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Astronomy &Astrophysics A&A 658, A135 (2022) https://doi.org/10.1051/0004-6361/202141662 © J. M. Winters et al. 2022 Molecules, shocks, and disk in the axi-symmetric wind of the MS-type AGB star RS Cancri? J. M. Winters1 , D. T. Hoai2 , K. T. Wong1 , W.-J. Kim3,4 , P. T. Nhung2 , P. Tuan-Anh2 , P. Lesaffre5 , P. Darriulat2, and T. Le Bertre6 1Institut de Radioastronomie Millimétrique (IRAM), 300 rue de la Piscine, Domaine Universitaire, 38406 St. Martin d’Hères, France e-mail: winters@iram.fr 2Department of Astrophysics, Vietnam National Space Center (VNSC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Ha Noi, Vietnam 3Instituto de Radioastronomía Milimétrica (IRAM), Av. Divina Pastora 7, Núcleo Central, 18012, Granada, Spain 4I. Physikalisches Institut, Universität zu Köln, Zülpicher Str. 77, 50937 Köln, Germany 5Laboratoire de Physique de l’École Normale Supérieure, 24 rue Lhomond, 75231 Paris, France 6LERMA, UMR 8112, CNRS and Observatoire de Paris, PSL Research University, 61 av. de l’Observatoire, 75014 Paris, France Received 29 June 2021 / Accepted 29 November 2021 ABSTRACT Context. The latest evolutionary phases of low- and intermediate-mass stars are characterized by complex physical processes like turbulence, convection, stellar pulsations, magnetic ﬁelds, condensation of solid particles, and the formation of massive outﬂows that inject freshly produced heavy elements and dust particles into the interstellar medium. Aims. By investigating individual objects in detail, we wish to analyze and disentangle the effects of the interrelated physical processes on the structure of the wind-forming regions around them. Methods. We use the Northern Extended Millimeter Array to obtain spatially and spectrally resolved observations of the semi- regular asymptotic giant branch (AGB) star RS Cancri and apply detailed 3D reconstruction modeling and local thermodynamic equilibrium radiative transfer calculations in order to shed light on the morpho-kinematic structure of its inner, wind-forming environment. Results. We detect 32 lines of 13 molecules and isotopologs (CO, SiO, SO, SO 2, H2O, HCN, PN), including several transitions from vibrationally excited states. HCN, H13CN, and millimeter vibrationally excited H 2O, SO,34SO, SO 2, and PN are detected for the ﬁrst time in RS Cnc. Evidence for rotation is seen in HCN, SO, SO 2, and SiO(v=1). From CO and SiO channel maps, we ﬁnd an inner, equatorial density enhancement, and a bipolar outﬂow structure with a mass-loss rate of 1107Myr1for the equatorial region and of2107Myr1for the polar outﬂows. The12CO/13CO ratio is measured to be 20on average, 242in the polar outﬂows and 193in the equatorial region. We do not ﬁnd direct evidence of a companion that might explain this kind of kinematic structure, and explore the possibility that a magnetic ﬁeld might be the cause of it. The innermost molecular gas is inﬂuenced by stellar pulsation and possibly by convective cells that leave their imprint on broad wings of certain molecular lines, such as SiO and SO. Conclusions. RS Cnc is one of the few nearby, low-mass-loss-rate, oxygen-rich AGB stars with a wind displaying both an equatorial disk and bipolar outﬂows. Its orientation with respect to the line of sight is particularly favorable for a reliable study of its morpho- kinematics. Nevertheless, the mechanism causing early spherical symmetry breaking remains uncertain, calling for additional high spatial- and spectral-resolution observations of the emission of different molecules in different transitions, along with more thorough investigation of the coupling among the different physical processes at play. Key words. stars: AGB and post-AGB – circumstellar matter – stars: mass-loss – stars: winds, outﬂows – stars: individual: RS Cnc – radio lines: stars 1. Introduction Mass-loss in red giants is due to a combination of stellar pulsations and radiation pressure on dust forming in dense shocked regions in the outer stellar atmosphere (e.g., Höfner & Olofsson 2018). Even if the basic principles are understood, a fully consistent picture – including the role of convection, the time-dependent chemistry, and a consistent description of dust formation – still needs to be developed. In particular, the contri- bution of transparent grains to the acceleration of matter close ?NOEMA data (FITS format) are only available at the CDS via anony- mous ftp to cdsarc.u-strasbg.fr (130.79.128.5 ) or via http: //cdsarc.u-strasbg.fr/viz-bin/cat/J/A+A/658/A135to the stellar photosphere (Norris et al. 2012) still needs to be assessed. The mechanisms shaping circumstellar environments around asymptotic giant branch (AGB) stars are vividly debated. Among them, magnetic ﬁelds (Matt et al. 2000; Duthu et al. 2017), bina- rity (Theuns & Jorissen 1993; Mastrodemos & Morris 1999; Decin et al. 2020), stellar rotation (Dorﬁ & Höfner 1996), and common-envelope evolution (Olofsson et al. 2015; Glanz & Perets 2018) have been considered. A major difﬁculty is to explain the observed velocity ﬁeld in axi-symmetrical sources, with larger velocities at high lati- tudes than at low latitudes (Hoai et al. 2014; Nhung et al. 2015b). Also, recent observations of rotating structures and streams bring additional conundrums (Tuan-Anh et al. 2019; Hoai et al. 2019). A135, page 1 of 27 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.	10.1051_0004-6361_202141662	page_0000
A&A 658, A135 (2022) We have concentrated our efforts on two relatively close (d150pc) sources that show composite proﬁles in CO rota- tional lines (Winters et al. 2003): EP Aqr (Winters et al. 2007, hereafter referred to as W2007), and RS Cnc (Libert et al. 2010). Data obtained at IRAM show that these two sources have an axi-symmetrical structure with a low-velocity ( 2 km s1) wind close to the equatorial plane, and faster ( 8 km s1) outﬂows around the polar axes (Hoai et al. 2014; Nhung et al. 2015b). For EP Aqr, W2007 ﬁnd a radial dependence of the density show- ing intermediate maxima. Additional data obtained with ALMA (Nhung et al. 2019b; Homan et al. 2018b) reveal a spiral structure explaining the earlier W2007 results. RS Cnc is one of the best examples of the interaction between the stellar wind from an AGB star and the surrounding interstel- lar medium (Hoai et al. 2014). Its high declination makes RS Cnc an ideal target for the Northern Extended Millimeter Array (NOEMA). Previous studies based on IRAM data show that it is a twin of EP Aqr, but observed at a different angle, with a polar axis inclined at about 30with respect to the line of sight (Libert et al. 2010; Hoai et al. 2014; Nhung et al. 2015b). This is favor- able for studying polar and equatorial structures simultaneously, whereas the different viewing angle between EP Aqr and RS Cnc can be exploited to discriminate between different models in explaining the observed composite CO line proﬁles (Le Bertre et al. 2016). In contrast to EP Aqr, technetium is detected in the atmosphere of RS Cnc (Lebzelter & Hron 1999), proving that it is evolving along the thermal pulsing asymptotic giant branch (TP-AGB) in the Hertzsprung-Russell (HR) diagram. From a chemical point of view, RS Cnc is in a slightly more advanced evolutionary stage on the AGB, as indicated by its spectral clas- siﬁcation as an MS star (see below) and by a higher photospheric ratio of12C/13C (35; Smith & Lambert (1986), but see Sect. 4.1 for an improved evaluation based on CO rotational lines from the circumstellar environment). RS Cnc is a semi-regular variable star with periods of 122 d and248 days (Adelman & Dennis 2005), located at a distance of150pc (Gaia Collaboration 2021; Bailer-Jones et al. 2021). It is listed as S-star CSS 589 in Stephenson (1984) based on its spectral classiﬁcation M6S given in Keenan (1954). With its weak ZrO bands, its chemical type is intermediate between M and S (Keenan 1954). The stellar temperature is estimated toT3200 K and its luminosity is L4950 L(Dumm & Schild 1998). From CO rotational line observations, two circum- stellar wind components were identiﬁed: an equatorial structure expanding at about 2 km s1and a bipolar outﬂow reaching a terminal velocity of vexp8km s1(Libert et al. 2010; Hoai et al. 2014), carrying mass-loss rates of 4108Myr1and 8108Myr1, respectively (see Sect. 4.1 for an improved value of the mass-loss rate derived here). Lines of12CO, 13CO, SiO, and HI were detected from previous observations at millimeter (mm) and radio wavelengths (Nyman et al. 1992; Danilovich et al. 2015; de Vicente et al. 2016; Gérard & Le Bertre 2003; Matthews & Reid 2007). NOEMA was recently equipped with the wide band cor- relator PolyFiX, covering a total bandwidth of 15.6 GHz and therefore offering the potential to observe several lines from different species simultaneously. In this paper we present new data obtained with NOEMA in D- and A-conﬁguration, com- plemented by short spacing observations obtained at the IRAM 30m telescope. Observational details are summarized in Sect. 2 and our results are presented in Sect. 3. Section 4 contains a discussion of the morphological structures and compares them to similar structures found in EP Aqr. Our conclusions are summarized in Sect. 5.2. Observations New observations of RS Cnc have been obtained in CO(2–1) with NOEMA/WideX in the (extended) nine-antenna A- conﬁguration in December 2016 (Nhung et al. 2018) and with NOEMA/PolyFiX in the (compact) nine-antenna D- conﬁguration during the science veriﬁcation phase of PolyFiX in December 2017 and in the ten-antenna A-conﬁguration in February 2020. The WideX correlator covered an instanta- neous bandwidth of 3.8 GHz in two orthogonal polarizations with a channel spacing of 2 MHz. Additionally, up to eight high-spectral resolution units could be placed on spectral lines, providing channel spacings down to 39 kHz. WideX was decom- missioned in September 2017 and replaced in December 2017 by the new correlator PolyFiX. This new correlator simultaneously covers 7.8 GHz in two sidebands and for both polarizations, and provides a channel spacing of 2 MHz throughout the 15.6 GHz total bandwidth. In addition, up to 128 high-spectral-resolution “chunks” can be placed in the 15.6 GHz-wide frequency range covered by PolyFiX for both polarizations, each providing a ﬁxed channel spacing of 62.5 kHz over their 64 MHz bandwidth. RS Cnc was observed with two individual frequency setups covering a total frequency range of 32 GHz in the 1.3 mm atmo- spheric window (see Fig. 1). We used the two quasars J0923+282 and 0923+392 as phase and amplitude calibrators; these were observed every20 min. Pointing and focus of the telescopes was checked about every hour, and corrected when necessary. The bandpass was calibrated on the strong quasars 3C84 and 3C273, and the absolute ﬂux scale was ﬁxed on MWC349 and LkHa101, respectively. The accuracy of the absolute ﬂux calibration at 1.3 mm is estimated to be better than 20%. In order to add the short spacing information ﬁltered out by the interferometer, in May and July 2020 we observed at the IRAM 30m telescope maps of 10by 10using the On-The-Fly (OTF) mode. This turned out to be necessary for the12CO(2– 1) and13CO(2–1) lines but was not needed for the SiO lines, whose emitting region was found to be smaller than 300. In the case of the12CO(2–1) and13CO(2–1) lines, the interferometer ﬁlters out large-scale structures that account for about two-thirds and three-quarters, respectively, of the total line ﬂux, informa- tion that is recovered by adding the short spacing data from the OTF map. A comparison of the respective line proﬁles is shown in Fig. A.1. The data were calibrated and imaged within the GILDAS1 suite of software packages using CLIC for the NOEMA data calibration and the uvtable creation, CLASS for calibrating the OTF maps, and the MAPPING package for merging and subse- quent uvﬁtting, imaging, and self-calibration of the combined data sets. Continuum data were extracted for each sideband of the two frequency setups individually by ﬁltering out spectral lines, and then averaging over 400 MHz bins to properly rescale theuvcoordinates to the mean frequency of each bin. Phase self-calibration was performed on the corresponding continuum data. The gain table containing the self-calibration solutions was then applied to the spectral line uvtables using the SELFCAL procedures provided in MAPPING. The resulting data sets were imaged applying either natu- ral weighting, or, on the high-signal-to-noise (S/N) cubes, by applying robust weighting with a threshold of 0.1 to increase the spatial resolution by typically a factor 2. The resulting dirty maps were then CLEANed using the Hogbom algorithm (Högbom 1974). 1https://www.iram.fr/IRAMFR/GILDAS A135, page 2 of 27	10.1051_0004-6361_202141662	page_0001
J. M. Winters et al.: Molecules, shocks, and disk in the axi-symmetric wind of the MS-type AGB star RS Cancri Table 1. Properties of the combined data sets for all detected lines. Line Frequency Eu=k Peak ﬂux FWHP beam size PA 1 noise vel.res Comments(a) (GHz) (K) (Jy) (arcsec) (arcsec2) (deg) (mJy beam1) ( km s1) 12CO(2–1) 230.538000 16.6 53.971 10.841 6.160.01 0.480.30 36 2.88 0.5 A+D+30m, rw 13CO(2–1) 220.398684 15.9 4.693 0.948 7.200.01 0.500.31 35 2.79 0.5 A+D+30m, rw SiO(v=0,5–4) 217.104919 31.3 17.464 3.523 1.710.01 0.510.32 36 3.38 0.5 A+D, rw, sc SiO(v=1,5–4) 215.596018 1800.2 0.105 0.025 0.190.02 0.580.43 38 1.71 1.0 A, nw, sc, Feb 2020: no maser SiO(v=1,5–4) 215.596018 1800.2 0.105 0.025 0.190.02 2.101.80 0 2.71 0.5 D, nw, sc, Dec 2017: maser SiO(v=2,5–4) 214.088575 3552.1 0.013 0.005 0.350.09 1.000.74 35 1.03 3.0 A+D, nw, double peak proﬁle (?) SiO(v=0,6–5) 260.518009 43.8 23.906 4.817 1.620.01 0.430.26 32 3.39 0.5 A+D, rw, sc SiO(v=1,6–5) 258.707324 1812.7 0.168 0.038 0.110.01 0.600.42 26 1.96 1.0 A+D, nw, sc 29SiO(v=0,5–4) 214.385752 30.9 5.372 1.083 1.190.01 0.520.32 37 1.12 3.0 A+D, rw, sc Si17O(v=0,6–5) 250.744695 42.1 0.340 0.076 0.880.04 1.901.50 36 4.15(b)3.0 D, nw, sc tentative identiﬁcation 29Si17O(v=0,6–5) 247.481525 41.6 0.020 0.008 0.730.44 1.901.50 26 2.10 3.0 D, nw, sc, tentative detection SO(5(5)–4(4)) 215.220653 44.1 0.455 0.093 0.790.01 0.510.32 36 1.16 3.0 A+D, rw, sc SO(6(5)–5(4)) 219.949442 35.0 0.634 0.130 0.800.01 0.500.31 36 1.17 3.0 A+D, rw, sc SO(6(6)–5(5)) 258.255826 56.5 0.870 0.178 0.740.01 0.430.27 32 1.59 3.0 A+D, rw, sc SO(7(6)–6(5)) 261.843721 47.6 1.168 0.238 0.780.01 0.430.26 32 1.38 3.0 A+D, rw, sc 34SO(6(5)–5(4)) 215.839920 34.4 0.030 0.009 0.920.11 0.910.80 69 0.86 3.0 A+D, nw 34SO(5(6)–4(5)) 246.663470 49.9 0.026 0.009 0.930.14 0.690.48 26 0.99 3.0 A+D, nw SO2(16(3,13)–16(2,14)) 214.689394 147.8 0.021 0.006 0.500.06 0.900.68 37 1.06 3.0 A+D, nw, sc SO2(22(2,20)–22(1,21)) 216.643304 248.4 0.023 0.007 0.380.05 0.890.67 36 1.11 3.0 A+D, nw, sc SO2(28(3,25)–28(2,26)) 234.187057 403.0 0.022 0.006 0.190.05 0.710.56 46 1.28 3.0 A+D, nw, sc SO2(14(0,14)–13(1,13)) 244.254218 93.9 0.043 0.011 0.430.03 0.690.49 27 1.00 3.0 A+D, nw, sc SO2(10(3, 7)–10(2, 8)) 245.563422 72.7 0.025 0.007 0.360.04 0.690.49 28 1.00 3.0 A+D, nw, sc SO2(15(2,14)–15(1,15)) 248.057402 119.3 0.015 0.005 0.260.06 0.690.48 27 1.07 3.0 A+D, nw, sc SO2(32(4,28)–32(3,29)) 258.388716 531.1 0.020 0.006 0.210.04 0.630.45 25 1.10 3.0 A+D, nw, sc SO2( 9(3, 7)– 9(2, 8)) 258.942199 63.5 0.026 0.008 0.490.05 0.640.45 26 1.08 3.0 A+D, nw, sc SO2(30(4,26)–30(3,27)) 259.599448 471.5 0.022 0.005 0.120.03 0.630.45 25 1.03 3.0 A+D, nw, sc SO2(30(3,27)–30(2,28)) 263.543953 459.0 0.019 0.006 0.160.04 0.610.42 26 1.25 3.0 A+D, nw, sc SO2(34(4,30)–34(3,31)) 265.481972 594.7 0.020 0.006 0.190.04 0.610.42 26 1.27 3.0 A+D, nw, sc H2O(v2=1,5(5,0)–6(4,3)) 232.686700(c)3462.0 0.0290.007 unresolved 0.71 0.57 47 1.17 3.0 A+D, nw, sc, JPL H2O(v2=1,7(7,0)–8(6,3)) 263.451357(d)4474.7 0.0210.005 unresolved 0.61 0.42 26 1.17 3.0 A+D, nw, sc, JPL HCN(3–2) 265.886434 25.5 1.116 0.234 0.760.01 0.420.26 32 4.80 0.5 A+D, rw, sc H13CN(3–2) 259.011798 24.9 0.041 0.011 0.710.05 0.640.45 26 0.95 3.0 A+D, nw, sc PN(N=5–4,J=6–5) 234.935694 33.8 0.028 0.009 0.800.10 0.700.56 47 1.00 3.0 A+D, nw, sc Notes. Line frequencies and upper level energies are from the CDMS (Müller et al. 2005), unless otherwise stated. The quoted ﬂux uncertainties include the rms of the ﬁts and the absolute ﬂux calibration accuracy of 20%, the uncertainties quoted for the source sizes refer to the rms errors of the Gaussian ﬁts (see text).(a)A: NOEMA A-conﬁguration, D: NOEMA D-conﬁguration, 30 m: short spacing data, rw: robust weighting, nw: natural weighting, sc: self-calibrated, JPL: Spectral line catalog by NASA/JPL (Pickett et al. 1998).(b)Increased noise at band edge.(c)Belov et al. (1987).(d)Pearson et al. (1991). The beam characteristics and sensitivities of the individual combined data sets from A- and D-conﬁguration (and including the pseudo-visibilities from the OTF maps, where appropriate) are listed in Table 1 for all detected lines. 3. Results The PolyFiX data, covering the frequency ranges 213–221 GHz (setup1, LSB), 228-236 GHz (setup1, USB), 243–251 GHz (setup2, LSB), and 258–266 GHz (setup2, USB) with two setups (see Fig. 1), showed different lines of CO and SiO, and, for the ﬁrst time, many lines of species like SO, SO 2, HCN, and PN and some of their isotopologs. Furthermore, the data conﬁrmed the H2O line at 232.687 GHz already detected serendipitously withWideX in 2016, with a second H 2O line at 263.451 GHz seen for the ﬁrst time in RS Cnc. All lines covered by the same setup (1 or 2, see Fig. 1) share the same phase-, amplitude-, and ﬂux calibration. All 32 detected lines are listed in Table 1. 3.1. Continuum Figure 2 shows the continuum map from A-conﬁguration only, using robust weighting to increase the spatial resolution to 0:39000:2200at PA 28. After self-calibration, S/N = 492 is obtained. The continuum source is unresolved, a point source ﬁt results in a ﬂux at 247 GHz of 23.65 4.7 mJy (where the quoted error accounts for the accuracy of the absolute ﬂux cali- bration of 20%) and a source position at RA = 09:10:38.780 and A135, page 3 of 27	10.1051_0004-6361_202141662	page_0002
A&A 658, A135 (2022) Fig. 1. Overview of the frequency ranges observed with PolyFiX using two spectral setups (setup1: red and setup2: blue, respectively). Lower diagrams : zoom onto the individual spectra covering 7.8 GHz each. Upper row : setup1, lower row : setup2. The central 20 MHz at the border between inner and outer baseband are blanked out, i.e., set to zero, as this region is contaminated by the LO2 separation of the 8 GHz-wide IF in the IF processor (“LO2 zone”). Dec = 30:57:46.62 in February 2020. All line data cubes dis- cussed in the remainder of this paper are re-centered on this continuum position. The source position is offset from the J2000 coordinates by 0.2600in RA and by0.6800in Dec, consistent with the proper motion of RS Cnc ( 10.72 mas yr1in RA and33.82 mas yr1 in Dec, Gaia Collaboration 2021; Bailer-Jones et al. 2021). From the PolyFiX data, spanning a total frequency range of about 53 GHz, we determine a spectral index of 1.99 0.09 for RS Cnc in the 1 mm range, which is fully consistent with a black body spectrum of the continuum (see also Libert et al. 2010). 3.2. Detected molecules and lines Within the total frequency coverage of about 32 GHz, we detect 32 lines of 13 molecules and isotopologs, including several tran- sitions from vibrationally excited states. All these lines are listed in Table 1 and are presented in the following sections. The peak ﬂux and FWHP of the line-emitting regions, as listed in Table 1,are determined by circular Gaussian ﬁts in the uv-plane to the central channel (if the source is (partially) spatially resolved) or by point-source ﬁts to the central channel (if the source is unre- solved). All line proﬁles shown in the following sections in Fig. 3 and Figs. 5 through 14 are integrated over square apertures whose sizes are given in each ﬁgure caption. Two-component proﬁles are seen in CO and13CO only, and not in any other of the lines detected here. We looked for but did not detect the vibrationally excited 12CO(v=1, 2–1) line, nor do we detect C18O(2–1), result- ing in 3upper limits for the line peaks of 6 mJy beam1and 3 mJy beam1, respectively (the12CO(v=1, 2–1) line was not covered in our A-conﬁguration data). 3.2.1. CO The proﬁles of12CO(2–1),13CO(2–1) (see Fig. 3), and12CO(1– 0) (see Libert et al. 2010) show a very distinct shape composed of a broad component that extends out to vlsr;8 km s1and A135, page 4 of 27	10.1051_0004-6361_202141662	page_0003
