282.9866 (calculated m/z = 282.9863) representing the loss of a methyl group (structure (3)). Although it is very difficult to predict which atom of cysteine, beside sulfur, acts as a nucleophilic site, the hard-soft acids-bases theory (HSAB) may be helpful to clear the structure. Nevertheless, a six membered ring structure of 5-amino-2-methyl-1,3,2oxathiabisman-6-one (see Fig. 1) seems to be more stable than one with only five members in case of bismuth–nitrogen interactions. In aqueous solution, also pH can affect the interaction of oxygen and nitrogen with bismuth. However, molecular drawings represent monocationic species and illustrate connectivity only; drawings of these complexes are aimed at the description of bonding features. Burford et al. (2003) showed that the non-methylated form of bismuth cysteine exists as bismuth dicysteine and verified its structure by electrospray mass spectrometry. In contrast, for the methylated form, a dicysteine (calculated mass 465.0350 amu) could not be detected in positive mode measurements. Data acquiring in the negative ion mode gave a minor impurity of tribromo methylbismuthate(III) (Fig. 3). Because of the typical isotopic pattern of three bromine atoms, the structure of [MeBiBr3 ]− can


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Acknowledgements. The authors acknowledge financial support provided by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, “Synthese und Analyse biologisch relevanter Bismutspezies”).

References

Fig. 3. Measured ESI-HR-TOF-MS spectrum (negative mode) of tribromo methylbismuthate(III): impurity of methyl bismuth cysteine after isolation and subsequent solution in water, (a) and the calculated spectrum of [MeBiBr3 ]− (b).

be derived. The measured spectrum (Fig. 3a) of this impurity is in good agreement with the calculated masses and isotopic patterns as shown in Fig. 3b. The system was controlled with the software packages BioTOF (version 2.2, Bruker Daltonics). Data and calculated masses in Fig. 3b were processed using a data analysis software (version 3.2, Bruker Daltonics). These results indicate that a possible complexation of bismuth increases the stability of methyl bismuth in aqueous solution. Further HPLC analysis of methyl bismuth cysteine and/or glutathione will allow in-vitro and in-vivo determination of these compounds, but such a study would in its complexity exceed the scope of this paper.

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