morphogens from dictyostelium discoideum

BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. 16, 353-355 (1988)

Morphogens from Dictyostefium discoideum

Mark S. Masento and Howard R. Morris Department of Biochemistry, Imperial College, London SW7 2AZ, UK

Graham W. Taylor

Robert R. Kay Laboratory of Molecular Biology, Hills Road, Cambridge, UK

Department of Clinical Pharmacology, Royal Postgraduate Medical School, London W12 OHS, UK

The morphogen, DIF-1, from Dicfyostelium discoideum has recently been characterized as 1~3,5dicblorc+2,6- dihydroxy4methoxyphenyI)-l-hexaaoae. Two related differentiation-induing factors, DIF-2 and DIF-3, have been identified as the butyl ketone and monocldoro analogues of DIF-1, respectively. These substances together with a number of structural analogues have been synthesized and subsequently analysed by mass spectrometry and bioassay.

INTRODUCTION

An understanding of the signalling processes in the morphogenesis of an organism has remained one of the major challenges in developmental biology. Consider- able attention has been focused towards the identifica- tion of these signals, or morphogens. Two strategies have emerged; one is to identify and clone genes affect- ing embryonic pattern development.' Alternatively, bio- assays can be used to detect morphogenically active signals in the embryo. In this way, the major differentiation-inducing factor, DIF- 1, was isolated from the slime mould Dictyostelium d i s ~ o i d e u m . ~ . ~ Using a stalk-cell induction bioassay for quantification: the major activity released during development, DIF-1, and a number of minor biological activities, DIF-2, DIF-3, DIF-4 and DIF-5, have been purified by solvent extrac- tion and reverse-phase high-performance liquid chromatography (HPLC).5 Here we describe the structure elucidation of DIF-1, DIF-2 and DIF-3.

EXPERIMENTAL

Chemicals were of Analar grade (BDH Chemicals) unless otherwise stated. HPLC solvents were obtained from Rathburn; water was of milliQ grade. Differentia- tion factors from Dictyostelium discoideum (DIFs 1-3) were purified as reported previ~usly.~ The stalk-cell induction assay was used for quantification? Direct probe electron impact (EI) mass spectrometry was per- formed on a Kratos MS 50; the electron energy was set at 70 eV and exponential scans (10 s) were obtained over the mass range 50-500 u. Samples were fractionally evaporated from a quartz tip over a temperature range of 100-300 "C. Accurate mass measurement was carried out on a VG ZAB HF using perfluorokerosene as reference. Derivatization and microchemical treat- ments were similar to those employed in the structure

0887-6 134/88/24O353-03 $05.00 0 1988 by John Wiley & Sons, Ltd.

elucidation of SRS-A. Gas chromatography/mass spectrometry (GC/MS) was carried out on a Finnigan 4500 quadrupole mass spectrometer. Samples were injected in octane onto an SE54 capillary column (30 m). Helium was used as the carrier gas and a temperature gradient of 10°C min-' from 100 to 280°C was applied. The gas chromatograph column was routed into the mass spectrometer for analysis in the EI mode. Proton nuclear magnetic resonance (NMR) spectra were obtained in deuterochloroform on a Bruker 250 MHz instrument. DIF-1 and its analogues were synthesized via the Hoesch reaction followed by chlorination (manuscript in preparation). The products were purified by reverse-phase HPLC.

RESULTS AND DISCUSSION

Early preparations of DIF-1 did not produce adequate material for mass spectrometric or NMR analysis; they were retrospectively shown to contain only picogram amounts. Therefore initial experiments were designed to attempt functional group analysis on picogram and nanogram quantities using strategies similar to those employed in the structure elucidation of SRS-A.6 These experiments demonstrated the absence of amino groups and the presence of a carbonyl function. Stability in acid and alkali precluded lactone, amide and ester functional groups. Treatment with diazomethane indicated the presence of phenolic or other acidic hydroxyl moi- eties. A scaled-up preparation allowed ultraviolet (UV) analysis. A broad absorbance band was produced with A,, between 273 nm and 280 nm, indicating the presence of a highly conjugated chromophore. EI mass spectrometric analysis, using a temperature-pro- grammed source to distil off impurities fractionally, produced a mass spectrum at 130°C (Fig. 1). Associated isotope peaks at m/z 308 and m/z 310 in the ratio of 9 : 6 : 1 gave definitive evidence of the presence of two chlorine atoms in the DIF-1 structure. The molecular

354 M. S. MASENTO ET AL.

(a)

H 3 C 0 cl$+-

c 1

OH 0

c1

250 300

Figure 1. Partial El mass spectrum of DIF-1 (130°C). The presence of two chlorine atoms in DIF-1 is defined by the 9 :6 :1 isotope ratio of the molecular ion (M+': m/z 306, 308, 310) and each of the major fragment ions at m/z 288 (M+ ' - H,O), m/z 263 (M+'-C,H,'), m/z 235 (M+'-C,H,,') and m/z 250 ( M + ' - C,H,), McLafferty rearrangement. Note the absence of fragment ions below m/z 208, indicating the presence of a dichlorinated stable ring structure.

weight was confirmed by desorption chemical ioniza- tion (M + H', m/z 307). Accurate mass measurement of the molecular ion (306.0424) defined the atomic composition of DIF-1 as CI3Hl6O4Cl2. Major fragment ions were observed at m/z 288, 263, 250, 235 and 220. The fragment ions were characterized by high- resolution mass measurement using perfluorokerosene as a reference standard (Table 1). The molecular ion shifted to m/z 334 on treatment with diazomethane, indicating the presence of two acidic hydroxyl (phenolic) groups.