J. M. Winters et al.: Molecules, shocks, and disk in the axi-symmetric wind of the MS-type AGB star RS Cancri Fig. 2. Continuum map around 247 GHz from A-conﬁguration. Con- tours are plotted in 100 steps, where 1 is 47.6 Jy beam1. The synthesized beam is indicated in the lower left corner. Fig. 3. CO line proﬁles, showing a two-component structure. Left: 12CO(2–1). Right :13CO(2–1). A-conﬁguration and D-conﬁguration are merged, OTF data are added, and the spectral resolution is 0.5 km s1. The CO emission is integrated over the central 22002200, i.e., over the full ﬁeld of view of the NOEMA antennas at 230 GHz. Fig. 4. Sketch of the geometrical structure of the wind components as inferred from the current data (see Sect. 4.1). The sketch is not to scale: there is a smooth transition between the equatorial enhancement and the polar outﬂows. a narrow component indicating velocities of 2 km s1with respect tovlsr;=7km s1. Velocity-integrated intensity maps of CO are shown in Fig. 18, indicating a clear kinematic structure in the north–south direction. In Fig. 4, we present a schematic representation of the geometrical structure of RS Cnc as implied by the data; see Sect. 4.1. The CO emitting region is spatially extended, consisting of a dense equatorial structure that corre- sponds to the low-velocity expansion and an inclined, bipolar Fig. 5. Proﬁles of SiO ground-state and ﬁrst vibrationally excited state lines. Left: SiO(6–5): upper :v=1,lower :v=0. A-conﬁguration and D- conﬁguration merged. Right : SiO(5–4): upper :v=1, D-conﬁguration (black) and A-conﬁguration (red), lower :v=0, A-conﬁguration and D- conﬁguration merged. The spectral resolution is 1 km s1for (v=1) and 0.5 km s1for the (v=0) lines, respectively. The emission is integrated over the central 500500aperture. structure corresponding to an outﬂow at a projected velocity of 8 km s1. These structures were discussed in Hoai et al. (2014) based on Plateau de Bure data obtained on12CO(2–1) and12CO(1–0) that had a spatial resolution of about 100. The model built by these latter authors was later reﬁned by Nhung et al. (2018) based on12CO(2–1) data obtained with the WideX correlator in NOEMA’s A-conﬁguration, providing a spatial res- olution of 0:44000:2800. Nhung et al. (2018) ﬁnd a position angle of the projected bipolar outﬂow axis of !=7(measured counter-clockwise from north) and an inclination angle of the outﬂow axis with respect to the line of sight of i=30. The CO distribution is further investigated in Sect. 4.1 below. Such a structure had already been found in the S-type star 1 Gru (Sahai 1992), which was later conﬁrmed by higher spatial resolution observations using ALMA (Doan et al. 2017). This object has a G0V companion (Feast 1953) and possibly a sec- ond, much closer companion (Homan et al. 2020). In Hoai et al. (2014), we reported for RS Cnc the possible presence of a com- panion seen in the12CO(1–0) channel maps at velocities around 6.6 km s1and located about 100west-northwest of the contin- uum source. The new data allow for a more detailed study of this feature, which is presented in Sect. 4.1. 3.2.2. SiO We detect a suite of28Si16O (henceforth SiO) transitions, includ- ing the vibrational ground-state lines of SiO(5–4) and SiO(6–5), the ﬁrst and second vibrationally excited state of SiO(5–4), and the ﬁrst vibrationally excited state of SiO(6–5). All SiO pro- ﬁles are shown in Figs. 5 and 6. The spatial region emitting the vibrational ground-state lines extends out to about 200from the continuum peak (see Table 1, Fig. 18, and Sect. 4.2). Interest- ingly, we detect a strong maser component on the SiO( v=1, J=5–4) line at vlsr14km s1in the data obtained in December 2017, which had completely disappeared when we re-observed RS Cnc in February 2020 (see Fig. 5). Such behav- ior is well known for pulsating AGB stars, and lends support to A135, page 5 of 27	10.1051_0004-6361_202141662	page_0004
A&A 658, A135 (2022) Fig. 6. Proﬁle around the SiO( v=2,J=5–4) line frequency. A- conﬁguration and D-conﬁguration merged. The spectral resolution is 1 km s1and the emission is integrated over the central 100100aperture. the idea that the SiO masers are excited by infrared pumping as opposed to collisional pumping (see, e.g., Pardo et al. 2004). The SiO(v=2,J=5–4) line is detected above the 3 level of 3 mJy beam1over a broad range of Doppler velocities from at least5 to 18 km s1(Fig. 6). Given its high excitation energy (3500 K), we expect this line to trace exclusively the inner- most region around RS Cnc, as was the case in oCet, where SiO(v=2) absorption and emission was spatially resolved by ALMA (Wong et al. 2016). Its broad line width suggests that it may trace the same high-velocity wings seen in other detected SiO lines (Sect. 3.3). However, our detection is too weak to allow for a detailed study of the morpho-kinematics of the emission. At the upper edge of the LSB of setup 2 at 250.744 GHz, we serendipitously detect a strong line that we identify as ground-state Si17O(6–5) at 250.7446954 GHz (Müller et al. 2013) from the Cologne Database of Molecular Spectroscopy (CDMS2, Müller et al. 2005); the proﬁle is shown in Fig. 7. This line and other transitions of Si17O have already been detected in a number of well-studied objects, such as the S-type star W Aql (De Beck & Olofsson 2020), the M-type star R Dor (De Beck & Olofsson 2018), and the evolved, high-mass-loss- rate oxygen-rich star IK Tau (Velilla Prieto et al. 2017). No other Si17O transitions are covered in our setups, but there is a highly excited H 2O line at 250.7517934 GHz ( v2=2,J(Ka;Kc)= 9(2,8)–8(3,5); Eu=k=6141 K) listed in the JPL catalog3and pre- dicted by Yu et al. (2012) from the Bending-Rotation approach analysis. If the detected line was H 2O emission, it would be redshifted from the systemic velocity by about 9 km s1. As indi- cated by the modeling of Gray et al. (2016), the 250.752 GHz line may exhibit strong maser action in regions of hot gas (Tkin=1500 K) with cool dust ( Td1000 K). While we can- not unequivocally exclude some contamination from a potential new, redshifted H 2O maser, we consider Si17O a more likely identiﬁcation of the 250.744 GHz emission. From the respective integrated line intensities of Si16O(6–5) and Si17O(6–5), which are163 Jy km s1and3 Jy km s1, and taking the difference of the Einstein coefﬁcients of the transitions into account, we estimate the isotopolog ratio16O/17O50, assuming equal exci- tation conditions for both transitions and optically thin emission of both lines. This value is much lower than the solar isotopic ratio of2700 (Lodders et al. 2009) due to dredge-up events (Karakas & Lattanzio 2014; Hinkle et al. 2016) and is broadly consistent with those obtained in the M-type star R Dor and the S-type star W Aql (61–74; De Beck & Olofsson 2018, 2020). The initial mass of RS Cnc is about 1:5M(Libert et al. 2010)4, 2https://cdms.astro.uni-koeln.de 3https://spec.jpl.nasa.gov/ftp/pub/catalog/catform. html 4As quoted in Libert et al. (2010), the value of 1:5Mwas esti- mated by Busso & Palmerini (their priv. comm.) using the FRANEC ).Fig. 7. Line proﬁles of SiO isotopologs. Upper left : proﬁle of the 247.482 GHz line, possibly29Si17O(6–5); D-conﬁguration, only. Lower left: Si17O(6–5): D-conﬁguration, only (line was not covered in A- conﬁguration). Right :29SiO(5–4); A-conﬁguration and D-conﬁguration merged. The spectral resolution in all cases is 3 km s1and the emission is integrated over the central 500500aperture. which is in the same range as R Dor ( 1:4M; De Beck & Olofsson 2018) and W Aql ( 1:6M; De Nutte et al. 2017) that gives a16O/17O ratio of<1000 (Hinkle et al. 2016). However, we note that the oxygen isotopic ratio (16O/17O) derived from the line intensity ratio is likely underestimated if the Si16O line is not optically thin, as has been shown in De Beck & Olofsson (2018), who obtained a value of 400in R Dor with radiative transfer modeling. Indeed, we demonstrate in Sect. 4.2 that the Si16O emission in RS Cnc is optically thick, especially within a projected radius of 100. A photospheric16O/17O ratio of 710 in RS Cnc (=HR 3639) was estimated by Smith & Lambert (1990) from the spectra of near-infrared overtone band transitions of C16O and C17O, which is probably a more realistic ratio. We do not cover C17O(2–1) in our setups and therefore cannot give an independent estimate of the16O/17O ratio. As Si18O(6–5) and C18O(2–1) are either not covered or not detected, there is not enough information from our data to obtain a meaningful con- straint on the initial stellar mass from oxygen isotopic ratios (e.g. from the17O/18O ratio; De Nutte et al. 2017). We detect a line at 247.482 GHz at low S/N that might be identiﬁed as29Si17O(v=0,J=6–5) at 247.4815250 GHz based on the line list by Müller et al. (2013) and used in the CDMS (see Fig. 7). However, in contrast to Si17O(6–5),29Si17O(6–5) has never been detected; only higher-J lines of29Si17O have been tentatively detected in R Dor ( J=7–6 and J=8–7, De Beck & Olofsson 2018). More speciﬁcally, the 247.482 GHz line is seen with an integrated line intensity of 0:08Jy km s1in our D-conﬁguration data only, observed in December 2017, but it does not show up in the A-conﬁguration data, taken in February 2020. This may largely be due to the much reduced brightness stellar evolution code (Cristallo et al. 2011) and the molecular abun- dances determined by Smith & Lambert. Smith & Lambert (1990) reported oxygen isotopic ratios of16O/17O=710 and16O/18O=440 in RS Cnc (their Table 9). The17O/18O ratio of 0.62 corresponds to an initial mass of 1.4–1.5 Min the comparative study of De Nutte et al. (2017), who investigated the17O/18O isotopic ratio as a sensitive function of initial mass of low-mass stars based on the models of Stancliffe et al. (2004), Karakas & Lattanzio (2014), and the FRANEC model. A135, page 6 of 27	10.1051_0004-6361_202141662	page_0005
J. M. Winters et al.: Molecules, shocks, and disk in the axi-symmetric wind of the MS-type AGB star RS Cancri ). Fig. 8. HCN line proﬁles. Left: HCN(3–2); A-conﬁguration and D- conﬁguration merged with a spectral resolution of 0.5 km s1.Right : H13CN(3–2); A-conﬁguration and D-conﬁguration merged with a spec- tral resolution of 3 km s1. The emission of both lines is integrated over the central 200200. sensitivity in the A-conﬁguration, which is a factor of approxi- mately 15 smaller because of the smaller synthesized beam area rather than some variable maser action in this line. Based on the D-conﬁguration data, the source position of the 247.482 GHz emission appears slightly offset toward the northwest direction from the Si17O(6–5) emission. Further data on29Si17O, possibly covering the J=6–5,J=7–6, and J=8–7 transitions, would be needed to draw any ﬁrm conclusion. 3.2.3. HCN We clearly detect the HCN(3–2) and H13CN(3–2) lines; the proﬁles are displayed in Fig. 8, and velocity-integrated inten- sity maps of both species are shown in Fig. B.1. Both lines are slightly spatially resolved and a circular Gaussian ﬁt to HCN(3–2) gives a peak ﬂux of 1.12 Jy and a FWHP size of 0.7600 on the merged data. To our knowledge, this is the ﬁrst detection of HCN and H13CN in RS Cnc (see Sect. 4.4). From the ﬁrst- moment map (shown in Fig. 17, left), a clear velocity pattern is evident that indicates possible rotation in the HCN-emitting region (see Sect. 3.4). Also, the velocity-integrated intensity maps presented in Fig. B.1 show a clear kinematic structure in the east–west direction. Formation of the HCN molecule in oxygen-rich environ- ments is further discussed in Sect. 4.4. A modeling using the 1D local thermodynamic equilibrium (LTE) radiative transfer code XCLASS (Möller et al. 2017, see Appendix D) gives a column density for HCN in RS Cnc of N HCN=1:61015cm2, cor- responding to an abundance of X(HCN/H 2)=6:6107. This value is well within the range found for other M- and S-type stars as modeled by Schöier et al. (2013), who ﬁnd X(HCN/H 2) equal to a few times 107(for more details see Sect. 4.4 and Appendix D). 3.2.4. H 2O The WideX spectrum obtained in A-conﬁguration in Decem- ber 2016 serendipitously revealed a line at 232.687 GHz that we ascribe to the J(Ka,Kc)=5(5,0)–6(4,3) transition of o-H 2O in thev2=1vibrational state. The H 2O source is weak and seems still unresolved within the synthesized beam of 0:5000:3400 obtained in the A-conﬁguration in February 2020, consistent with its high upper-state energy of 3462 K. The line proﬁle is shown in Fig. 9. With the follow-up observations employing PolyFiX in D-conﬁguration and A-conﬁguration we also cov- ered and detected the 263.451 GHz o-H 2Ov2=1,J(Ka,Kc)= 7(7,0)–8(6,3) line (Fig. 9, right; Eu=k=4475 K). Both lines are resampled to a resolution of 3 km s1, data are merged from A-conﬁguration and D-conﬁguration, and the emission is Fig. 9. H2O line proﬁles. Left: H 2O line at 232.687 GHz. Right : H 2O line at 263.451 GHz. Data are merged from A-conﬁguration and D- conﬁguration, the spectral resolution is 3 km s1, and the emission of both lines is integrated over the central 100100aperture. integrated over an aperture of 100100. Intensity maps of both lines are shown in Fig. B.2, testifying to the compactness of the H2O-emitting region. These are the ﬁrst detections of millimeter vibrationally excited H 2O emission in RS Cnc. We note that the 22 GHz H2O maser in the ground state was tentatively detected by Szymczak & Engels (1995) in one of the two epochs they cov- ered, but the 22 GHz line is not detected in other observations (Dickinson et al. 1973; Lewis 1997; Han et al. 1995; Yoon et al. 2014). RS Cnc also shows clear photospheric H 2O absorption at 2:7m (Merrill & Stein 1976; Noguchi & Kobayashi 1993), and at1:3m (7500 cm1; Joyce et al. 1998), although the H 2O band near 900 nm is not detected (Spinrad et al. 1966). Both the 232 and 263 GHz water lines have upper levels belonging to the so-called transposed backbone in the v2=1 vibrationally excited state of H 2O, that is Ka=JandKc=0or 1 (see Fig. 1 of Alcolea & Menten 1993). The 232 GHz line was ﬁrst detected in evolved stars together with the 96 GHz line from another transposed backbone upper level by Menten & Melnick (1989) toward the red supergiant VY CMa and the AGB star W Hya. The latter is an M-type star with a similar mass-loss rate to RS Cnc. The authors ﬁnd that the 232 GHz line emission in both stars may be of (quasi-)thermal nature while the 96 GHz line clearly showed maser action. The (unpublished) detection of the 263 GHz line was mentioned in Alcolea & Menten (1993), who also described a mechanism that may lead to a system- atic overpopulation of the transposed backbone upper levels in thev2=1state of H 2O in the inner region of circumstellar envelopes. If the vibrational decay routes (to the ground state) of the transposed backbone upper levels become more optically thick than the lower levels in the v2=1state, then differential radiative trapping may cause population inversion of these lines. Additional vibrationally excited H 2O emission lines from trans- posed backbone upper levels were predicted and later detected in VY CMa by Menten et al. (2006) and Kami ´nski et al. (2013). We observed the 232 GHz line in RS Cnc at three epochs (December 2016, December 2017, and February 2020) and the 263 GHz line at the latter two epochs, and the emission appears to be stable in time for both lines. The proﬁles appear to be very similar, both are broad, even broader than the (ground-state) lines of other species reported here, and there is no sign for any narrow com- ponent in either of the two proﬁles at any of the epochs. As the lines should arise from a region very close to the star – compat- ible with their broad widths; see Sect. 3.3 – one might expect to see time variations due to the varying density and radiation ﬁeld caused by the stellar pulsation, in particular if the emission were caused by maser action, as seen on the SiO( v=1;5–4) line observed in December 2017 (see Fig. 5). Also, the model- ing of Gray et al. (2016) shows only very little inversion of the A135, page 7 of 27	10.1051_0004-6361_202141662	page_0006
A&A 658, A135 (2022) Fig. 10. Proﬁles of the four detected SO lines, with A-conﬁguration and D-conﬁguration merged. The spectral resolution is 3 km s1and the emission is integrated over the central 200200aperture. Fig. 11. Proﬁles of the two34SO lines detected here, with A- conﬁguration and D-conﬁguration merged. The spectral resolution is 3 km s1and the emission is integrated over the central 200200aperture. involved level populations for the 263 GHz H 2O transition. We therefore think that both lines could be thermally excited. A def- inite assessment of the nature of the vibrationally excited H 2O emission would however require some detailed modeling of the emission, together with high-sensitivity monitoring of the line proﬁles with high spectral resolution, possibly including other H2O lines from transposed backbone upper levels and/or known maser lines for comparison, which is beyond the scope of the present paper. 3.2.5. SO Four lines of SO are detected (see Fig. 10) along with two lines of the isotopolog34SO (Fig. 11). These represent the ﬁrst detections of SO and34SO in RS Cnc. SO has been observed in several M- type stars, including R Dor and W Hya, (Danilovich et al. 2016)), but remains undetected in S-type stars (e.g., W Aql, Decin et al. 2008; De Beck & Olofsson 2020). All SO lines detected here are slightly spatially resolved with a FWHP around 0:800and therefore seem to be emitted from the same region as HCN. Velocity-integrated intensity maps of SO are shown in Fig. B.3. The SO lines show the same velocity pattern (indicating rota- tion) as HCN, although the velocity resolution of the SO lines is only 3 km s1; see Fig. B.3 and the ﬁrst-moment map in the right panel of Fig. 17. Using the integrated line strengths of SO(6(5)–5(4)) and34SO(6(5)–5(4)) found here ( 4.69 Jy km s1and 0.20 Jy km s1, respectively) and taking the difference of the Einstein coefﬁcients of the transitions into account, weestimate the isotopolog ratio32SO/34SO23, assuming equal excitation conditions for both transitions and optically thin emission of both lines. This value is in good agreement with the values of 21.68:5and 18.55:8derived from the radiative transfer models for M-type stars by Danilovich et al. (2016, 2020), respectively. We note that, for the S-type star W Aql, an Si32S/Si34S isotopolog ratio of 10.6 2:6was derived by De Beck & Olofsson (2020). As32S is mainly produced by oxygen burning in massive stars and, to a lesser extent, in type Ia supernovae, and as34S is formed by subsequent neutron capture (e.g., Nomoto et al. 1984; Wilson & Matteucci 1992; Timmes et al. 1995; Hughes et al. 2008), the32S/34S isotopic ratio remains virtually unaltered during AGB evolution (see, e.g. tables in the FRUITY5database, Cristallo et al. 2011) and there- fore should reﬂect the chemical initial conditions of the natal cloud from which the star has formed. The spread in the isotopic ratio seen among the different AGB stars mentioned above would then rather be indicative of the Galactic environment in which the star has formed (see, e.g., Chin et al. 1996; Humire et al. 2020) instead of reﬂecting any evolutionary effect. For the low-mass-loss-rate M-type stars R Dor and W Hya, Danilovich et al. (2016) reproduce their observed line proﬁles best with centrally peaked SO (and SO 2) distributions, consistent with the maps presented in Fig. B.3. 3.2.6. SO 2 In SO 2, 11 lines are detected; their parameters are summarized in Table 2, and all proﬁles are shown in Fig. C.1. These are the ﬁrst detections of SO 2in RS Cnc. A previous survey with the IRAM 30 m telescope by Omont et al. (1993) did not detect SO2in RS Cnc with an rms noise of 0.052 K (or 0:25Jy at 160.8 GHz). As an example, we show the SO 2(14(0,14)– 13(1,13)) line at 244.3 GHz, only in Fig. 12. A ﬁrst-moment map of the SO 2(14(0,14)–13(1,13)) line is shown in Fig. 17 in the middle left panel. Although the source remains barely resolved (source size 0:4300) by the beam ( 0:69000:4900), there is a signature of a rotating structure in SO 2, as was also seen in EP Aqr (Homan et al. 2018b; Tuan-Anh et al. 2019). Inte- grated intensity maps of three SO 2lines (SO 2(9(3, 7)–9(2, 8)), which has the lowest upper level energy of the SO 2lines detected here ( Eu=64K); SO 2(14(0,14)–13(1,13)), the strongest line, and SO 2(34(4,30)–34(3,31)), which has the highest upper level energy of the detected lines, Eu=595K) are shown in Fig. B.4. All lines show kinematic structure in the E–W direction, approx- imately orthogonal to the outﬂow structure seen in CO and SiO, cf. Fig. 18. We derive the rotational temperature and column density of the SO 2-emitting region with a population diagram analysis (Sect. 3.6) and by an XCLASS modeling (Appendix D). Both methods give a similar rotational temperature of 320350K and a column density of 3:51015cm2. 3.2.7. PN We detect a line at 234.936 GHz that we ascribe to the PN molecule, which would be the ﬁrst detection of PN in RS Cnc. PN has been detected in several M-type stars (e.g., De Beck et al. 2013; Ziurys et al. 2018), and in the C-rich envelopes of IRC +10216 and CRL 2688 (Guélin et al. 2000; Cernicharo et al. 2000; Milam et al. 2008). The presence of PN in an MS-type star therefore does not seem to come as a surprise. However, RS Cnc 5http://fruity.oa-teramo.inaf.it/ A135, page 8 of 27	10.1051_0004-6361_202141662	page_0007
J. M. Winters et al.: Molecules, shocks, and disk in the axi-symmetric wind of the MS-type AGB star RS Cancri Table 2. Parameters of the detected SO 2lines used for the population diagram analysis. Frequency WI=R S(v)dv g ulog10(Aul) Eu=kab (GHz) (Jy km s1) (s1) (K) (arcsec2) 214.6894 0.1410.0385 33 –4.0043 147.843 0.90 0.68 216.6433 0.166 0.0434 45 –4.0329 248.442 0.89 0.67 234.1871 0.1600.0439 57 –3.8401 403.033 0.71 0.56 244.2542 0.293 0.0698 29 –3.7855 93.901 0.69 0.49 245.5634 0.170 0.0451 21 –3.9240 72.713 0.69 0.49 248.0574 0.119 0.0333 31 –4.0939 119.328 0.69 0.48 258.3887 0.153 0.0396 65 –3.6773 531.100 0.63 0.45 258.9422 0.192 0.0524 19 –3.8800 63.472 0.64 0.45 259.5994 0.182 0.0448 61 –3.6835 471.496 0.63 0.45 263.5440 0.152 0.0448 61 –3.7227 459.038 0.61 0.42 265.4820 0.168 0.0448 69 –3.6426 594.661 0.61 0.42 Notes. Data are merged from A-conﬁguration and D-conﬁguration. Quoted errors include the rms errors of the Gaussian ﬁts in the uvplane and the absolute ﬂux calibration accuracy of 20%. The SO 2line parameters are retrieved from the CDMS and are based on the calculations by Lovas (1985) and Müller & Brünken (2005). Fig. 12. Proﬁle of SO 2(14(0,14)–13(1,13)) with A-conﬁguration and D- conﬁguration merged, a spectral resolution of 3 km s1, and emission integrated over the central 200200aperture. Fig. 13. Proﬁle of PN( N=5–4,J=6–5) with A-conﬁguration and D- conﬁguration merged, a spectral resolution of 3 km s1, and emission integrated over the central 200200aperture. appears to be the source with lowest mass-loss rate in which this molecule has been reported so far. The PN line proﬁle is shown in Fig. 13. The line is spatially resolved at 0:800, which places it in about the same region as HCN and SO. The ﬁrst-moment map of this line also shows signatures of rotation but due to the weak- ness of the line, the evidence is low. An integrated intensity map of PN is presented in Fig. B.5, showing that the line-emitting region is slightly spatially resolved. The 3feature seen about 1:500south of the phase center should not be considered as a detection but rather as a noise peak, as long as this structure is not conﬁrmed by higher sensitivity observations. Fig. 14. Line wings in SiO(5–4) and SiO(6–5) compared to CO(2–1). The emission is integrated over the central 500500aperture. Fig. 15. High velocities close to the line of sight as seen in SiO. PV maps are shown in the Vzvs.Rplane for SiO(5–4) ( left) and SiO(6–5) (right ). The horizontal black line indicates the wind terminal velocity as traced in CO and the white scale bar indicates the spatial resolution. R=p (Dec)2+(RA)2,jVzj=jvlsrvlsr;j: 3.3. High-velocity wings in SiO, and in other molecules In SiO, ﬁve lines in three different vibrational states ( v=0,1,2) are detected (see Figs. 5 and 6). The vibrational ground-state lines clearly indicate the presence of material at velocities much higher than the wind terminal velocity of 8 km s1as traced by CO lines at this stellar latitude (see Sect. 4.1). This is illustrated in Fig. 14, and in Fig. 15 where we deﬁne vz=vlsrvlsr;, the Doppler velocity relative to the star. The high-velocity region is centered on the line of sight and is conﬁned to the inner 0:300; see Fig. 15. A similar feature was seen in high-spatial- resolution observations of other oxygen-rich, low-mass-loss-rate A135, page 9 of 27	10.1051_0004-6361_202141662	page_0008
A&A 658, A135 (2022) AGB stars, such as W Hya (Vlemmings et al. 2017), EP Aqr (Tuan-Anh et al. 2019), oCet (Hoai et al. 2020), R Dor (Decin et al. 2018; Nhung et al. 2019a, 2021), and in 15 out of 17 sources observed in the ALMA Large Program ATOMIUM (Decin et al. 2020; Gottlieb et al. 2022), calling for a common mechanism causing high-velocity wings in this type of object. In the case of EP Aqr, where the bipolar outﬂow axis almost coincides with the line of sight (with an inclination angle of i10), the high- velocity wings were interpreted in terms of narrow polar jets. For R Dor and oCet, which do not show obvious signs of axial symmetry in their winds, such an interpretation could not be retained and it was argued instead that the high-velocity wings were caused by (a mixture of) turbulence, thermal broadening, and some effect of shocks, acting at distances below some 10 to 15 AU from the central star. The presence of broad wings in the SiO lines emitted from RS Cnc, whose symmetry axis is inclined by30with respect to the line of sight (see Sect. 4.1), lends sup- port to the latter type of interpretation and casts serious doubts on the polar jet interpretation proposed earlier for EP Aqr, which shows a morpho-kinematics similar to that of RS Cnc (Nhung et al. 2015b). Indeed, if the broad line widths are present regard- less of the orientation of a possible symmetry axis with the line of sight, they must be caused by a mechanism of nondirectional (accounting for the resolving beam) nature. A possible candi- date, whose action is limited to the close vicinity of the star, is pulsation-driven shocks that dissipate their energy relatively close to the star and imply positive and negative velocities in the shocked region that can be much higher than the terminal out- ﬂow velocity of the wind. Such structures could be explained by the B-type models discussed in Winters et al. (2000b) as presented in Winters et al. (2002); see their Fig. 3. Recent 3D model calculations that self-consistently describe convection and fundamental-mode radial pulsations in the stellar mantle would provide the physical mechanism that leads to the development of such shocks close to the star surface (e.g., Freytag et al. 2017) and could therefore replace the simpliﬁed inner boundary condi- tion (the so-called “piston approximation”) that was used in the earlier 1D models mentioned above. In the data presented here, wings at high Doppler velocity are seen in nearly all lines detected with sufﬁcient sensitivity to probe the proﬁle over at least vlsr;10km s1. This is illus- trated in Fig. 16, where vzproﬁles are integrated over a circle of radius 0:200centered on the star. Gaussian proﬁles centered at the origin are shown as visual references (not ﬁts), showing how absorption produces asymmetric proﬁles. A major differ- ence is seen between vibrational ground-state lines, which have a Gaussian FWHM of 10km s1, and vibrationally excited- state lines, which have a Gaussian FWHM of 14km s1. Such a difference is not surprising, assuming that the high- velocity wings are formed in the inner layer of the circumstellar envelope (CSE), which is preferentially probed by the ( v=1) lines. In this context, we note that Rizzo et al. (2021) recently reported the detection of a narrow SiO( v=1, 1–0) maser line in RS Cnc at a velocity of +14 km s1with respect to the star’s lsr velocity. The effect of shocks on line proﬁles was ﬁrst observed in the near-infrared range on CO ro-vibrational lines, probing the stellar photosphere and the innermost circumstellar region within10R(e.g.,Cyg, an S-type star, Hinkle et al. 1982). Very-high-angular-resolution observations obtained over the past decade using VLT, VLTI, and ALMA show that the effect of shocks from pulsations and convection cell ejections is conﬁned within some 10 AU from the star (see, e.g., Khouri et al. 2018; Höfner & Olofsson 2018; Ohnaka et al. 2019, and references therein). Rotation, when observed, is instead found Fig. 16. Line proﬁles of different molecules on a logarithmic intensity scale. Gaussian proﬁles are shown for comparison, FWHM =10km s1 for the ground-state lines of all molecules, and FWHM =14km s1 for the (v=1) lines of SiO. All observed proﬁles are integrated over R<0:200. to extend beyond this distance, typically up to 20 AU (e.g., Vlemmings et al. 2018; Homan et al. 2018a; Nhung et al. 2021). The angular resolution of the present data is insufﬁcient to detect such differences directly; however, the effect of rotation and shocks on lines of sight contained within a beam centered on the star depends on the region probed by each speciﬁc line: lines that probe the inner layers exclusively, such as the ( v=1) lines, are mostly affected by shocks, and somewhat by rotation; CO lines, for which the probed region extends very far out, see little effects of rotation and even less effects of shocks because the emission from the inner envelope provides too small a fraction of the total emission. Between these two extremes, the relative importance of the contributions of shocks and rotation depends on the radial extent of the region probed by the line. Such an interpretation is consistent with the data displayed in Fig. 16. 3.4. Rotation In Fig. 17, we present ﬁrst-moment maps of HCN(3–2) (left), SO2(14(0,14)–13(1,13)) (middle left), SiO( v=1, 6–5) (middle right), and SO(7(6)–6(5)) (right). At projected distances from the star not exceeding 0:500, all four tracers display approximate anti-symmetry with respect to a line at PA10. This is sugges- tive of the presence of rotation in the inner CSE layer around an axis that projects on this line in the plane of the sky. Such a morpho-kinematic structure has also been observed in other stars, notably R Dor (Vlemmings et al. 2018; Homan et al. 2018a; Nhung et al. 2021). The angular resolution of the present data does not allow for a detailed exploration of this region, which prevents us from commenting on its possible cause. Neverthe- less, the anti-symmetry axis of the velocity pattern projected on the plane of the sky at a PA that approximately coincides with the projected symmetry axis of the polar outﬂows (see Sect. 4.1) A135, page 10 of 27	10.1051_0004-6361_202141662	page_0009
J. M. Winters et al.: Molecules, shocks, and disk in the axi-symmetric wind of the MS-type AGB star RS Cancri Fig. 17. First-moment maps of different lines, indicating a possibly rotating structure (see Sect. 3.4). Left: HCN(3–2), middle left : SO 2(14(0,14)– 13(1,13)), middle right : SiO(v=1,6–5), right : SO(7(6)–6(5)). The black ellipses indicate the synthesized beam. is remarkable and suggests that rotation is taking place about this same polar axis in the inner CSE layer. The line-of-sight velocities of these structures are small, on the order of the velocities derived from CO for the equatorial region, and we interpret them here as possible signs of rotation (rather than indicating another bipolar outﬂow oriented perpen- dicular to the larger scale outﬂow traced in CO and SiO ( v=0) lines). We note that out of these four lines, the HCN(3–2) line is detected with the highest S/N (S/N =233in the line peak, cf. Table 1). The mean Doppler velocity hvzi, averaged over the inner 0:500, of the HCN line can be ﬁt in position angle !, measured counter-clockwise from north, by hvziHCN=0:19 km s1+1:0 km s1sin(!19); (1) whereas the SiO( v=1, 6–5) velocity is well ﬁt by hvziSiO (v=1;65)=0:37 km s1+0:46 km s1sin(!26):(2) The small offsets of 0:3km s1on average are within the uncertainty attached to the measurement of the star’s LSR velocity. The coefﬁcients of the sine terms measure the pro- jected rotation velocity, namely the rotation velocity divided by the sine of the angle made by the rotation axis with the line of sight. Assuming that the rotation axis is the axi-symmetry axis of the CSE, this angle is i30(see Sect. 4.1), meaning rotation velocities of2and1km s1for HCN and SiO respectively. Observations of higher angular resolution are needed to conﬁrm the presence of rotation within a projected distance of 0:500from the star and we prefer to summarize the results presented in this section in the form of an upper limit to the mean rotation velocity of a few km s1. 3.5. Global outﬂow structure traced by CO and SiO The detailed structure of the morpho-kinematics of the CSE has been studied using observations of the12CO(1–0) and12CO(2–1) molecular line emission. The analyses of Hoai et al. (2014) and Nhung et al. (2015b) conﬁrmed the interpretation of the two- component nature of the Doppler velocity spectrum originally given by Libert et al. (2010). The CSE is axi-symmetric about an axis making an angle of i30with the line of sight and projecting on the plane of the sky at a position angle !7east of north (see also the sketch in Fig. 4). The expansion velocity reaches8to9km s1along the axis – we refer to this part of the CSE as bipolar outﬂow – and 3to4km s1in the plane perpendicular to the axis – we refer to this part of the CSE as equatorial enhancement. The transition from the equator to thepoles of the CSE is smooth. Section 4.1 below, using observa- tions of the12CO(2–1) and13CO(2–1) molecular lines, conﬁrms and signiﬁcantly reﬁnes this picture. The right panels of Fig. 20 show projections of the CSE on the plane containing the axis and perpendicular to the plane of the sky, which give a good qualitative idea of the global structure. Velocity-integrated channel maps of the CO(2–1) and SiO(6–5) observations analyzed in the present article are dis- played in Fig. 18. They clearly show the bipolar outﬂows, inclined toward the observer in the north and receding in the south. We note that the red wings are brighter than the blue wings as a result of absorption (see Sects. 4.1 and 4.2) The SiO- emitting region is seen to be signiﬁcantly more compact than the CO-emitting region; this is in conformity with observations of many other oxygen-rich AGB stars and is generally interpreted as the result of SiO molecules condensing on dust grains and being ultimately dissociated by the interstellar radiation at some 200 AU from the star, well before CO molecules are dissociated (see e.g., Schöier et al. 2004). 3.6. Temperature and SO 2abundance In this section, we use the 11 detected SO 2lines to derive an approximate temperature and column density of the SO 2- emitting region by means of a population diagram. Following Goldsmith & Langer (1999), in the optically thin case, the col- umn density of the upper level population Nuof a transition u->l can be expressed as Nu=8k2 hc3AulZ Tbdv: (3) Nuis the column density of the upper level population of the transition, kandhare the Boltzmann and Planck constant, respectively, is the line frequency, cthe speed of light, Aulis the Einstein coefﬁcient for spontaneous emission of the transi- tion, andR Tbdvis the velocity-integrated main-beam brightness temperature. The latter is converted to the surface brightness distribution of the source Sper beam, measured by the inter- ferometer, by means of Tb=2 2k bS; (4) where=c is the observing wavelength, and b=ab 4 ln 2witha andbbeing the major and minor axis of the synthesized beam. We determineR S(v)dv=:WIfrom a circular Gaussian ﬁt to the velocity-integrated emission in the uv-plane, where the inte- gration is taken from (vlsr,*4:5)km s1to(vlsr;+4:5)km s1, that is over the three central channels of the SO 2lines. A135, page 11 of 27	10.1051_0004-6361_202141662	page_0010
A&A 587, A91 (2016) about their formation paths during the past evolution of the core. In particular, their degree of deuterium fractionation is a relic of the conditions that were prevailing in earlier, colder stages (e.g.Taquet et al. 2012 ,2014 ;Aikawa et al. 2012 ). Apart from methanol, relativ ely little is known about deuter- ation of complex organic molecules in the interstellar medium,in particular in high mass star-forming regions. A few detec- tions or tentative detections of deuterated (complex) organic molecules have been reported toward Orion KL ( Gerin et al. 1992 ;Daly et al. 2013 ;Esplugues et al. 2013 ;Neill et al. 2013 ; Coudert et al. 2013 ), but no systematic study of the deuteration of complex organic molecules in high mass star-forming regionshas been reported so far. Sagittarius B2, hereafter Sgr B2, is one of the most mas- sive star-forming regions in the Galaxy. It is located in the cen-tral molecular zone, close to the Galactic center. Its current star formation rate qualiﬁes Sgr B2 as a mini-starburst region (see, e.g., Appendix A of Belloche et al. 2013 ). The cloud contains several sites of ongoing, high-mass star formation. One of these sites, Sgr B2(N), contains two hot molecular cores that we des- ignate as Sgr B2(N1) and Sgr B2(N2) 2. Their angular separation is 5/prime/primein the north-south direction, corresponding to 0.2 pc in projection at a distance of 8.3 kpc ( Reid et al. 2014 ). Their ve- locities projected along the line of sight di ﬀer by 9–10 km s−1. Both are characterized by extremely high H 2column densities (>1025cm−2over few arcsec, see Belloche et al. 2008 ,2014 ; Qin et al. 2011 ). They are both in an early stage of star forma- tion when a (massive) protostar has already formed and startedto heat up its circumstellar envelope. The high kinetic temper- atures of the hot cores ( ∼150–200 K) lead to the sublimation of molecules that formed in the ice mantles of dust grains dur-ing the prestellar phase and the warming-up period of the proto- stellar phase. As a result of both the high temperatures and col- umn densities, numerous complex organic molecules have beendetected toward Sgr B2(N), many of these for the ﬁrst time in the interstellar medium, since the beginning of radio astronomy nearly ﬁve decades ago. Following up a molecular line survey of Sgr B2(N) per- formed with the IRAM 30 m telescope that led to the ﬁrst detection in space of a few new complex organic molecules(Belloche et al. 2008 ,2009 ,2013 ), we performed a system- atic line survey of Sgr B2(N) in the 3-mm atmospheric win- dow at high angular resolution and sensitivity with the Atacama Large Millimeter/submillimeter Array (ALMA) in its Cycles 0 and 1. This survey is called EMoCA, which stands for Exploring Molecular Complexity with ALMA, and it aims to investigatemolecular complexity in the interstellar medium. One of the ini- tial results of EMoCA was the ﬁrst interstellar detection of a branched alkyl molecule ( Belloche et al. 2014 ). Here, we take advantage of this sensitive survey to explore, for the ﬁrst time in a systematic way, the deuterium fractionation of complex or- ganic molecules in Sgr B2(N2). We focus on Sgr B2(N2) ratherthan the main hot core Sgr B2( N1) because the former has rel- atively narrow linewidths ( ∼5k ms −1) at the angular resolution of EMoCA (∼1.8/prime/prime), while the latter still has prominent linew- ings like in our previous single-dish survey. A companion paper reports on the detection of alkanols and alkanethiols based on EMoCA ( Müller et al. 2016b ). The article is structured as follows. The observational setup and the process of data reduction are described in Sect. 2. Section 3explains the method employed to model the observed 2They were named P1 and P2 in Belloche et al. (2008 )a n dS M A 1a n d SMA2 in Qin et al. (2011 ).spectra in the approximation of l ocal thermodynamic equilib- rium (LTE) and Sect. 4gives some details about the spectro- scopic predictions used to generate the synthetic spectra. Theresults of the analysis are reported in Sect. 5and discussed in Sect. 6. Section 7gives our conclusions about deuterium fractionation of complex organic molecules in Sgr B2(N2). 2. Observations and data reduction 2.1. Observations We used ALMA to perform a complete spectral line survey to-ward Sgr B2(N) between 84.1 and 114.4 GHz. The ﬁeld was cen- tered at (α,δ) J2000=(17h47m19.87s,−28◦22/prime16/prime/prime), halfway be- tween Sgr B2(N1) and (N2), which are separated by 4 .9/prime/primein the north-south direction. The size (HPBW) of the primary beam of the 12 m antennas varies between 69/prime/primeat 84 GHz and 51/prime/prime at 114 GHz ( Remijan et al. 2015 ). The spectral line survey was divided into ﬁve spectral setups. Each setup was observed in one polarization and delivered fourspectral windows, two per sideba nd. The separation between the centers of the lower and upper sidebands is 12 GHz. Each spec- tral window has a bandwidth of 1875 MHz and a channel spacingof 244.141 kHz, but the spectra were smoothed to a spectral res- olution of 488.3 kHz (1.7 to 1.3 km s −1). Each pair of adjacent spectral windows has an overlap of about 50 MHz. Details aboutthe frequency coverage, the date of observation, the number of antennas, the range of baselines, the on-source integration time, and the bandpass, amplitude, and phase calibrators are given inTable 1. Setups S1 to S4 were observed in Cycle 0 while setup S5 was observed in Cycle 1. As reported in Table 1, setups S1 and S5 were observed only once, but setups S2, S3, and S4 wereobserved on several days, between two and four times each. 2.2. Data reduction The data was calibrated and imaged with the CommonAstronomy Software Applications package (CASA). We used version 4.2.0 (r28322) for setups S1 to S4 and version 4.2.1 (r29047) for setup S5. We used the standard procedures providedby the Joint ALMA Observatory to apply the bandpass, ampli- tude, and phase calibrations. The deconvolution was performed with the csclean imager mode and a Briggs weighting scheme with a robust parameter of 0.5. The cell size was set to 0 .3 /prime/prime. In addition, three or four iterations of self-calibration were per- formed using a strong spectral line detected toward Sgr B2(N1) in each setup. This signiﬁcantly improved the dynamical range in the resulting images. The spectra toward Sgr B2(N1) and (N2) are full of lines and close to the confusion limit. It is thus di ﬃcult to separate the line emission from the continuum emission in a systematic way forthe full data cubes, but it is a necessary step to produce sepa- rate line and continuum maps. For each spectral window of each setup, we selected six groups of a few channels that seemed to befree of strong line emission. A ﬁrst-order baseline was ﬁtted to these selected channels and the result of the ﬁt was used to split each data cube into two cubes: one for the line emission and onefor the continuum emission. Given the di ﬀerence in systemic ve- locity between the two hot cores ( ∼10 km s −1,s e e Belloche et al. 2013 ), we selected diﬀerent sets of channels for the northern and southern parts of the ﬁeld. This process of baseline subtraction was performed with the CLASS software3. 3Seehttp://www.iram.fr/IRAMFR/GILDAS A91, page 2 of 66	10.1051_0004-6361_201527268	page_0001