NMR analysis of the final DIF-1 preparation (about 50 pg) indicated the presence of an isolated aryl methoxy group and methylenes adjacent to the carbonyl moiety. Collisional activation tandem mass spectral data (courtesy of Professor W. Richter) confirmed the phenol moiety, and overall, the mass spectral, NMR and microchemical data defined DIF-1 as a hexanophenone, with dichloro-dihydroxy-methoxy sub- stitution. A number of synthetic analogues were pre- pared : DIF-1 was defined as 1-(3,5-dichloro-2,6- dihydroxy-4-methoxypheny1)- 1 -hexanone (Fig. 2(a) by

Table 1. Mass measurement of the molecular ion and major fragment ions for DIF-1

Elemental Ion composition Measured Fragment

( m p ) C H 0 CI mass loss

306 13 16 4 2 306.0424 M+' 288 13 14 3 2 288.0320 -H,O 263 10 9 4 2 262.9876 -C,H,' 250 9 a 4 2 249.9798 -c,H, 235 a 5 4 2 234.9563 -c,H,; 220 7 2 4 2 219.9329 -&Hi,

The ions were manually mass measured against a perfluorokerosene reference. The measured masses of each of the ions (with the exception of m/z 220) were within 1 ppm of the calculated mass.

OH 0

H 3 C 0 OH

C 1

Figure 2. The structure of the differentiation-inducing factors, DIF-1 (a), DIF-2 (b) and DIF-3 (c).

comparison of synthetic products with natural DIF by HPLC retention, bioactivity, mass spectral, NMR and other analyses.

The EI mass spectrum of DIF-2 was analogous with that of DIF-1; the 9 : 6 : 1 isotopic pattern of the major ions again defined the presence of a dichlorinated species. The molecular ion was observed at m/z 292; accurate mass measurement defined the chemical formula as C1 2H 1404C12 (292.0269). Fragment ions were observed at m/z 263 (-C2H5*), 250 (-C3H6) and 235 (-C4H9'). This suggested that DIF-2 was a butyl homologue of DIF-1, and synthesis confirmed this as the n-butyl compound : 1-(3,5-dichloro-2,6-dihydroxy-4- methoxypheny1)- 1-pentanone (Fig. 2(b)).

The molecular ion of DIF-3 was m/z 272 and the spectrum indicated that only one chlorine atom was present (isotope ratio of 3: 1). Fragment ions were observed at m/z 229 (-C3H7'), 216 (-C4H,) and 201 (-C5Hlls), which is analogous to the DIF-1 mass spectrum less one chlorine atom. Therefore DIF-3 was defined as monochloro DIF-1: 1-(3-chloro-2,6- dihydroxy-4-methoxypheny1)- 1-hexanone (Fig. 2(c)).

A number of other analogues were synthesized by the Hoesch reaction, including the isopentyl isomer, C3-C8 alkyl ketone analogues and monochloro species (manuscript in preparation); they were characterized by EI mass spectrometry and NMR spectroscopy. The relative biological activities of these substances is cur- rently under investigation.

CONCLUSIONS ~~~ ~~ ~ ~~~ ~ ~

The chemical structures of the Dictyostelium morphogens were determined by a combined microchemical, spectroscopic and synthetic strategy. The chemical structure of DIF-1 was determined as 1-(3,5-dichIoro-2,

MORPHOGENS FROM D I C T Y O S T E L I U M DISCOIDEUM 355

6-dihydroxy-4-methoxyphenyl)-l-hexanone. This rep- its function in the pattern-forming processes during resents a new type of biologically active chemical compound and a new class of effector molecule. DIF-2 is the butyl ketone analogue of DIF-1 and DIF-3 is monochloro DIF-1. Knowledge of the structure and avail- ability of synthetic DIF-1 and its analogues should allow a full analysis of both the biosynthesis of DIF and

Dictyostelium development.

Acknowledgements

We are grateful to Professor W. Richter for tandem mass spectrometric analysis and to Ms S. Johnson for NMR analysis.

REFERENCES

H. G. Frohnhofer and C. Nusslein-Volhard, Nature 324, 120 (1 986). 51 (1983). C. D. Town, J. D. Gross and R. R. Kay, Nature 262, 717 (1976). 285,104 (1 980). C. D. Town and E. Stanford, Proc. Nat. Acad. Sci. 76, 308 (1979). J. J. Brookman, C. D. Town, K. A. Jermyn and R. R. Kay, Devl. Biol. 91, 191 (1982).

5. R. R. Kay, B. Dhokia and K. A. Jermyn, Eur. J . Biochem. 136,

6. H. R. Morris, G. W. Taylor, P. J. Piper and J. R. Tippins, Nature

7. H. R. Morris, G. W. Taylor, M . S. Masento, K. A. Jermyn and R. R. Kay, Nature 328, 81 1 (1 987).

morphogens from dictyostelium discoideum

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