A. Belloche et al.: Deuterated complex organic molecules in Sgr B2(N2) Table 1. Observational setups of the EMoCA survey. Setup Frequency range Date of tstartaNabBaseline tintdCalibratorsePeakf LSB USB observation (UTC) rangecBA P ΔαΔδ GHz GHz yyyy-mm-dd hh:mm m min/prime/prime /prime/prime S1 84.1–87.8 96.2–99.8 2012-08-27 01:05 26 17–400 54.7 1 3 2 0.2 −2.4 S2 87.7–91.4 99.7–103.4 2012-06-05 09:03 20 14–392 24.2 1 3 2 1.2⋆−2.4 2012-07-01 07:04 13 25–393 40.0 1 3 2 1.2⋆−2.6 2012-07-03 06:36 21 14–395 48.4 1 3 2 0.6⋆−2.3 2012-09-28 21:57 25 20–387 44.1 1 4 2 0.2 −2.5 S3 91.4–95.1 103.4–107.1 2012-06-06 08:20 18 15–395 40.2 1 3 2 0.7⋆−2.4 2012-06-18 07:29 22 15–395 40.4 1 3 2 0.6⋆−2.2 S4 95.0–98.7 107.0–110.7 2012-07-04 05:38 21 17–398 8.1 1 3 2 0.1 −2.3 2012-08-01 02:32 24 19–442 34.9 1 3 2 0.1 −2.4 2012-08-10 00:45 26 21–400 35.0 1 3 2 0.2 −2.4 S5 98.7–102.4 110.7–114.4 2014-04-05 06:22 38 15–413 24.4 2 4 5 0.2 −2.4 Notes.(a)Start time of observation.(b)Number of ALMA 12 m antennas.(c)Minimum and maximum projected baseline separations.(d)On-source integration time.(e)Bandpass (B), amplitude (A), and phase (P ) calibrators. The calibrators are: 1: B1730-130, 2: J 1700-2610, 3: Neptune, 4: Titan, 5: J1744-3116.(f)Oﬀset of the continuum peak position of Sgr B2(N1) with respect to the phase center, in equatori al coordinate system (J2000). Measurement sets with Δαdiﬀering from 0.1/prime/primeby more than 0.2/prime/prime(marked with a⋆) are believed to be a ﬀected by an astrometric problem. We checked the accuracy of the relative astrometry between the ten measurement sets by ﬁtting the peak position of thecontinuum emission toward Sgr B2(N1) in selected channels of the line+continuum data cubes that appeared to be free of line emission. It turns out that the ﬁrst three measurement sets ofsetup S2 and both measurement sets of setup S3 are a ﬀected by an astrometric problem: the continuum peak of Sgr B2(N1) is shifted by 0.6 /prime/primeto 1.2/prime/primein right ascension with respect to all other measurement sets (see Table 1). The dispersion of the peak position in declination is also a bit higher for the af- fected measurement sets compared to the nona ﬀected measure- ment sets. The average peak position of Sgr B2(N1) in all non- aﬀected measurement sets is at ( Δα,Δδ)=(0.15/prime/prime,−2.40/prime/prime), i.e. (α,δ)J2000=(17h47m19.881s,−28◦22/prime18.40/prime/prime). The ﬁve aﬀected measurement sets were obtained after transit when the sourcewas setting and the phase calibrator was at low elevation, which leads us to believe that the shift of the a ﬀected measurement sets may be due to an inacurrate calibration of the atmospheric phaseﬂuctuations. As a result, we ignored the a ﬀected measurement sets of setup S2 and used only its fourth measurement set. For setup S3, both measurement sets were used but the o ﬀset was approximately compensated for by modifying the visibilities of the phase calibrator with the CASA task ﬁxvis before the phase calibration. After this correcti on, the relative positional accuracy of all measurement sets selected for this work is on the order of±0.1 /prime/primein both right ascension and declination. The measurement sets of setup S3 were merged into one sin- gle measurement set with the CASA task concat before imag- ing. The same was carried out for setup S4. Only one measure- ment set was used for the other three setups. The size (HPBW)of the synthesized beam and the rms noise level in the ﬁnal cubes are given in Table 2. The noise level of each spectral win- dow corresponds to the median of the noise levels measured inall channel maps using the procedure go noise in GREG 3.The noise levels reported in Table 2were measured in the continuum- subtracted datacubes. They are typically a factor ∼2 higher in the line+continuum datacubes. Based on the redundancies of the measurement sets and the spectral overlap between the setups, we estimate the relative cal-ibration uncertainty on the ﬂux density to be on the order of 15%.3. Radiative transfer modeling of the line survey We used the input parameters of our LTE model of theIRAM 30 m spectrum of Sgr B2(N) ( Belloche et al. 2013 )a s a starting point to assign the lines detected in the ALMA spec- tra and model the emission of the detected molecules. Given the high H 2densities of Sgr B2(N1) and (N2) ( ∼108cm−3at arc- second scale, Belloche et al. 2008 ,2014 ;Qin et al. 2011 ), the LTE approximation is appropriate. We used the software Weeds (Maret et al. 2011 ) to produce synthetic LTE spectra that take into account the radiative transfer and the (spectral-window- and measurement-set-dependent) angular resolution of the observa- tions. We performed the modeling for each species separately,and then we linearly added the contributions of all detected species to the emitted spectra to obtain the ﬁnal synthetic spec- trum (hereafter called the full LTE model). This approximation is valid for optically thin lines that overlap in frequency space orfor (optically thick or thin) lines of species that are emitted from separated regions within the beam, but it is no longer correct for frequency-overlapping optically thick lines of species that arecospatial or aligned along the line of sight. In such cases, the synthetic spectrum overestimates the actual line ﬂux density. The model of each species is deﬁned by ﬁve parameters: angular size of the emitting region assumed to be Gaussian, column density, rotational temperature, velocity o ﬀset with re- spect to the assumed systemic velocity of the source, and linewidth (FWHM). For a given species, the source size was de- rived from two-dimensional Gau ssian ﬁts to the integrated in- tensity maps of all transitions that were well detected and found to be free of contamination (based on the full LTE model). The source size was set to the median deconvolved size of all suchtransitions. The other four parameters were optimized by eye. We constructed population diagrams based on the transitions that are well detected and not severe ly contaminated by transitions of other species. In the case where a transition was partially con- taminated, the contributions of the contaminating species was re- moved from the measured integrated intensities, on the basis ofthe full LTE model. Each population diagram was also corrected for optical depth following the method described in Goldsmith & Langer (1999 ), using the opacities delivered by Weeds. We used the population diagrams to verify that the rotational temperature A91, page 3 of 66	10.1051_0004-6361_201527268	page_0002
A&A 587, A91 (2016) Table 2. Beam sizes and noise levels. Setup SPWaFrequency Synthesized beam rmsc range HPBW PAbmJy MHz/prime/prime×/prime/prime ◦beam−1K S1 0 84 091–85 966 2 .1×1.5−85 3.0 0.16 1 85 904–87 779 2 .0×1.5−83 2.7 0.14 2 96 154–98 029 1 .8×1.4−85 3.0 0.16 3 97 904–99 779 1 .8×1.3−85 3.1 0.16 S2 0 87 729–89 604 1 .9×1.7 86 3.1 0.15 1 89 554–91 429 1 .8×1.6 52 2.8 0.15 2 99 728–101 602 1 .6×1.4 48 2.7 0.14 3 101 552–103 427 1 .6×1.4 49 2.7 0.14 S3 0 91 368–93 242 2 .9×1.5 84 3.4 0.12 1 93 193–95 067 2 .8×1.5 83 3.1 0.10 2 103 365–105 239 2 .5×1.3 82 3.4 0.11 3 105 189–107 064 2 .5×1.3 82 3.6 0.12 S4 0 95 021–S96 896 1 .9×1.4−82 1.9 0.10 1 96 846–98 720 1 .8×1.3−82 1.9 0.10 2 107 019–108 893 1 .7×1.2−83 2.2 0.11 3 108 843–110 718 1 .6×1.2−82 2.3 0.12 S5 0 98 672–100 546 1 .8×1.4−76 2.8 0.14 1 100 496–102 370 1 .7×1.4−76 2.7 0.13 2 110 669–112 543 1 .6×1.3−72 3.5 0.17 3 112 494–114 368 1 .6×1.2−77 4.9 0.24 Notes.(a)Spectral window.(b)Position angle east from north.(c)Median rms noise level measured in the channel maps of the continuum-removed data cubes. derived in the course of the (manual) modeling with Weeds made sense. In the population diagrams corrected for optical depth and contamination, the residual dispersion of the synthetic data-points (red crosses) results in part from the frequency boundaries set to integrate the intensity. These boundaries are a compromise between covering the line as much as possible and limiting thecontamination from other species emitting at nearby frequencies as much as possible. In addition, the correction for optical depth is an approximation and may also introduce some bias. Finally,another limitation of this ﬁt is that it can be biased by residualcontamination that remains even after removal of the contribu- tion of the identiﬁed contamina ting species. Therefore, we be- lieve that the formal errors on the rotational temperature derivedfrom the ﬁt to the population diagrams do not necessarily rep- resent the true errors on this temperature and should be viewed with caution. The emission of vibrationally excited states of a given molecule were modeled independentely of the vibrational ground state. The emission of isotopologues of a given moleculewere also modeled separately. The physical structure of the source assumed for the model- ing is uniform. This may sound simplistic given that temperature and density gradients are expect ed in the envelope of Sgr B2(N2) (e.g., Rolﬀs et al. 2011 ). It turns out that, even with such a sim- ple assumption, the spectra of most complex organic molecules detected toward Sgr B2(N2) can be well reproduced so we areconﬁdent that the rotational temperatures and column densities derived from our analysis are reliable. In the following, we count a line of a given species as a de- tected line if its frequency, peak intensity, and width are wellreproduced by our model and the line is not (or barely) con- taminated by emission from other species. As a counter exam- ple, a synthetic line that is consistent with the observed spec-trum, i.e., that has a peak intensity simply below the intensity ofthe detected signal, but would still remain consistent if it were shifted by a frequency o ﬀset typically equal to its linewidth is not counted as detected. We emphasize that our deﬁnition of a de- tected line is very conservative but we believe that it is requiredto avoid unsecure molecule detections. The complete list of transitions identiﬁed in our survey is presently not available but the list of lines identiﬁed in our pre- vious single-dish survey of Sgr B2(N) can be taken as reference (Belloche et al. 2013 ). 4. Spectroscopic predictions The origin of the spectroscopic predictions used to model the emission of the species reported in Sect. 5is provided here. Predictions for the three singly deuterated species of ethyl cyanide were taken from the catalog of the Cologne Databasefor Molecular Spectroscopy (CDMS 4,Müller et al. 2001 ,2005 ; tags 56 509, 56 510, and 56 511, all version 1). They are based onMargulès et al. (2009 ). All other ethyl cyanide data were also taken from the CDMS. The main species predictions are based onBrauer et al. (2009 ) with published data in the range of our survey from Fukuyama et al. (1996 ). Transition frequencies of the isotopologues containing one13C were taken from Richard et al. (2012 ), those for the15N isotopologue from Margulès et al. (2009 ). Vibrational corrections to the rotational partition func- tion, and thus to the column density, were derived for the main isotopologue from Heise et al. (1981 ) and applied to all species. Only limited isotopic data are available. It is safe to assume thatdiﬀerences among the isotopologues are small, most likely not exceeding a few percent because of the large number of heavy atoms in the molecule. Predictions for singly deuterated methyl cyanide were taken from the CDMS catalog (tag 42 511, version 2). This entry is 4Seehttp://www.cdms.de A91, page 4 of 66	10.1051_0004-6361_201527268	page_0003
A. Belloche et al.: Deuterated complex organic molecules in Sgr B2(N2) based on Nguyen et al. (2013 ). Predictions for CH 3CN in its /v18=1 and 2 excited states are based on preliminary results from Müller et al. (2015 ), those in /v14=1 are preliminary data from a subsequent study (Müller et al., in prep.). Transition frequen- cies in the range of our study are from Bauer & Maes (1969 ) andBauer (1971 )f o r /v18=1 and 2, respectively. Predictions for isotopic species with15N or one or two13C in their ground vibra- tional states are from the CDMS. They are based on Müller et al. (2009 ) with transition frequencies in the range of our survey from Demaison et al. (1979 ). Predictions for13C isotopologues in their /v18=1 states are based on preliminary data from Müller et al. (2016a ). Vibrational corrections to the partition function were included in the private entries for the main isotopic species.T h e ya r en o wa v a i l a b l ei n Müller et al. (2015 ). As the correc- tions are small, the error using the values from the main isotopic species for the other isotopologues as well is very small, evenfor CH 2DCN. Predictions for deuterated vinyl cyanide CH 2CDCN were taken from the molecular spectroscopic database of the Jet Propulsion Laboratory (JPL5,Pickett et al. 1998 ; tag 54 004, ver- sion 2). We prepared predictions for cis-CHDCHCN and trans - CHDCHCN. All predictions are based on Colmont et al. (1997 ). Predictions for C 2H3CN and isotopic species with one13Co r with15N were taken from the CDMS and are based on Müller et al. (2008 ). Transition frequencies in the range of our survey are mostly from that study. For the main species, they are, toa large extent, also from Baskakov et al. (1996 ). Predictions for excited states of vinyl cyanide used in the present work are based on Cazzoli & Kisiel (1988 ) and unpublished data from one of us (HSPM). These data included vibrational corrections which are essentially complete at 200 K. The vibrational ener- gies were gathered from several sources. A recent compilation of low-lying vibrational states is available in Kisiel et al. (2015 ). It is safe to assume that di ﬀerences among the isotopologues are small, most likely not exceedin g a few percent because of the large number of heavy atoms in the molecule. Predictions for deuterated c yanoacetylene were taken from the CDMS catalog (tag 52 508, version 2). This entry is based on Spahn et al. (2008 ). All other cyanoacetyl ene predictions were also taken from the CDMS. The /v1 7=1 predictions of the main species are based on Thorwirth et al. (2000 ) with data in the range of our survey from Yamada & Creswell (1986 ). All pre- dictions of isotopologues containing one or two13Ca r eb a s e d onThorwirth et al. (2001 ), and those for HC15 3No n Fayt et al. (2004 ). Ground state transition frequencies for singly substituted species in the range of our survey were taken from Creswell et al. (1977 ). Vibrational contributions to the partition functions of HC 3Na n dD C 3N can be evaluated from the study of their low-lying vibrational states by Uyemura et al. (1982 ). Isotopic shifts, in particular of the lowest ν7mode, are much smaller for 13Co r15N species. Therefore, using vibrational corrections of the main isotoplogue introduces small errors for these species. Predictions for deuterated methanol CH 2DOH were taken from the JPL catalog (tag 33 004, version 1). They are based on Pearson et al. (2012 ) with rest frequencies almost entirely from that study. With the use of torsional energies from Lauvergnat et al. (2009 ), we estimate a vibrational correction factor to the partition function of 1.15 at 160 K. For CH 3OD, we prepared a catalog entry based on Anderson et al. (1988 ), with frequen- cies updated to the values published in Duan et al. (2003 ). We estimated the partition function to be 11 770 at 150 Kand 25 550 at 225 K, taking torsional energies of CH 3OD in 5Seehttp://spec.jpl.nasa.govAnderson et al. (1988 ) into account. Details on other methanol isotopologues are given in Müller et al. (2016b ). Predictions for all singly deuterated species of ethanol were taken from the CDMS (tags 47 515 to 47 518, all version 1). They are based on Walters et al. (2015 ) with rest frequencies almost entirely from that study. All other ethanol analyses weretaken from Müller et al. (2016b ), and further details can be found there. Conformational and vibrational corrections to the parti- tion function were taken from the main isotopologue for whichonly data were available. This assumption is reasonable, though errors may not be completely negligible. They are, however, diﬃcult to evaluate. Predictions for both conformers of CH 2DOCHO were ex- tracted from Table 7 of Coudert et al. (2013 ) and split into two separate entries. The partition function was taken from theirTable 6. It is identical for both entries. Contrary to the CDMS en- tries for CH 2DCH 2CN and CH 2DCH 2OH, this treatment means that the two entries represent a single species with a statisticaldistribution (the out-of-plane conformer being twice as abun- dant as the in-plane one). This means that the column density derived for each conformer represents the total column densityof the molecule. However, we assumed a statistical distribu- tion (2:1) to compute and report individual column densities in Sect. 5. Predictions for the main isotopologue were taken from the JPL catalog. The entry is based on Ilyushin et al. (2009 ). Vibrational corrections to the partition function were derived from the 13C species ( Favre et al. 2014 ). These authors pro- vide rotational partition function values at di ﬀerent temperatures as well as detailed vibrational corrections that are complete up to 300 K. The correction factors are 1.59 and 1.23 at 150 K forthe deuterated and main isotopic species, respectively. These val- ues diﬀer because values for the deuterated species refer to the ground state only whereas contributions of the ﬁrst excited stateswere already included for the main isotopologue. 5. Results In this section, we report the detection or tentative detectionof deuterated complex organic molecules toward Sgr B2(N2).Column density upper limits are also reported for several non- detections. Each subsection ﬁrst presents the LTE model de- rived for the main isotopologue and its 13Ca n d/or15N isotopo- logues. This model is then used to obtain constraints on the col- umn density of the deuterated species. The rotational tempera- tures derived from ﬁts to the population diagrams are reportedin Table 3and the parameters of the LTE model used to ﬁt the spectra are listed in Table 4. The analysis toward Sgr B2(N2) was performed at the o ﬀset position (Δα,Δδ)=(−0.1 /prime/prime,2.6/prime/prime), i.e. (α,δ)J2000=(17h47m19.86s,−28◦22/prime13.4/prime/prime). 5.1. Deuterated ethyl cyanide CH 2DCH 2CN and CH3CHDCN About 154, 54, 38, and 37 lines of ethyl cyanide and its singly substituted13C isotopologues,13CH 3CH 2CN, CH 313CH 2CN, and CH 3CH 213CN, respectively, are detected toward Sgr B2(N2) (Figs. A.1–A.4). The15N isotopologue is also detected un- ambiguously, with nine detected lines (Fig. A.5). The best-ﬁt LTE model ﬁts very well all detected transitions, except the very optically thick lines of the main isotopologue, which itsigniﬁcantly underestimates. We ignored the lines with opti- cal depth higher than ∼2.5 to construct the population diagram of this species (Fig. A.6), while all lines of the 13C isotopo- logues that matched the criteria deﬁned in Sect. 3were used A91, page 5 of 66	10.1051_0004-6361_201527268	page_0004
A&A 587, A91 (2016) Table 3. Rotational temperatures derived from population diagrams of selected (complex) organic molecules toward Sgr B2(N2). Molecule StatesaTﬁtb (K) CH 3CN /v18=1,/v18=2,/v14=1 253 (15) 13CH 3CN /v1=0,/v18=1 168 (13) CH 313CN /v1=0,/v18=1 165.5 (3.3) CH 2DCN /v1=0 136 (14) C2H5CN /v1=0 137.3 (1.6) 13CH 3CH 2CN /v1=0 138.3 (7.5) CH 313CH 2CN /v1=0 112 (11) CH 3CH 213CN /v1=0 150 (40) C2H3CN /v1=0,/v111=1,/v115=1,/v111=2 199.5 (3.4) 13CH 2CHCN /v1=0 255 (101) CH 213CHCN /v1=0 140 (31) CH 2CH13CN /v1=0 278 (126) H13CCCN /v1=0,/v17=1 171.1 (3.2) HC13CCN /v1=0,/v17=1 167.7 (5.5) HCC13CN /v1=0,/v17=1 177 (18) CH 3OCHO /v1t=0,/v1t=1 142.4 (4.4) Notes.(a)Vibrational states that were taken into account to ﬁt the popu- lation diagram.(b)The standard deviation of the ﬁt is given in parenthe- ses. As explained in Sect. 3, these uncertainties should be viewed with caution. They may be underestimated. for their population diagrams (Figs. A.7–A.9). The results of the linear ﬁt to the population diagrams of all four isotopologues are given in Table 3. The rotational temperature is well con- strained to∼140 K for both C 2H5CN and13CH 3CH 2CN. This value is consistent with the result of the ﬁt for the two other iso- topologues within the uncertainties. The temperature derived in this way depends on the model used to make the opacity correc-tion. With an earlier best-ﬁt model that assumed a temperature of 170 K (instead of 150 K here), the ﬁt to the population di- agrams of both C 2H5CN and13CH 3CH 2CN yielded a tempera- ture of∼150 K. This is the reason why we decided to assume a rotational temperature of 150 K for ethyl cyanide and all its isotopologues. The median source size derived for the selected lines of C2H5CN is about 1.15/prime/prime, but there seems to be a trend of de- creasing size with increasing upper level energy, from ∼1.3/prime/primeat low energy to∼0.8/prime/primeatEu∼700 K (Fig. 1). Similar results are obtained for13CH 3CH 2CN, with a median size of ∼1.25/prime/primeand a hint of a decrease down to ∼1.0/prime/primeatEu∼120 K. Our model does not treat such gradients. As a compromise, we used a source size of 1.2/prime/prime. With this source size and a rotational temperature of 150 K, we obtain an excellent ﬁt to all emission lines of the four iso- topologues, except for the very optically thick lines of C 2H5CN (τmax∼60), as mentioned above. A better ﬁt to these lines would be obtained by increasing the temperature and /or assuming a larger source size. Increasing the size to 1 .4/prime/primeturns out to be insuﬃcient. A larger size would be inconsistent with the mea- sured sizes. Increasing the temperature to 200 K and the size to 1.3/prime/primeyields peak temperatures of the optically thick lines sim- ilar to those observed, but the synthetic lines look too saturatedcompared to the observed lines, and the ﬁt to the optically thin lines becomes worse; lines with high upper level energies be- come overpredicted. A more complex model with nonuniformphysical parameters would probably be needed to reproduce the intensity and shape of the very optically thick lines. Assuming the same source size, rotational temperature, linewidth, and velocity o ﬀset as derived for C 2H5CN and its13Ca n d15N isotopologues, we looked for emission of the singly deuterated isotopologues, CH 3CHDCN and CH 2DCH 2CN. The former is a chiral molecule b ecause the carbon atom in the middle of the chain is linked to four di ﬀerent atoms or func- tional groups. Both isotopologues are tentatively detected to- ward Sgr B2(N2) with 1 and 2 line(s), respectively (Figs. A.10 andA.11 ), the latter isotopologue in its out-of-plane conforma- tion only. For the in-plane confomer of CH 2DCH 2CN, we derive an upper limit only. This upper lim it is uncertain because the ap- parent inconsistency between the synthetic spectrum and the ob-served one around ∼101 190 MHz may result from a slight over- estimate of the baseline, at the 3 σlevel (Fig. A.12 ). Owing to the limited signal-to-noise ratios, the source size derived from theintegrated intensity maps of the uncontaminated lines assigned to CH 3CHDCN and CH 2DCH 2CN is uncertain, varying between unresolved and∼2/prime/prime. The emission looks compact in the maps. Thus, assuming the same source size as the other isotopologues looks reasonable. 5.2. Deuterated methyl cyanide CH 2DCN Methyl cyanide is clearly detected in its vibrational ground statetoward Sgr B2(N2) but its transitions are very optically thick (τ max∼50) and cannot be properly ﬁtted in the framework of our simple model (Fig. A.13 ). Transitions from within its vibra- tionally excited states /v18=1a n d /v18=2 are also clearly detected, withτmax∼2.7 and 0.3, respectively (Figs. A.14 andA.15 ). We also ﬁnd four transitions from within /v14=1 around 91 520 MHz and 109 820 MHz ( τmax∼0.06), but they partially su ﬀer from blends with other species (Fig. A.16 ). The assignment looks reasonable, but the detection should be considered tentative. The singly substituted13C isotopologues are very well de- tected, both in their vibrational ground state and in their ﬁrst vibrationally excited state /v18=1( F i g s . A.17 –A.20 ). The ﬁt to their population diagrams yields rotational temperatures of about 170 K (see Table 3and Figs. A.21 andA.22 ). The analysis of the integrated intensity maps of the13C isotopologues deliv- ers a source size of ∼1.4/prime/prime. For the main isotopologue, it seems that the source size decreases with the vibrational energy ( ∼1.2/prime/prime for/v18=1a n d∼0.8/prime/primefor/v18=2). With the assumption of a source size of 1 .4/prime/primeand a temper- ature of 170 K, our LTE modeling yields excellent and consis- tent ﬁts to the13C isotopologues (both /v1=0a n d /v18=1) and to the /v18=1 transitions of the main isotopologue. However, it was necessary to increase the column density and linewidth to ﬁt the transitions of the /v18=2a n d /v14=1 states. The ﬁt to the population diagram of the main isotopologue includingthe three vibrationally excited states suggests a temperature of ∼250 K (Fig. A.23 ). This explains why our 170 K model needs higher column densities to reproduce the intensities of the /v1 8=2 and /v14=1 transitions. Here again, a more complex model with a nonuniform physical structur e would be necessary to ﬁt all transitions in a consistent way. However, given that our simpleLTE model yields a good ﬁt to the /v1 8=1 transitions of the main isotopologue and all transitions of the13C isotopologues with a single set of parameters, we consider that the derived12C/13C column density ratios are reliable. On the basis of the LTE model obtained above, we were able to identify emission from the doubly substituted13C isotopologue of methyl cyanide,13CH 313CN. One transition at 107 108 MHz is well detected, and a group of transitions at 89 270 MHz is relativel y well detected (Fig. A.24 ). Given that the column density ratio of the singly to doubly substi-tuted isotopologues is very close to the ratio between the main A91, page 6 of 66	10.1051_0004-6361_201527268	page_0005
A. Belloche et al.: Deuterated complex organic molecules in Sgr B2(N2) Table 4. Parameters of our best-ﬁt LTE model (or upper limit) of selected (complex) organic molecules toward Sgr B2(N2). Molecule StatusaNdetbSizecTrotdNeCfΔVgVoﬀh Nref Ni30 mj (/prime/prime)( K ) ( c m−2)( k m s−1)( k m s−1) CH 3CN, /v18=1⋆d 20 1.40 170 2.2 (18) 1.00 5.4 -0.5 1 y /v18=2 d 8 1.40 170 7.5 (18) 1.00 6.5 –0.5 0.29 y /v14=1 t 1 1.40 170 2.0 (19) 1.00 6.5 –0.5 0.11 y 13CH 3CN, /v1=0 d 8 1.40 170 1.1 (17) 1.18 5.4 –0.5 21 y /v18=1 d 3 1.40 170 1.1 (17) 1.18 5.4 –0.5 21 y CH 313CN, /v1=0 d 7 1.40 170 1.1 (17) 1.18 5.4 –0.5 21 y /v18=1 d 9 1.40 170 1.1 (17) 1.18 5.4 –0.5 21 y 13CH 313CN d 1 1.40 170 4.7 (15) 1.18 5.4 –0.5 466 n CH 3C15N t 0 1.40 170 8.3 (15) 1.18 5.4 –0.5 266 n CH 2DCN d 6 1.40 170 8.3 (15) 1.18 5.4 –0.6 266 n C2H5CN⋆d 154 1.20 150 6.2 (18) 1.38 5.0 –0.8 1 y 13CH 3CH 2CN d 54 1.20 150 1.9 (17) 1.38 5.0 –0.8 32 y CH 313CH 2CN d 38 1.20 150 1.9 (17) 1.38 5.0 –0.8 32 y CH 3CH 213CN d 37 1.20 150 1.9 (17) 1.38 5.0 –0.8 32 y C2H5C15N d 9 1.20 150 1.2 (16) 1.38 5.0 –0.8 500 n CH 2DCH 2CN (out of plane) t 2 1.20 150 3.0 (15) 1.38 5.0 –0.8 2045 n CH 2DCH 2CN (in plane) n 0 1.20 150 <1.5 (15) 1.38 5.0 –0.8 >4091 n CH 3CHDCN t 1 1.20 150 3.0 (15) 1.38 5.0 –0.8 2045 n C2H3CN, /v1=0⋆d 44 1.10 200 4.2 (17) 1.00 6.0 –0.6 1 y /v111=1 d 30 1.10 200 4.2 (17) 1.00 6.0 –0.5 1 y /v115=1 d 20 1.10 200 4.2 (17) 1.00 6.0 –0.5 1 y /v111=2 d 6 1.10 200 4.2 (17) 1.00 6.0 –0.5 1 y 13CH 2CHCN d 10 1.10 200 2.1 (16) 1.38 6.0 –0.6 20 y CH 213CHCN d 9 1.10 200 2.1 (16) 1.38 6.0 –0.6 20 y CH 2CH13CN d 8 1.10 200 2.1 (16) 1.38 6.0 –0.6 20 y C2H3C15N n 0 1.10 200 <3.4 (15) 1.38 6.0 –0.6 >122 n cis-CHDCHCN n 0 1.10 200 <3.4 (15) 1.38 6.0 –0.6 >122 n trans- CHDCHCN n 0 1.10 200 <3.4 (15) 1.38 6.0 –0.6 >122 n CH 2CDCN n 0 1.10 200 <2.1 (15) 1.38 6.0 –0.6 >203 n HC 3N,/v17=1⋆d 6 1.30 170 3.5 (17) 1.44 5.0 –0.7 1 y H13CCCN, /v1=0 d 2 1.30 170 1.7 (16) 1.44 5.0 –0.7 20 y /v17=1 d 4 1.30 170 1.7 (16) 1.44 5.0 –1.0 20 y HC13CCN, /v1=0 d 3 1.30 170 1.7 (16) 1.44 5.0 –0.7 20 y /v17=1 d 3 1.30 170 1.7 (16) 1.44 5.0 –1.0 20 y HCC13CN, /v1=0 d 3 1.30 170 1.7 (16) 1.44 5.0 –0.7 20 y /v17=1 d 3 1.30 170 1.7 (16) 1.44 5.0 –1.0 20 y H13C13CCN t 1 1.30 170 7.2 (14) 1.44 5.0 –0.7 480 n H13CC13CN t 0 1.30 170 7.2 (14) 1.44 5.0 –0.7 480 y HC13C13CN t 1 1.30 170 7.2 (14) 1.44 5.0 –0.7 480 n HC 315N t 0 1.30 170 1.2 (15) 1.44 5.0 –0.7 300 y DC 3N t 0 1.30 170 3.0 (14) 1.51 5.0 –0.5 1144 n CH 3OH, /v1t=1⋆d 16 1.40 160 4.0 (19) 1.00 5.4 –0.2 1 y CH 2DOH t 2 1.40 160 4.8 (16) 1.15 5.4 –0.5 828 n CH 3OD n 0 1.40 160 <2.6 (16) 1.05 5.4 –0.5 >1524 n C2H5OH⋆d 168 1.25 150 2.0 (18) 1.24 5.4 0.0 1 y CH 3CH 2OD n 0 1.25 150 <3.0 (16) 2.96 5.4 0.0 >67 n CH 3CHDOH n 0 1.25 150 <3.0 (16) 2.96 5.4 0.0 >67 n CH 2DCH 2OH (out of plane) n 0 1.25 150 <3.0 (16) 2.96 5.4 0.0 >67 n CH 2DCH 2OH (in plane) n 0 1.25 150 <2.1 (16) 2.96 5.4 0.0 >96 n CH 3OCHO, /v1t=0⋆d 90 1.50 150 1.2 (18) 1.23 4.7 –0.4 1 y /v1t=1 d 35 1.50 150 1.2 (18) 1.23 4.7 –0.4 1 y CH 2DOCHO (out of plane) n 0 1.50 150 <2.5 (16) 1.07 4.7 –0.4 >50 n CH 2DOCHO (in plane) n 0 1.50 150 <7.3 (15) 0.52 4.7 –0.4 >167 n Notes.(a)d: detection, t: tentative detection, n: nondetection.(b)Number of detected lines (conservative estimate, see Sect. 3). One line of a given species may mean a group of transitions of that species that are blended together.(c)Source diameter ( FWHM ).(d)Rotational temperature.(e)Total column density of the molecule. X(Y) means X×10Y.(f)Correction factor that was applied to the column density to account for the contribution of vibrationally or torsionally excited states or other conformers (e.g., gauche for ethanol), in the cases where this contribution was not included in the partition function of the spectroscopic predictions. For deuterated methyl formate, it is the scaling factor used to compute the column densityof each conformer as if it were an independent species. (g)Linewidth ( FWHM ).(h)Velocity oﬀset with respect to the assumed systemic velocity of Sgr B2(N2), Vlsr=74 km s−1.(i)Column density ratio, with Nrefthe column density of the previous reference species marked with a ⋆.(j)Detected (y) or not detected (n) toward Sgr B2(N) (N1 and /or N2) with the IRAM 30 m telescope ( Belloche et al. 2013 ). A91, page 7 of 66	10.1051_0004-6361_201527268	page_0006
A&A 587, A91 (2016) Fig. 1. Deconvolved major, minor, and mean sizes (FWHM) derived for uncontaminated C 2H5CN transitions detected toward Sgr B2(N2) and plotted as a function of upper level energy. The symbols code for the spectral setup (S1 to S5, like in Table 2). In each panel, the dashed line indicates the median value. isotopologue and those that are singly substituted, we consider the identiﬁcation of13CH 313CN as secure. Our LTE modeling indicates that the15N isotopologue con- tributes signiﬁcantly to the emission detected at 107 054 MHz and 107 060 MHz (Fig. A.25 ). Since there is no clearly de- tected line, we do not consider this identiﬁcation as secure. Thederived column density is, therefore, relatively uncertain andshould rather be considered as an upper limit. Finally, using the same parameters as for the other isotopo- logues, we obtain a secure identiﬁcation of singly deuteratedmethyl cyanide, CH 2DCN, with about six transitions clearly de- tected (Fig. A.26 ). The rotation temperature derived from the population diagram is consistent with the assumed temperaturewithin 2.4σ(Table 3and Fig. A.27 ). The source size can be measured for three of the detected transitions and is found to be consistent with the size derived from the 13C isotopologues. 5.3. Deuterated cyanoacetylene DC 3N Cyanoacetylene is detected toward Sgr B2(N2) in its vibrational ground state (Fig. A.28 ) as well as in many vibrationally excited states: /v17=1( F i g . A.29 ),/v17=2( F i g . A.30 ),/v16=1( F i g . A.31 ), /v15=1a n d /v17=3( F i g . A.32 ), and /v16=/v17=1( F i g . A.33 ). In addition, emission from within the following excited states istentatively detected: /v1 4=1 (one detected line, Fig. A.34 ),/v17=4 and /v15=/v17=1 (signiﬁcantly contributes to detected signal, but no line individually detected, Fig. A.35 )6,a n d /v16=2 (one detected line, Fig. A.36 ). The three singly substituted13C isotopologues of cyanoacetylene are also clearly detected in their vibrational ground state (Figs. A.37 –A.39 )a n di n /v17=1( F i g s . A.40 –A.42 ). HC13CCN is also detected in /v17=2( F i g . A.43 ) while the two other isotopologues are only tentatively detected in this state(Figs. A.44 andA.45 ). HC 13CCN and HCC13CN are tentatively detected in /v16=1 with one detected line each (Figs. A.46 and 6The current model is somewhat inconsistent with the observed spec- trum at 92 129 MHz (blend of /v17=41 0−2–9 2and 10 4–9 4)a n d 100 431 MHz ( /v15=/v17=1l=0−110–1 0 0) but this is most likely due to resonant interactions between /v17=4a n d /v15=/v17=1, which are not well accounted for in the spectroscopic predictions. The frequen-cies of these transitions may well be o ﬀb yaf e wM H z( s e eC D M S documentation and Sect. 4.4.33 of Belloche et al. 2013 ).A.47 ). Emission of H13CCCN in /v16=1 signiﬁcantly contributes to the detected signal, but this state cannot be unambiguously identiﬁed (Fig. A.48 ). Two doubly substituted13C isotopologues of cyanoacety- lene, H13C13CCN and HC13C13CN are tentatively detected in their vibrational ground state with one line each (Figs. A.49 andA.50 ). The third, H13CC13CN has no clearly detected line, but the model using the same parameters as the former two iso- topologues is fully consistent with the signal detected around 105 328 MHz (Fig. A.51 ). Therefore we consider this species as tentatively detected too. The15N isotopologue HC 315N is not unambiguously de- tected in its vibrational ground state, but if we assume a 14N/15N isotopic ratio of 300, it contributes signiﬁcantly to the detected signal at 88 334 MHz and 105 999 MHz and is there- fore included in our model (Fig. A.52 ). The column density of this isotopologue should rather be considered as an upper limit. The ﬁts to the integrated intensity maps suggest that the size of the emission decreases with increasing energy of the vibra- tional state from within which the lines are emitted. Since our model cannot account for a nonunifo rm physical structure, we deﬁned two groups of vibrational states: /v1=0a n d /v17=1 were modeled with a source size of 1 .3/prime/primewhile the higher ex- cited states were modeled assuming 0 .9/prime/prime. The ﬁts to the population diagrams of the singly substituted 13C isotopologues including both /v1=0a n d /v17=1 yield rota- tional temperatures of ∼170–180 K (Table 3,F i g s . A.53 –A.55 ). With a temperature of 170 K and a source size of 1 .3/prime/prime,t h e emission of all isotopologues reported above is well ﬁtted up to/v17=1, except for the vibrational ground state of HC 3N: its transitions are very optically thick ( τmax∼30) and cannot be reproduced with our simple model. For the vibrationally excited states of the main and singly substituted13C isotopologues above /v17=1, we assume a source size of 0 .9/prime/primeand obtain a very good ﬁt to the observed spectra with a temperature of 200 K and a unique column density (divided by 20 for the13C isotopologues) that is 1.5 times higher than for the model of the lower states. Assuming the same parameters as derived above for the vi- brational ground state, we looked for emission of deuterated cyanoacetylene DC 3N. The molecule seems to contribute at a level of∼70% to the signal detected at 101 315 MHz (Fig. A.56 ). The rest of the emission comes from a transition of CH 2CO in its A91, page 8 of 66	10.1051_0004-6361_201527268	page_0007
A. Belloche et al.: Deuterated complex organic molecules in Sgr B2(N2) vibrationally excited state /v19=1. The detection of DC 3N is only tentative, and its column density should rather be considered as an upper limit. 5.4. Deuterated methanol CH 2DOH Methanol and its13Ca n d18O isotopologues are well detected toward Sgr B2(N2). The detected lines and detailed modeling of these species is presente d in a companion paper ( Müller et al. 2016b ). We report in Table 4the parameters derived in that paper for the main isotopologue based on the analysis of allisotopologues. Assuming the same source size and rotational tempera- ture, we obtain a tentative detection of CH 2DOH, with two lines detected at 91 587 MHz (4 1,3e0–40,4e0) and 99 672 MHz (61,5e0–60,6e0), two lines tentatively detected at 85 600 MHz (62,4e1–61,6o1) and 94 563 MHz (1 1,0o1–10,1o1), and a few other lines contributing signiﬁcantly to the detected signal(Fig. A.57 ). The line appearing at 91 589 MHz in the full syn- thetic model with no counterpart in the observed spectrum corre- sponds to two transitions of acetone (23 18,6–23 17,7of the EE state and 23 18,6–23 17,7of the AE state). While acetone is unambigu- ously detected in our ALMA spectrum of Sgr B2(N2), a signif- icant number of predicted lin es of acetone do not match the ob- served spectrum. The spectrosc opic predictions are not accurate enough for this set of problematic lines, the line at 91 589 MHz being one of those. The ALMA spectrum suggests that the truefrequency could be 91 592 MHz for this acetone line. The source size derived from the maps of the two detected CH 2DOH lines is uncertain but the emission looks compact in the integrated in-tensity maps and is consistent with the source size assumed forthe modeling. 5.5. Upper limits 5.5.1. Deuterated methanol CH 3OD CH 3OD is not unambiguously detected toward Sgr B2(N2). It may signiﬁcantly contribute to the emission detectedat 90 743 MHz (blend of 10 1,1–92,1and 2 1,0–11,0), 110 951 MHz (41,0–40,0), 111 846 MHz (5 1,0–50,0), and 113 352 MHz (6 1,0– 60,0), but there is no clearly detected line (Fig. A.58 ). Assuming the same parameters as for methanol (Sect. 5.4), we derive a col- umn density upper limit that is a factor 1.8 times lower than the column density tentatively derived for CH 2DOH (Table 4). This upper limit corresponds to the synthetic spectrum shown in red in Fig. A.58 . 5.5.2. Deuterated vinyl cyanide CHDCHCN and CH 2CDCN Many lines of vinyl cyanide are detected in its ground state and vibrationally excited states /v111=1,/v115=1, and /v111=2 (Figs. A.59 –A.62 ). The sizes derived from the corresponding in- tegrated intensity maps tend to decrease with increasing energy, from∼1.2/prime/primeforEup<100 K to∼0.8/prime/primefor higher energy tran- sitions. As a compromise we adopt a source size of 1 .1/prime/prime. With this source size, the analysis of the population diagram yields a temperature of∼200 K (Table 3and Fig. A.63 ). Transitions from within even higher vibrationally excited states are also detectedtoward Sgr B2(N2), but we do not report about these states. Transitions of all three singly substituted 13C isotopologues of vinyl cyanide are also clearly detected (Figs. A.64 –A.66 ;s e e also Müller et al. 2008 for a previous single-dish detection). Only a few lines are su ﬃciently free of contamination to allowfor a size measurement in the corresponding integrated inten- sity maps. The outcome is more uncertain than for the main iso- topologue, but is consistent with the source size adopted above.Because of the smaller number of detected lines, the population diagrams have a higher dispersion than for the main isotopo- logue and the rotational temperature is less well constrained butthe ﬁts to all three diagrams are consistent with a temperature of about 200 K (Figs. A.67 –A.69 ). As a result of this analysis, we adopt a source size of 1 .1 /prime/prime and a temperature of 200 K for vinyl cyanide and its isotopo- logues. With these parameters, we do not detect the15Ni s o - topologue. We also looked for the singly deuterated species cis- CHDCHCN, trans -CHDCHCN, and CH 2CDCN, but did not de- tect them. Column density upper limits are reported in Table 4. 5.5.3. Deuterated ethanol CH 3CH 2OD, CH 3CHDOH, and CH 2DCH 2OH Ethanol and its13C isotopologues are well detected toward Sgr B2(N2). The detected lines and detailed modeling of these species is presented i n a companion paper ( Müller et al. 2016b ). We report in Table 4the parameters derived in that paper for the main isotopologue based on the analysis of all isotopologues. Assuming the same LTE parameters as for the main isotopo- logue, we searched for all singly deuterated isotopologues ofethanol. None is detected. Upper limits to their column densities are reported in Table 4. 5.5.4. Deuterated methyl formate CH 2DOCHO Methyl formate is clearly seen toward Sgr B2(N2), with dozens of transitions detected in both its ground and ﬁrst torsionalstates (Figs. A.70 andA.71 ). We derive a median source size of 1.5 /prime/primefrom ﬁts to the integrated intensity maps of its numer- ous uncontaminated lines. The formal ﬁt to its population di-agram including both states yields a rotational temperature of ∼140 K (Fig. A.72 and Table 3). We used a temperature of 150 K in our model, which ﬁts the ALMA spectrum very well,apart from a few discrepancies that we describe now. The rea- son why the synthetic spectrum of the ground state poorly ﬁts the ALMA spectrum at 100 080 MHz is unclear. It may be dueto the nearby HC 3N 11–10 transition at 100 076 MHz, which is probably aﬀected by self-absorption and /or spatial ﬁltering and is by far overestimated by our simple LTE model. Thediscrepancy around 110 226 MHz is due to contamination by dif- fuse cloud absorption in 13CO 1–0 that is not yet included in our full model. Similar contamination by c-C3H2absorption features not yet implemented in our full model likely explains the smalldiscrepancies for the /v1 t=1 transitions around 85 370 MHz. Assuming the same LTE parameters as for the main isotopo- logue, we searched for the in-plane and out-of-plane conformersof CH 2DOCHO toward Sgr B2(N2) but none of them is detected. Upper limits to their individual column densities are reported in Table 4. 6. Discussion 6.1. Comparison to other observations The levels of deuterium fractionation derived in Sect. 5for (com- plex) organic molecules toward Sgr B2(N2) are summarized inTable 5and shown in Fig. 2. A91, page 9 of 66	10.1051_0004-6361_201527268	page_0008
A&A 587, A91 (2016) Table 5. Deuterium fractionation of selected (complex) organic molecules toward Sgr B2(N2) compared to predictions of astrochem-ical models. Molecule Statusa[XD]/[XH] N2bT14cA12d %% % CH 2DCN d 0.38 3.6–0.15 2.3–6.3 CH 2DCH 2CN (oop) t 0.05 – – CH 2DCH 2CN (ip) n <0.024 – – CH 3CHDCN t 0.05 – – cis-CHDCHCN n <0.8 – – trans- CHDCHCN n <0.8 – – CH 2CDCN n <0.5 – – DC 3N t 0.09 – 5.4–1.1 CH 2DOH t 0.12 5.5–0.51 2.4–2.4e CH 3OD n <0.07 3.5–0.3 –e CH 3CH 2OD n <1.5 – – CH 3CHDOH n <1.5 – – CH 2DCH 2OH (oop) n <1.5 – – CH 2DCH 2OH (ip) n <1.0 – – CH 2DOCHO (oop) n <2.0 14–0.43f– CH 2DOCHO (ip) n <0.6 7–0.22f– Notes. The notations oop and ip describe the position of the deuterium and stand for out of plane and in plane, respectively.(a)d: detection, t: tentative detection, n: nondetection.(b)Deuterium fractionation mea- s u r e dt o w a r dS g rB 2 ( N 2 ) .(c)Deuterium fractionation predicted by the model of Taquet et al. (2014 ) in the hot corino at the beginning and end of the Class 0 phase.(d)Deuterium fractionation predicted by the model ofAikawa et al. (2012 ) in the hot corino at the beginning and end of the Class 0 phase.(e)The model of Aikawa et al. (2012 ) was not de- signed to predict the abundance ratios of deuterated isomers: it assumesstatistical branching ratios. (f)The model of T a q u e te ta l . (2014 ) does not distinguish between the in-plane and out-of-plane conformers. Thevalues listed here assume a statistical distribution (2:1). 6.1.1. Deuterated methyl cyanide The detection of CH 2DCN toward Sgr B2(N2) is the most se- cure among the deuterated species reported here (Sect. 5.2and Fig.A.26 ). On the basis of the LTE modeling of methyl cyanide and its various isotopologues, we derive a deuterium fraction-ation of 0.4% for this molecule. This is a factor 2.6 lower than the fractionation reported by Gerin et al. (1992 )t o w a r d Orion KL (1%). Along with this ﬁrst interstellar detection, theseauthors also reported a tentative detection toward the hot core G34.26+0.15 that, if true, would indicate a similar level of deu- terium fractionation as toward Orion KL. The di ﬀerence with the level measured in Sgr B2(N2) is probably not signiﬁcant because the Orion KL and G34.26 +0.15 values may su ﬀer, as mentioned by these authors, from a lack of knowledge of the source sizeand opacity of the lines of the main isotopologue. A detection of deuterated methyl cyanide toward the Class 0 (low-mass) protostar IRAS 16293–2422 was also reported in Taquet et al. (2014 ) based on an unpublished analysis. They quote a deuterium fractionation of 1 .3%, a factor 3.4 higher than the one obtained for Sgr B2(N2). 6.1.2. Deuterated ethyl cyanide Both deuterated isotopologues of ethyl cyanide are tenta-tively detected toward Sgr B2(N2) (Sect. 5.1and Figs. A.10 andA.11 ). We derive a deuterium fractionation of ∼0.05% for both CH 2DCH 2CN (in its out-of-plane conformation) and the chiral molecule CH 3CHDCN. The upper limit obtained for theFig. 2. Deuterium fractionation of (complex) organic molecules toward Sgr B2(N2). Secure detections are indicated with a ﬁlled square, ten-tative detections with an empty square, and upper limits with an arrowpointing to the left. The notations oop and ip describe the position ofthe deuterium and stand for out of plane and in plane, respectively. in-plane conformer of CH 2DCH 2CN (Fig. A.12 ) is still consis- tent with the expectation that it should be half as abundant asthe out-of-plane conformer. If we assume this expected ratio,then the total deuterium fractionation for CH 2DCH 2CN would be∼0.075%, a factor 1.5 times higher than for CH 3CHDCN. This would be consistent with th e statistical expectation because the methyl group at the end of the carbon chain has three equiva- lent hydrogen atoms while the middle chain group has only two. The deuterium fractionation derived for ethyl cyanide to- ward Sgr B2(N2) is nearly one order of magnitude lower thanfor methyl cyanide (0.4%), but similar within a factor two to methanol (0.12%) and cy anoacetylene (0.09%). Margulès et al. (2009 ) reported a detection of the 15Ni s o - topologue of ethyl cyanide toward Orion KL but obtained onlyan upper limit for CH 2DCH 2CN. They derived a column density ratio [CH 2DCH 2CN (oop)]/[C2H5C15N]<0.33, which trans- lates into [CH 2DCH 2CN (oop)]/[C2H5CN]<0.2% using the 14N/15N isotopic ratio of 148 ±74 derived by Daly et al. (2013 ). Daly et al. (2013 ) claimed tentative detections of both deuter- ated isotopologues of ethyl cyanide with a deuterium fractiona-tion of 2% based on the same survey of Orion KL. No detected transitions are shown in that study, though, and their Table 3 ac- tually reports upper limits for the deuterated species. Given thelower deuterium fractionation obtained by Gerin et al. (1992 ) for methyl cyanide toward Orion KL (1%) and the order of magnitude diﬀerence in deuterium fractionation between methyl cyanide and ethyl cyanide obtained here toward Sgr B2(N2), a deuterium fractionation of 2% for ethyl cyanide in Orion KL sounds unlikely and questions the tentative detection of Daly et al. (2013 ). 6.1.3. Deuterated methanol CH 2DOH is tentatively detected toward Sgr B2(N2) (Sect. 5.4 and Fig. A.57 ). The deuterium fractionation we derive for this isotopologue is 0.12%, a factor ∼3 lower than the value we A91, page 10 of 66	10.1051_0004-6361_201527268	page_0009
A. Belloche et al.: Deuterated complex organic molecules in Sgr B2(N2) obtain for methyl cyanide, the d euterated species of which is securely identiﬁed in our ALMA spectrum of Sgr B2(N2) (Sect. 6.1.1 ). Therefore, even if the di ﬀerence in deuterium fractionation between methanol and methyl cyanide is a priori surprising, the fact that it is lower for methanol gives us more conﬁdence in the detection of CH 2DOH. The deuterium frac- tionation derived for CH 2DOH is a factor ﬁve lower than to- ward the Compact Ridge in Orion KL (0.58%, Neill et al. 2013 ), about one order of magnitude lower than toward the high-massprotostellar objects surveyed by Fontani et al. (2015 )a n dt h e intermediate-mass protostar NGC 7129 FIRS 2 (2%, Fuente et al. 2014 ), and more than two orders of magnitude lower than toward Class 0 protostars which have values between 19%and 33% ( Parise et al. 2006 ) 7. Evidence for the presence of CH 3OD is too tenuous in our ALMA spectrum of Sgr B2(N2) to claim a detection, even a tentative one (Sect. 5.5.1 ). Still, we cannot completely exclude that CH 3OD is present at the level indicated by our upper limit. The synthetic spectrum shown in Fig. A.58 indicates that a large fraction (>50%) of the ﬂux density detected at 90 744, 99 964, 110 951, and 113 352 MHz may well be emitted by this molecule. If this is true, the deuterium fractionation of methanolwould then be∼0.07% for CH 3OD. This would be nearly one order of magnitude lower than toward the Compact Ridge in Orion KL (0.5%, Neill et al. 2013 ) and about 50 times lower than toward Class 0 protostars (1.6%–4.7%, Parise et al. 2006 ). A detection of CH 3OD toward Sgr B2 was reported by Gottlieb et al. (1979 ) with the 36 foot radio telescope of the National Radio Astronomy Observatory at Kitt Peak ( HPBW∼ 74/prime/prime). These authors detected a line at the frequency ex- pected for the pair of partially blended transitions 2 −1–1−1E (90 703.6 MHz, Eu/kB=11.3K )a n d2 0–10A (90 705.8 MHz, Eu/kB=6.5 K), but they did not detect the nearby 2 1–11Et r a n - sition (90 743.5 MHz, Eu/kB=15.6K ) ,w h i c hi si nf a c te x - pected to be partially blended with the 10 1–92A transition (90 741.7 MHz, Eu/kB=124 K). The former two transitions are blended with deep HNC absorption features produced bydiﬀuse clouds along the line of sight in our ALMA spectrum of Sgr B2(N2). This prevents th eir detection in our spectrum (see Fig. A.58 ). Our LTE model shows that, for a temperature of 160 K, the latter two transitions are expected to be as strong as the former two. We conclude from this that either the assignment of the 90 704 MHz line to CH 3OD in the Kitt Peak spectrum was not correct, or the line reported by Gottlieb et al. (1979 ) traces low-excitation emission of CH 3OD. Given that the line detected in emission in the Kitt Peak sp ectrum dominates over the ab- sorption features, opposite to what is seen in the ALMA spec-trum, this emission line, if real, must come from a region more extended than the Sgr B2 continuum emission that is absorbed by the diﬀuse clouds along the line of sight. Such an extended emission would be ﬁltered out in our ALMA spectrum. Gottlieb et al. (1979 ) derived a ratio [CH 3OD]/[13CH 3OH]∼0.18 for Sgr B2. This translates into [CH 3OD]/[CH 3OH]∼0.7%, assuming a12C/13C isotopic ratio of 25 as derived for methanol toward Sgr B2(N2) in the companion paper Müller et al. (2016b ). This is an order of 7There was an issue with the spectroscopic predictions used in the early studies reporting CH 2DOH column densities (B. Parise, priv. comm.). We compared the Sμ2values listed in Table 1 of Parise et al. (2002 ) ,w h i c hw e r ea l s ou s e di n Parise et al. (2006 ), with the cur- rent JPL catalog. The new values of the selected transitions are a factor2.1±0.4 times higher on average than the old values. The partition func- tion is the same in both cases. As a result, the column densities reportedfor CH 2DOH in both articles were overestimated by a factor of ∼2.magnitude higher than our upper limit of 0.07% derived in Sect. 5.4. This discrepancy seriously questions the detection of CH 3OD reported by Gottlieb et al. (1979 )t o w a r dS g rB 2 , unless deuteration of methanol is more e ﬃcient by one order of magnitude on large scales in the Sgr B2 cloud compared to the embedded hot cores. Comito et al. (2003 ) derived an abundance ratio [HDO]/[H2O]∼0.06% toward the Sgr B2 hot cores and their T<100 K envelope, and even lower values of ∼0.013% and∼0.02% (uncertain within a factor two) were obtained for [DCN]/[HCN] and [DCO+]/[HCO+] in the molecular ridge close to Sgr B2(M) ( Jacq et al. 1999 ). Deuterium fractionation thus does not appear to be generally more e ﬃcient on larger scales in Sgr B2, which again questions the detection of CH 3OD reported by Gottlieb et al. (1979 ). 6.1.4. Deuterated cyanoacetylene The detection of DC 3N reported toward Sgr B2(N2) is only ten- tative (Sect. 5.3and Fig. A.56 ). We obtain a deuterium fraction- ation of 0.09%, similar to the values obtained for methanol and ethyl cyanide. DC 3N was ﬁrst detected toward TMC 1 with a deuterium fractionation of 2–8% ( Langer et al. 1980 ), revised to a lower value of 1.5% by Turner (2001 ). High values were reported with single dish telescopes for a number of other cold dense cores (5%–10%, Howe et al. 1994 )a sw e l la sf o rap r o t o - star in a stage of “Warm Carbon-Chain Chemistry” ( ∼3%,Sakai et al. 2009 ). A tentative detection toward the Compact Ridge and the Hot Core of Orion KL was recently reported with a deu-terium fractionation of 1 .5%±0.9% ( Esplugues et al. 2013 ). A tentative detection was also recently reported toward the high- mass protostar NGC 2264 CMM3 (1.8% ±1.5%, Watanabe et al. 2015 ). The deuterium fractiona tion of cyanoacetylene ten- tatively derived toward Sgr B2(N2) is thus at least one order of magnitude lower than in Orion and NGC 2264 CMM3 (if con-ﬁrmed) and even two orders of magnitude lower than in cold dense gas. 6.1.5. Deuterated vinyl cyanide The column density upper limits reported in Sect. 5.5.2 yield deuterium fractionations <0.8%,<0.8%, and<0.5% for cis-CHDCHCN, trans- CHDCHCN, and CH 2CDCN, respectively. We are not aware of any reliable detection of deuterated vinyl cyanide in the interstellar medium. 6.1.6. Deuterated ethanol The column density upper limits reported in Sect. 5.5.3 yield deuterium fractionations <1.5%,<1.5%,<1.5%, and<1.0% for CH 3CH 2OD, CH 3CHDOH, and the out-of-plane and in- plane conformers of CH 2DCH 2OH, respectively. The latter two translate into a total deuterium fractionation <2.5% for CH 2DCH 2OH. These upper limits are about one order of mag- nitude higher than the deuterium fractionation measured for methanol (Sect. 6.1.3 ). They are thus not very constraining. We are not aware of any detection of deuterated ethanol in theinterstellar medium. 6.1.7. Deuterated methyl formate The column density upper limits reported in Sect. 5.5.4 yield deuterium fractionations <2.0% and<0.6% for the out-of-plane and in-plane conformers of CH 2DOCHO, respectively. This A91, page 11 of 66	10.1051_0004-6361_201527268	page_0010
174901-3 P.-M. Lam and Y. Zhen J. Chem. Phys. 143, 174901 (2015) g(f)+1 2Asin4α r2+U(r)−sin 2α 2rM=0. (10) Three equations (8)–(10) can be solved to obtain the remaining three unknowns quantities α,r, and M. In the high-force limit, Neukirch and Marko28have given the solutions in closed form. Define a quantity Kas K= 9π/8ν2LBkBT/g(f). (11) The plectoneme radius rand angleαare given by r=logK/(2κD), (12) α=2r2g(f)/(3A)1/4. (13) The torque Mis given by M=2A rsin3αcosα cos 2α. (14) A Taylor expansion of this expression for small αand substituting the results, Eqs. (12) and (13), for αandryields M≈[(32/27)A]1/4g(f)3/4/√κD × log 9π/8ν2LBkBT/g(f)1+α2.(15) Another quantity of experimental interest is the slope of the average extension q=∂⟨X⟩/∂∆Lk=−∂2G/∂f∂∆Lk =−2π∂M/∂f. Using Eq. (10) for M, this becomes q =−4πrg′(r)/sin 2α. Taylor expanding this for small αand substituting Eqs. (12) and (13) for αandryields q=(6A κD2g(f))1/4 log 9π/8ν2LBkBT/g(f) ×g′(f)(1+2α2/3). (16) Using Eqs. (15) and (16) and g(f)=f−kBT f/A, the slope and torque calculated are in qualitative agreement with experiment.28Experimental values of A/kBT=46, 47, 44, 45 nm at 50, 100, 200, and 500 nM salt and C/kBT=94 nm are used in the calculation. We will show in Sec. III that using a more accurate form of g(f)can significantly improve on the agreement with experiment. III. CALCULATION USING AN IMPROVED FREE ENERGY In this section, we give our calculation of the slope and the torque using an improved form of the untwisted free energy. The force-extension curve in the worm like chain (WLC) model is given by the widely used interpolation formula26 f=(kBT) LpX L+1 4( 1−X L)−2 −1 4, (17) where Lphere is the persistence length, related to the bending rigidity A, byA=kBT Lp, and Xis the extension. The negative of the free energy per unit length g(f)is obtained by a Legendre transform Lg(f)=f X−W(X), (18)where W(X)=X 0dX′f(X′) (19) is the work done in extending the polymer. The functions gandWdepend also on the persistence length Lp. From Eq. (17), the extension Xis an implicit function of the force f. Since the extension is a single-valued, monotonic increasing function of f, we can define the inverse function Xf(f)which gives the extension Xas a function of the force f. Even though this function cannot be obtained analytically, it can be calculated numerically to high accuracy. Substituting Eq. (17) into Eq. (19), the function Wcan be calculated analytically, W(X(f)) =LkBT 4LpXf(f) L( 2Xf(f) L−1) +( 1−Xf(f) L)−1.(20) From Eq. (18), the negative of the free energy per unit length is given as a function of the force fby g(f)=1 Lf Xf(f)−kBT 4Lp ×Xf(f) L( 2Xf(f) L−1) +( 1−Xf(f) L)−1. (21) We will use this form of the free energy in Eqs. (15) and (16) to calculate the torque Mand slope q. From Eq. (16), in order to calculate the slope q, the derivative of gwith respect to fis needed. From Eqs. (18) and (19), this is given by Lg′(f)=Xf(f). (22) In Fig. 2, we show our calculation of the slope of the average extension q=∂⟨X⟩/∂∆Lkobtained using Eq. (16), FIG. 2. Comparison of experimental and theoretical slopes q=∂⟨X⟩/∂∆Lk of the average extension, as a function of the applied force, for 50, 100, 200, and 500 mM salt (top to bottom). Circles are experimental data. Full lines are our theoretical results using a better form of the free energy. Dashed lines are theoretical results using approximate form of the free energy.	10.1063_1.4934988	page_0003
174901-4 P.-M. Lam and Y. Zhen J. Chem. Phys. 143, 174901 (2015) FIG. 3. Comparison of experimental and theoretical torque as a function of the applied force, for 50, 100, 200, and 500 mM salt (top to bottom). Circles are experimental data. Full lines are our theoretical results using a better form of the free energy. Dashed lines are theoretical results using approximate form of the free energy. withgandg′given by Eqs. (21) and (22), together with results obtained using the approximate forms for gandg′. The experimental data are directly taken from Fig. 2 of Ref. 28. The data in Ref. 28 are obtained from Ref. 21. The slope in Ref. 21 is a dimensionless quantity defined as ˜ q=L−1∂⟨X⟩/∂σ, with σ=(Lk−Lk0)/Lk0, where Lk0≈1500 is the linking number of the DNA molecule under no external tension or torque. Our slope qis related to ˜ qbyq=(L/Lk0)˜q=(5.4µm/1500 )˜q =(3.6 nm )˜q. The experimental data given in Ref. 28 are actually a factor (−3.6 nm )times the data in Ref. 21. In Figure 3, we present the results of our calculation for the torque M, using Eqs. (15) and (21), together with results obtained using the approximate form, compared with the experimental data, taken from Fig. 3 of Ref. 21. We can see that this better form of the free energy improves significantly the agreement with experiment. The agreement with experiment is now surprisingly good, except for low salt concentrations. IV. CONCLUSION We have shown that by using a better form of the free energy for the stretched but untwisted part of the DNA, the Neukirch-Marko model can give quantitative agreement with experimental results. There is still some disagreement at low salt concentration, but this is probably due to the inadequacy of the Debye–Hückel approximation of the Poisson-Boltzmann equation, which results in imperfect screening of the elec- trostatic potential at these low salt concentrations. It was mentioned in Ref. 28 that the disagreement with experiment may be due to the neglect of confinement entropy.36Since our results using a better free energy already yield quantitative agreement with experiment, the e ffect of confinement entropy is probably small. Our calculation is based on the model of Neukirch and Marko.28This theory is an analytic theory, with analyticexpressions for the slope and torque as functions of the tension. In order to arrive at this theory, several reasonable simplications have been introduced. It does not incorporate thermal fluctuations in plectoneme. The argument is that at least at higher tensions, the fluctuations are small and can as a consequence be neglected. It also neglects multi- plectoneme e ffects. The use of a two-cylinder repulsion in the Debye–Hückel regime is a rough approach not taking into account the e ffect on plectoneme angle as was shown to be important by Ubbink and Odijk.35More recent models37,38 have taken these e ffects into account. In Ref. 37, the authors give results of the slope versus tension, in very good agreement with experiment. However, for this quantity, the original theory of Neukirch and Marko also gives good agreement with experiment. It is the torque versus tension results in the Neukirch-Marko theory that show the largest disagreement with experiment, especially for low tension and low salt concentrations. For the torque versus tension result, the result of Ref. 37 is not so good. In more recent work,38Marko and Neukirch have also incorporated the above mentioned e ffects in their model, but unfortunately they do not give any new torque versus tension results. Notwithstanding the clearly better agreement between theory and experiment achieved in this work, one notes, how- ever, that it holds as far as the Debye–Hückel approximation of the Poisson-Boltzmann theory remains valid, i.e., for high screening /salt concentration only. As one can see from Figs. 2 and 3, the agreement with experiment deteriorates at low salt concentration for both the slope and the torque. A closer inspection of the q-f variation, shown in Fig. 2, indicates that the agreement with experiment at higher applied tensions (when f >3 pN at 500 mM and f >1 pN at 200 mM). This is puzzling because the expressions for the free energy g(f)and twist modulus Cs(f)should be correct for large fand the fluctuations in plectoneme and multi-plectoneme e ffects neglected in the model should also decrease with tension. Dhar and Chaudhuri39and Samuel and Sinha40have explored e ffects that go beyond the high force limit ( g(f)=f−kBT f/A). At these high forces, such e ffects may be relevant. It should also be pointed out that the force-extension formula given in Eq. (17) is only an interpretation formula which is convenient for calculation and should not be considered as a substitute for analytical or semi-analytic theoretical models in Refs. 39 and 40. In particular, the force-extension curve given by Eq. (19) did not take into account the entropy of the chain, even when no external force is applied, as pointed out by Neumann.41Also, in this high tension limit, one would have to include the e ffects of thermal fluctuations on DNA elasticity, as studied by Kulic et al.42,43and Sinha and Samuel.40,44Finally, the Legendre transform (Eqs. (18) and (19)) was used because we are considering the long DNA limit. If one were to look at shorter chains, with chain lengths comparable to the persistence length of 50 nm, one would need to work with Laplace transforms instead.41,45,46This is because such short chains are not in the thermodynamic limit and one has to distinguish between the isometric ensemble in which the chain ends are held fixed and the applied force is allowed to fluctuate and the isotensional ensemble in which the applied force is held fixed and the chain lengths are allowed to fluctuate. Only in the thermodynamic	10.1063_1.4934988	page_0004
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J. Chem. Phys. 145, 114901 (2016); https://doi.org/10.1063/1.4962516 145, 114901 © 2016 Author(s).On the influence of the intermolecular potential on the wetting properties of water on silica surfaces Cite as: J. Chem. Phys. 145, 114901 (2016); https://doi.org/10.1063/1.4962516 Submitted: 17 June 2016 • Accepted: 29 August 2016 • Published Online: 16 September 2016 E. Pafong , J. Geske and B. Drossel ARTICLES YOU MAY BE INTERESTED IN A reactive molecular dynamics simulation of the silica-water interface The Journal of Chemical Physics 132, 174704 (2010); https://doi.org/10.1063/1.3407433 Contact angles from Young’s equation in molecular dynamics simulations The Journal of Chemical Physics 147, 084708 (2017); https://doi.org/10.1063/1.4994088 TRAVIS—A free analyzer for trajectories from molecular simulation The Journal of Chemical Physics 152, 164105 (2020); https://doi.org/10.1063/5.0005078	10.1063_1.4962516	page_0000
THE JOURNAL OF CHEMICAL PHYSICS 145, 114901 (2016) On the influence of the intermolecular potential on the wetting properties of water on silica surfaces E. Pafong,a)J. Geske, and B. Drossel Institut für Festkörperphysik, Technische Universität Darmstadt, Hochschulstr. 6, 64289 Darmstadt, Germany (Received 17 June 2016; accepted 29 August 2016; published online 16 September 2016) We study the wetting properties of water on silica surfaces using molecular dynamics (MD) simu- lations. To describe the intermolecular interaction between water and silica atoms, two types of interaction potential models are used: the standard BródkA and Zerda (BZ) model and the Gulmen and Thompson (GT) model. We perform an in-depth analysis of the influence of the choice of the potential on the arrangement of the water molecules in partially filled pores and on top of silica slabs. We find that at moderate pore filling ratios, the GT silica surface is completely wetted by water molecules, which agrees well with experimental findings, while the commonly used BZ surface is less hydrophilic and is only partially wetted. We interpret our simulation results using an analytical calculation of the phase diagram of water in partially filled pores. Moreover, an evaluation of the contact angle of the water droplet on top of the silica slab reveals that the interaction becomes more hydrophilic with increasing slab thickness and saturates around 2.5–3 nm, in agreement with the experimentally found value. Our analysis also shows that the hydroa ffinity of the surface is mainly determined by the electrostatic interaction, but the van der Waals interaction nevertheless is strong enough that it can turn a hydrophobic surface into a hydrophilic surface. Published by AIP Publishing. [http: //dx.doi.org /10.1063 /1.4962516] I. INTRODUCTION Water is an essential material in our everyday life and is the most used solvent for chemical and biological reactions. Water molecules are highly polar, forming hydrogen-bonded networks and sharing hydrogen bonds with other molecules. In particular, water confined within nanoscale geometries of hydrophilic surfaces is subject to two competing interactions: the hydrophilic interactions between water molecules and those between water and surface molecules. One of the standard systems for studying hydrophilic interactions with water is silica nanopores such as sols- gels,1mesoporous silica (MCM-41),2–7Vycor-glasses,8–10 controlled pore glasses (CPGs).11–15Water confined in silica nanopores or near silica flat surfaces is a topic which has attracted considerable attention,16–22mainly because of the relevance of the water-silica interaction in understanding the water transport in porous rocks,23 nanofluidic devices,24heterogeneous catalysis in mesoporous materials,15,25and permeation through membrane channels.26 Experimental investigations of water in silica nanopores have been carried out using NMR spectroscopy,2,12X-ray and neutron di ffraction,5,6,9,10,27,28quasi-elastic neutron scattering,3,4Small-angle neutron scattering (SANS),8and optical Kerr-e ffect spectroscopy,1showing that the dynamics of water in such pores is slow in comparison to the dynamics of bulk water. This originates from the strong binding or trapping of water molecules by silica surfaces as found by experimental measurements conducted with MCM-41 as well as CPG pores2,12resulting in a complete coverage of the pore a)priscel@fkp.tu-darmstadt.desurface at even moderate hydration levels. Accordingly, the intermolecular interactions between water and silica surfaces in MD simulations should be set up such that the experimental results are reproduced. The influence of the filling ratios on the wetting properties of water in silica nanopores has been studied by previous MD research.29–32Such investigations are motivated by the fact that in experiments the fluid is placed on top of a porous surface and flows to enter the pores. In the mentioned MD simulations, it was demonstrated that at all hydration levels water molecules are absorbed by the Vycor material. However, they have not shown to what extent the pore surface is wetted, whether it is only partially wetted or rather completely wetted (as expected from measurements2,12). Moreover, the confinement near such hydrophilic surface was found to substantially alter the dynamic behaviour of water, depending on the filling ratio,19,33but it has not been checked how this relates to the configurations that water can take inside the pore. In all these previous MD analyses, the (12-6) Lennard-Jones (LJ) potential and the partial charges assigned to each silica atom site are chosen according to BródkA and Zerda (BZ).34In this model, the LJ potential parameters for silica oxygen atoms are approximated from the Kirkwood-Mueller formula,35while no LJ interaction centers are assigned to silicon and hydrogen (of the silanol groups) as they are small in size and possess a low polarizability. In the present investigation, we have found that water molecules do not completely wet the BZ silica model surface at intermediate hydration levels. For this reason, the silica model recently introduced by Gulmen and Thompson36(GT) has been tested. The GT36potential is defined similarly to the silica potential by BZ,34however, a weak short-ranged interaction for silicon and hydrogen 0021-9606/2016/145(11)/114901/9/$30.00 145, 114901-1 Published by AIP Publishing.	10.1063_1.4962516	page_0001
114901-2 Pafong, Geske, and Drossel J. Chem. Phys. 145, 114901 (2016) atoms has been added and the partial charges on each silica atom are increased. We probed the performance of both silica models by analyzing the wetting behaviour of water on silica surfaces and comparing to the experimental results.2,12,37–39 In the following, we present the results of MD simulations of water in a cylindrical silica nanopore of roughly 4 nm diameter and 6 .1 nm height. Additionally, MD simulations of water droplets wetting silica slabs of varying thicknesses were performed. The silica nanopores and slabs were created in our group, with the silanols molecules uniformly distributed on the surface. For water in the silica pore, we evaluate the minimum number of water layers necessary to completely wet a silica surface by looking at the radial and angular density distribution, as well as the number of hydrogen bonds formed for di fferent filling ratios of water in the pore. Furthermore, a phase diagram of the surface tension of the different configurations adopted by water molecules in the nanopore is calculated analytically, providing deeper insights into the relation between the interaction energies and the water arrangement in the pore. To complete the work, the contact angle of a water droplet on a silica flat slab is evaluated and compared between the two model surfaces. Previous MD investigations have used the contact angle investigation to approximate LJ parameters between water and silica atoms40,41but they have not stated clearly whether the simulations were performed in such a way that the periodic images provided by the periodic boundary conditions do not influence the contact angle evaluated. In our investigation, we run the simulations without periodic boundary conditions to avoid this issue. Previous experimental results showed that the wetting properties do not only involve atoms of layers in the vicinity of the interface but also the atoms located deeply inside the slab material.37–39,42 Therefore, we measure how the contact angle changes with the thickness of the slab, showing that a thickness of 2.5–3 nm is sufficient for MD simulations. In order to disentangle the contributions of the interfacial electrostatic and van der Waals (VdW) interactions on the contact angle, we varied these two contributions in our simulations, showing that the influence of the electrostatic interaction is considerably larger than that of the VdW interaction. II. SIMULATION DETAILS Classical MD simulations were performed with the NAMD432.10 simulation package. An amorphous cylindrical nanopore of roughly 4 nm of diameter and silica slabs of different thicknesses were fabricated in our group. To create the pore, a crystalline cell of SiO 2with a box length of approximately 6 nm was built, the system was melted at 5000 K and cooled to room temperature with the method described in Ref. 44, and then a cylindrical cavity of ∼4 nm diameter was cut. The process of fabrication of the silica cylindrical pore and the silica slab is explained in detail in a separate paper.44The surface concentration of hydroxyl groups on the surface is 7 .5 nm−2corresponding to highly hydrated silica surfaces.34The silica slabs were created following a similar procedure.TABLE I. LJ potential parameters for silica interaction centers. Parameters Sites σ(nm) ε(kcal/mol) q (e) BZ34Si 0.0178a0.000 00 1.283 OSi 0.27 0.457 056 94 −0.629 OH 0.3 0.457 056 94 −0.533 H 0.0178a0.000 00 0.206 GT36Si 0.25 0.000 1 1.28 OSi 0.27 0.457 −0.64 OH 0.307 0.17 −0.74 H 0.1295 0.000 365 7 0.42 aValues were not mentioned in the model and were chosen arbitrarily small. The silica nanopore and slab contain two types of oxygen atoms depending on the number of silicon atoms to which they are connected. There are bridging oxygens (O Si) bonded to two adjacent silicons and nonbridging oxygens (O H) on the surface attached to only one silicon. Hydrogen atoms are attached to the O Hin order to form the silanols groups (SiOH, Si(OH) 2). The bonded interaction parameters for silica atoms were obtained from Hill and Sauer.45Apart from the hydrogen atoms of the silanols groups that are allowed to rotate, all atoms in the silica pore and slab are immobile, constrained to a fixed position, whereas water molecules are free to move within the pore. Liquid water is defined using 2 models: a set of 3 rigid sites given by the SPC /E46model and a set of 4 sites provided by the TIP4P200547model. The atoms of the silica substrate are allowed to interact with the water sites by means of the Coulomb potential and LJ potential in Eq. (1), ULJ=4ϵ* ,σ12 r12 i,j−σ6 r6 i,j+ -(1) which implements the VdW interaction. LJ parameters and fractional charges for the SiO 2sites are given in Table I. All simulations were made with the NVT ensemble with a fixed room temperature T=298 K using a Langevin thermostat43with a coupling coe fficient of 1.0 ps−1and with the hydrogen atoms included in the Langevin dynamics. An integration time step of 2 fs was utilized and the simulations were run for at least 20 ns. Periodic boundary conditions were set for the simulations of water in the nanopore allowing the calculation of the long-range Coulombic electrostatic interactions with the particle-mesh Ewald sum, using a cut-o ff of 1.2 nm. No periodic boundary conditions were defined for the simulation of water wetting silica slabs in order to allow the calculation of the full electrostatic and VdW interactions between all the water droplet atoms and silica slab atoms. III. RESULTS I: WATER IN PARTIALLY FILLED SILICA PORES In the following, we investigated the configuration of water in a partially filled silica pore for the two di fferent models using MD simulations. Furthermore, we performed an analytical calculation of the di fferent possible phases of water in a cylindrical pore that allows us to interpret the findings.	10.1063_1.4962516	page_0002
114901-3 Pafong, Geske, and Drossel J. Chem. Phys. 145, 114901 (2016) FIG. 1. Top view of the two initial con- figurations used in the MD simulations, labelled in the subsequent figures with “center” (a) and “surface” (b). Here the hydration level is 30%. Si, O Si,H, H are drawn in yellow, blue, and green, while water atoms O, H are indicated by red and white, respectively. The pic- tures were generated using the VMD program.48 A. MD simulation results We evaluated the equilibrated configurations of water in the pore for the GT and BZ surfaces, using di fferent filling ratios and di fferent starting configurations. The pore filling ratios are in the range 30%–97%, based on the estimated number of molecules for 100% filling ratio, which is 2700.30The equilibrium configurations were analyzed by calculating the radial density profile, the distribution of water molecules on the interior pore surface, and the number of hydrogen bonds among water molecules and between water and silica molecules. In order to see how far the final configurations depend on the initial configuration, we used the two di fferent initial configurations shown in Fig. 1, where water is concentrated around the cylinder axis and at the pore surface, respectively. There is thus a void between the water droplet and the silica surface in the first configuration, and a void in the pore center for the second configuration. Fig. 2 shows the radial density profile of water molecules inside the pore as a function of the distance to the pore center, averaged over 15 ns after at least 5 ns of equilibration for each simulation. One can see that water is closer to the GT surface. The GT density profile shows only one peak for a filling ratio of 30%, indicating that all water molecules are in contact with the pore surface. Only after the first layer is completed, a second layer is formed, as is visible for the curves for filling ratios between 40% and 55%. At 65% filling ratio, thewater molecules can also be found in the interior of the pore, indicating a configuration with a completely wetted surface and a compact water droplet in the pore interior. The density profiles for the BZ surface show several layers of water, with a peak height that depends on the filling ratio. Furthermore, the density profile depends on the initial configuration for intermediate filling ratios, with the initial configurations at the surface leading to final configurations with rather flat density profiles. This suggests that for the initial configuration at the boundary, the water droplet forms a “plug” in the pore interior, while for the initial configuration in the center, water forms some type of droplet sitting at the surface. Since the pore surface is rough, some water molecules can also be found inside the silica pore material. In order to test the intuition obtained for the water configurations based on the density profiles, we evaluated the distribution of water molecules within a distance of 0.3 nm of the surface. Fig. 3 shows the resulting surface density profiles, using cylinder coordinates. This figure confirms that for the GT surface, the water droplet first wets the surface completely, before filling the interior. For the BZ surface, the pore surface is only partially covered with water, and the final configuration depends on the starting configuration for intermediate filling ratios. For instance, for 40% filling ratio with the “center” starting configuration, water molecules are concentrated in one angular segment of the surface but are covering the whole length, while for the “surface” starting configuration they FIG. 2. Radial density profile of water in the silica pore for di fferent filling ratios and starting configurations, for the (a) BZ surface and the (b) GT surface. The radius of the pore is 2 nm. The gray area indicates the silica pore surface and is arbitrarily scaled for a clear visibility.	10.1063_1.4962516	page_0003
114901-4 Pafong, Geske, and Drossel J. Chem. Phys. 145, 114901 (2016) FIG. 3. The water density at the pore surface, showing water molecules that are within a distance of 0.3 nm of the silica surface. For the GT surface ((e)-(h)), only filling ratios of 30% and 40% are shown, since for larger filling ratios the surface is completely wetted. For the BZ surface, the top ((a)-(b)) graphs correspond to the “surface” starting configuration and the bottom graphs ((c)-(d)) correspond to the “center” starting configuration, (i) represents the density at 65% filling ratio. occupy only part of the zrange, but all angles. These final configurations for intermediate filling ratios are in fact very plausible if one tries to imagine how the initial configurations can evolve with time in a situation where the water-surface interaction is not strong enough that the entire surface is wetted. When the initial configuration has a water cylinder in the pore center, the entire cylinder gets attracted by the silica molecules under the influence of electrostatic and VdW interaction and moves as a whole towards the pore surface, wetting a specific angular region of the surface. When the initial configuration sits at the pore surface, the water film may rupture along an angular line, and the water will contract to form a plug. Even if one of the two final configurations has a lower free energy, this free energy di fference will not be large, and the transition between them will involve a barrier that is so large that it is not overcome during the simulation time. When the filling ratio is lower (as can be seen for 30%), the plug is not observed for either initial configuration, indicating that there is only one stable configuration. The two di fferent final configurations merge also for larger filling ratios (as can be seen for 65%), where the void left by the water droplet takes the shape of a droplet that sits at the pore surface. Finally, we evaluated the average number of hydrogen bonds formed between water molecules, and between water molecules and silica molecules. This shows to what extent the stronger hydroa ffinity of the GT model a ffects the formationof molecular bonds. We considered two oxygen atoms to be connected via a hydrogen bond if the angle between the intramolecular O—H vector and the intermolecular O ···O vector is less than 30◦, provided that the O ···O separation is less than 0 .335 nm. The results are shown in Fig. 4. For the BZ surface, the number of hydrogen bonds between water molecules reaches the bulk value in the inner part of the pore for a filling ratio larger than 40% with the “surface” initial configuration. Also for the “center” initial condition, the bulk value is reached for the water molecules in the interior of the water droplet. In the GT surface, the bulk value is reached only for filling ratios above 60%. This illustrates the fact that the BZ surface disrupts the water structure more than the GT surface. Accordingly, the number of hydrogen bonds formed between the silica surface and the water molecules is larger for the GT surface. For both models, the maximum number of water-silica hydrogen bonds is already reached at 40% filling ratio, confirming that one and half layer of water molecules is sufficient to completely wet the GT silica surface. It is at first surprising that for the BZ surface, the number of water-silica hydrogen bonds does not increase for filling ratios larger than 40% and stays considerably below the value of the GT surface. This can only be explained by di fferent water orientations near the surface in the two models. In the supplementary material we show that near the BZ surface the OH bonds of	10.1063_1.4962516	page_0004
114901-5 Pafong, Geske, and Drossel J. Chem. Phys. 145, 114901 (2016) FIG. 4. Average number of hydrogen bonds per water molecule for water–silica ((a)-(b)) and water–water ((c)-(d)) contacts, for di fferent initial conditions and filling ratios, as indicated in the legends. The results for the GT silica surface are given on the left-hand side, and those for the BZ silica surface on the right-hand side. water molecules have a preferred orientation, while this is not the case near the GT surface. This means that only part of the water molecules can act as a hydrogen bond donor or acceptor near a BZ silica molecule, while near the GT surface all the water molecules can share hydrogen bonds with the surface atoms. B. Theoretical evaluation of the phase diagram In order to better understand the dependence of the water droplet configuration in the pore on the interaction energies and the filling ratio, we performed a theoretical analysis that is based on surface energy minimization. Denoting the surface area between the water droplet and vacuum with A1, the surface area between the water droplet and the pore material with A2, andγ1as the surface tension between water and vacuum,γ2as the di fference between the surface tension of silica and water with the surface tension of silica and vacuum. The total surface energy of the wetting droplet can be written as ES=γ1·A1+γ2·A2. (2) If we assume that the entropy does not change much between different phases, the configuration of the water droplet in the pore can be obtained by minimizing ESfor a given filling ratio. In order to perform the calculation mostly analytically, we approximated the di fferent possible phases using simple geometrical shapes, so that the energy minimization can be performed by varying at most 2 parameters that characterize the phase. We fixed the ratio of the pore radius and pore length to the value R/L=2/6.1 used in the simulations. We determined the phase diagram in dependence of the filling ratio and the ratio between the two surface energies. We allowed for hydrophilic ( γ2<0) as well as for hydrophobicsurfaces (γ2>0). The surface tension γ1is a positive quantity. Fig. 5 shows the eight phases and the phase diagram obtained from minimizing ES. Phases 1, 2, and 4 are translationally invariant along the cylinder axis and represent the cases of partial, full, and no wetting of the silica surface. Phase 3 represents a plug in the shape of a cylinder that is shorter than the pore. Phases 5-8 describe the cases where the water or the vacuum forms a spherical droplet in the interior or one that intersects with the pore surface. The calculation of the surface energy for phases 1-6 is a straightforward analytical calculation. For phase 1, we had to use mathematica to evaluate the final expression. In order to evaluate phases 7 and 8, we had to resort to a numerical evaluation. We first created a database by FIG. 5. (a) The eight di fferent phases used for energy minimization and (b) the phase diagram in dependence of the ratio of the water-vacuum and water-silica surface energies and of the filling ratio. The aspect ratio r/l between radius and length of the cylindrical pore is 2 /6.1.	10.1063_1.4962516	page_0005
114901-6 Pafong, Geske, and Drossel J. Chem. Phys. 145, 114901 (2016) calculating numerically the volume and surface of the cut droplet for over 1 ×106different combinations of the sphere radius and the distance of the sphere center from the cylinder axis. Then, we used this database to find for a given filling ratio the values that minimize ES. The phase diagram shows clearly three qualitatively different regions that depend on the ratio γ2/γ1. For γ2/γ1.−1, the energy is minimized by having maximum surface area with the pore surface. The water wets the pore completely. Correspondingly, the phases 2 and 6 occur. With increasing filling ratio, the volume of the vacuum becomes smaller, and eventually a free volume that does not touch the surfaces fits into the pore. For su fficiently large filling ratio, the droplet clearly has the smaller surface area with the vacuum and therefore the lower energy. (Our simple calculation did not take into account that the droplet could be stretched, and therefore the position of the phase boundary between phases 2 and 6 should in fact be at a lower filling ratio.) Forγ2/γ1&1, the pore material is highly hydrophobic, and the phases 4 and 5, which have no contact between water and silica, have the lowest energy, depending on the filling ratio. The phase boundary between phases 5 and 4 moves downwards with decreasing aspect ratio r/l, because a vacuum droplet that does not touch the pore wall takes a smaller proportion of the total volume when r/lis smaller. (See the supplementary material for phase diagrams with other aspect ratios.) In the intermediate parameter region −1.γ2/γ1.1, we observe phases 7, 1, and 8 as the filling ratio is increased. These are the phases that have surfaces with the pore and with the vacuum. Since the (absolute value of) water-vacuum energy is larger than that of the water-silica energy, these phases are to a large extent a ffected by the condition that the water-vacuum interface shall be minimum. The transition from phase 7 to phase 1 occurs for lower values of γ2/γ1at smaller filling ratios than for larger γ2/γ1, because a larger surface area to the pore is energetically favorable for negative γ2. For the same reason, phase 8 wins over phase 1 for high filling ratios and negative γ2, because phase 8 has more surface area between water and the pore. The phase boundary to phase 8 moves upwards and the boundary to phase 7 moves downwards with decreasing aspect ratio r/l, because droplets that touch the pore wall only at one side take a smaller proportion of the total volume when r/lis smaller. The droplet phases will also vanish when the ratio r/lbecomes large, as the system then is e ffectively two-dimensional and shows only the three phases that are translationally invariant along the z-axis. (See supplementary material for phase diagrams with other aspect ratios.) Phase 3 does not occur in the phase diagram. It will certainly occur when the aspect ratio r/lbetween the radius and length of the pore becomes smaller, because it has then smaller surface area than phase 1. (See the supplementary material.) In our simulations with the BZ potential, we found this phase for intermediate filling ratios, where it coexists with phase 1. Phase 3 thus might well be metastable. On the other hand, it is also possible that phase 3 is indeed stable in part of the phase diagram due to entropic e ffects, which were not taken into account when calculating the phase diagram. Since in the canonical NVT ensemble the free energy F=E−T Shas to be minimized, phases with larger entropy become more favored when entropy is taken into account. This will shift the phase boundaries somewhat. For instance, when phase 2 contains only two layers of water molecules, its entropy per molecule is smaller than in bulk water. Similarly, the entropy per water molecule is larger in phase 3 than in phase 1, since the water in phase 3 is more bulk-like. With the insights gained from these analytical calcula- tions, we can interpret the results of the MD simulations shown in Figs. 2–4: For the BZ surface, the water wetted the silica surface only partially for all simulated filling ratios, and we observed the phases 1, 3, and 8 depending on the filling ratio. The transition to phase 8 occurs at a filling ratio of approximately 60%. For smaller filling ratios below 15%, we also see phase 7 (see the supplementary material). This means that the ratio of surface energies γ2/γ1is in the interval (−1,0). (Since the surface is hydrophilic, we have γ2<0.) With the GT surface, we observed a complete wetting of the silica surface (phases 2 and 6) for all simulated filling ratios, with a transition between these two phases at a filling ratio around 60%. This means that γ2/γ1<−1. This appears to be the more realistic scenario, as it agrees well with experimental results.2 In order to obtain an additional perspective on the di fferent interaction between water and a silica surface in the two models, we will in Sec. IV investigate the contact angle of water on top of a flat silica slab using both models. IV. RESULTS II: WATER ON TOP OF A SILICA SLAB A good tool to examine the performance of silica potentials is the evaluation of the contact angle of a water droplet wetting the surface. We performed MD simulations of a water droplet on a flat surface of amorphous silica and measured the contact angle. We did not use periodic boundary conditions in order to remove the influence of the neighbouring water periodic images. Instead, the Coulombic and the VdW (LJ) interaction energies between all atoms in the water droplet and the silica slab were calculated exactly. In order to evaluate the contact angle θ, the density profile of all horizontal water layers of 0 .05 nm thickness was determined, and from these a contour plot of the density was obtained. The contour plot was fitted to a circular segment, and the contact angle was deduced from the tangential line to the base of the circular segment. The result is shown in Fig. 6 for both types of potentials, with a slab of thickness t=2.5 nm. For the GT model, the droplet covers the entire surface and has a very small contact angle of 7◦. When we performed the same simulation with periodic boundary conditions, the water layer became completely flat. In contrast, the contact angle of the water droplet on top of the BZ surface is 25◦. These results confirm the findings of Subsection III B, that the GT silica surface is so hydrophilic that water wets it completely, while the BZ surface is less hydrophilic. The contact angle is closely related to the surface tensions that we used for evaluating the phase diagram. The condition that the total surface energy ( ES) of water wetting a silica surface must be minimal for an equilibrated droplet of constant	10.1063_1.4962516	page_0006
A&A 641, A54 (2020) Fig. 6. Abundances relative to H2of MF, DE, F, and EC (Table 10) as a function of the total luminosity/mass ratio (see Sect. 6.3 for details) for the sources of our sample. The linear best-ﬁt to the data is shown for each molecule. luminosity and is not affected by any distance-induced observa- tional bias, since we have checked that molecular abundances are independent from source mass and distance. PC and most HMS- FRs are in very good agreement with the trend observed in our sample, while hot corinos, IMSFRs and some of the other HMS- FRs show slightly higher values. Since the results of the latter are based on interferometric data, this discrepancy could be due to the different angular resolution. Although we accounted for beam dilution effects as consistently as possible (see Sect. 4), lower resolution (single-dish) observations may still result in slightly underestimated molecular column densities. Figure 8 summarises the main result of this analysis, showing the average abundances of the four molecules with respect to the evolutionary stage of the sources. For molecules detected at mul- tiple stages (MF, DE, and EC), average values increase with the evolution, namely from protostellar to intermediate until UCHII regions, preserving the mutual molecular ratios. The increasing trend is particularly evident for MF and DE. Average abundances increasing with time were also found by Gerner et al. (2014) for less complex molecules CH 3OH(methanol), CH 3CN(methyl cyanide), and other simpler molecules, and were predicted by Choudhury et al. (2015) for COMs including MF and DE through evolutionary models of HMCs. 6.4. Implications for the chemistry of COMs The abundances of MF, DE, and EC are very well correlated (r0:92, Fig. 2) and their mutual molecular ratios are nearly constant (Figs. 3–4). The result is very robust since it is based on a sample with good statistics (20 sources in our sample plus 59 sources from literature overall), covering several orders of magnitude in abundance and source luminosity. In some cases, this may indicate a chemical link between the species. This is most likely the case of MF and DE, show- ing the strongest correlations in many parameters (abundance, source size, and FWHM) and a constant 1ratio over a remark- able9orders of magnitude in source luminosity (Fig. 3, upper panel), with a limited scatter both in a large sample of low- to high-mass star-forming regions and among different interstellar environments (Fig. 4). The link may consist in a common forma- tion pathway or in one species being the precursor of the other. Fig. 7. Same as Fig. 6, but for individual molecules MF ( upper panel ), DE (middle panel ), and EC ( lower panel ). The evolutionary classiﬁca- tion is shown for the sources of our sample (different colours), while black symbols represent different interstellar sources taken from litera- ture for comparison (see Table F.1 for references). The black lines ﬁt the data of the sources included in this work. The ﬁrst scenario is indeed predicted by the theoretical model of Garrod & Herbst (2006) and Garrod et al. (2008), who propose a common formation route through surface chemistry on dust grains at low temperatures ( 50K), from the methoxy precursor CH 3O(see also Allen & Robinson 1977): CH 3O+HCO!CH 3OCHO; (MF) CH 3O+CH 3!CH 3OCH 3: (DE) A54, page 12 of 25	10.1051_0004-6361_202038212	page_0011
A. Coletta et al.: Evolution of COMs in star-forming regions Fig. 8. Average abundances relative to H2(with respective standard errors) of MF, DE, F, and EC (different colours), as a function of the evolutionary stage. Balucani et al. (2015) present instead a gas-phase route able to efﬁciently form MF from DE at low temperatures ( 10K) through reactions involving the radical CH 3OCH 2: CH 3OCH 3+F!CH 3OCH 2+HF; CH 3OCH 3+Cl!CH 3OCH 2+HCl; CH 3OCH 2+O!CH 3OCHO +H: In addition, the correlated FWHM of the lines (middle panel of Fig. 5), the similar overall range of excitation temperatures (Sect. 5.3), and the spatial coexistence derived from interfero- metric observations (e.g. Brouillet et al. 2013; Bøgelund et al. 2019; El-Abd et al. 2019) suggest that MF and DE could trace the same gas in various environments and evolutionary stages. However, also in the case of species for which a chemical link is not so clear (EC and MF, or EC and DE, showing slightly higher dispertion in molecular ratios, Fig. 3, bottom two panels) a clear abundance trend is observed. A potential link between these molecules may involve the methyl radical CH 3as a com- mon precursor. EC could indeed form through a sequence of gas-phase and grain-surface reactions mainly involving the CN andCH 3radicals (Garrod et al. 2017). We cannot exclude either the existence of a chemical link with formamide, consistent with the abundance correlations ( >0:9) found in Sect. 6.2.1, but the poor statistics obtained for this molecule prevents conclusive considerations, and needs to be improved by further targeted observations. Although the formation paths of formamide are still under debate (see e.g. Bisschop et al. 2007; Barone et al. 2015; Codella et al. 2017; Skouteris et al. 2017; Ligterink et al. 2018; Quénard et al. 2018; López-Sepulcre et al. 2019), recent works propose that it would form more efﬁciently on icy dust grains during the cold phases of star formation (Jones et al. 2011; López-Sepulcre et al. 2015; Fedoseev et al. 2016). It has to be noted, however, that abundance correlations between molecules do not necessarily imply the existence of a chemical link, as recently proved by Quénard et al. (2018) for formamide andHNCO (isocyanic acid), and conﬁrmed by Belloche et al. (2020) in a sample of hot corinos. These observational corre- lations seem to be a necessary but not sufﬁcient condition to claim a chemical link. Nevertheless, observations are needed to test models and understand how molecules are formed. This work shows, in fact, that between molecules whose chemistry isexpected to be related (such as MF and DE) the correlations are tighter. Furthermore, a clear trend of increasing molecular abun- dances with L=M(mainly governed by L) emerges for all species, spanning up to4orders of magnitude in abundance and 6in L=M, which implies also a trend with the evolutionary stage of the sources (Figs. 6–8). Besides suggesting potential individual links between the COMs, these results allow us to formulate a general, most likely scenario for their formation and evolution. The fact that the molecular ratios are nearly constant across the whole star forma- tion process and among different types of sources is particularly interesting, because the physical conditions in these environ- ments (especially in the case of MF/DE, Fig. 4) are different: pre-stellar cores, shock-dominated regions (protostellar shock and GC clouds), thermal-dominated regions (cores in low- to high-mass star-forming regions), and comets (whose chemical composition is thought to be presolar, see e.g. Rivilla et al. 2020). This seems to reveal a rather universal chemistry for COMs, mainly developed at the cold earliest stages of star for- mation and then essentially preserved through the evolution, being only marginally altered by the evolving physical condi- tions. In more detail, molecules may be formed in pre-stellar cores, possibly in gas phase or on the surface of dust grains, from which they can desorb thanks to non-thermal mechanisms such as cosmic rays (see e.g. Shingledecker et al. 2018; Bonfand et al. 2019; Willis et al. 2020). This would explain the detec- tion and the relative (low) abundances in the pre-stellar cores and the comets. The lack of molecular detections (at least at 2mm) among our 11 HMSCs may be due to the fact that they are tipically much more distant than the observed PCs (which can be resolved even with relatively low resolutions, see e.g. Jiménez-Serra et al. 2016), and thus more affected by beam dilution. Later on, in star-forming regions and GC molecular clouds, other mechanisms are able to massively (and more efﬁ- ciently) desorb the molecules formed on grains: thermal heating and shock-induced heating. This has the effect to signiﬁcantly increase the observed gas-phase molecular abundances and thus the expected number of detections. This scenario is consistent with the trend we ﬁnd between abundances and L=M(proxy for the evolutionary stage), as well as with the number of detections we report for each evolutionary group (Sect. 5.1). Moreover, while low luminosity sources (pre-stellar and hot corinos) are usually isolated (or at most binary) systems, high-mass star- forming regions are clustered environments. In these regions, the thermal and shock energy injected to the medium strongly increases with time due to the protostellar activity (heating and protostellar outﬂows). This produces more and more desorption, accordingly increasing the gas-phase abundances of COMs with evolution. Therefore, the proposed scenario supports the forma- tion of COMs on grain surfaces, indicating that the majority of COMs observed in star-forming regions could be produced by the desorption from icy grain mantles. However, it is still possi- ble that gas-phase formation pathways (see e.g. Balucani et al. 2015; Codella et al. 2017; Skouteris et al. 2019), though not expected to signiﬁcantly affect the molecular ratios (based on our results), could contribute to the abundance of COMs in cold regions. Moreover, our results suggest that O- and N-bearing COMs may behave similarly in star-forming regions at all stages, shar- ing the same physical conditions (or even direct chemical links) for their formation. This has been found also by Fontani et al. (2007) in hot cores, whereas other authors noticed differences between O- and N-bearing COMs in both the spatial distribution (e.g. Liu 2005; Csengeri et al. 2019) and the radial velocities A54, page 13 of 25	10.1051_0004-6361_202038212	page_0012
A&A 641, A54 (2020) (Blake et al. 1987). We also note that, given the increasing abun- dance trend, molecular destruction routes seem to be less efﬁ- cient than formation/desorption mechanisms, especially at later evolutionary stages (i.e. higher luminosities). However, destruc- tion routes represent a less investigated but non-negligible topic, as they can in principle affect the predicted molecular abun- dances (see e.g. Garrod 2013; Shingledecker et al. 2019; Ascenzi et al. 2019 and refs. therein). Lastly, we stress that the angular resolution of our data (Table 2) is larger than the size of the observed sources. Although this issue has been taken into account through the beam dilution factor applied in the line ﬁtting procedure (see Sect. 4), we are still not able to spatially resolve the inner struc- ture of the targets, which is often fragmented into multiple smaller objects in potentially diverse evolutionary stages. The observed emission could hence include contributions from both the inner hot core and its cooler outer envelope, preventing a clear distinction between nearby emission zones, and causing sometimes potentially misleading correlations among differently distributed molecules. High angular resolution interferometric observations would be able to conﬁrm more robustly the pro- posed scenario for the formation of COMs, as they can more accurately identify spatial correlations and resolve the poten- tial protostellar multiplicity within a region (see e.g. Murillo et al. 2018). Nevertheless, we do not ﬁnd relevant differences by comparing our results to interferometric data from literature, seemingly indicating that the observed chemistry is almost the same across different spatial scales within star-forming regions. 7. Summary and conclusions In this work we have analysed spectra at 3,2, and 0:9mm of 39 selected high-mass star-forming regions at different evolution- ary stages (HMSCs to UCHIIs) obtained with the IRAM-30m telescope, searching for rotational transitions of the complex O- bearing molecules CH 3OCHO (MF) and CH 3OCH 3(DE), and N-bearing molecules NH 2CHO (F) and C2H5CN(EC). We have reported molecular detections in 20 sources, performing a line ﬁtting procedure to derive the main physical parameters for each molecule. We summarise below the main results of this study: – The highest number of detections was reported in UCHII regions ( 45%, 9 out of 20 sources). DE was detected in 19 sources, while MF in 13, EC in 9, and F in 5. – We observe relevant discrepancies between the total molec- ular column densities obtained at different wavelengths (up to 2 orders of magnitude between 0:9and 3mm), so that in all sources N3(3mm)>N2(2mm)>N1(0:9mm)and N2=N1>N3=N2. This can be interpreted as an effect of the differential attenuation caused by dust opacity at each fre- quency (d/), proving that dust properties have indeed to be considered when dealing with young, tipically dust-rich star-forming regions at multiple wavelengths. Therefore, we chose the 2mm data for our analysis (being the band that reported the most detections) and found source-averaged col- umn densities ranging from 1015to1018cm2for MF, DE, and EC, and from 1014to1017cm2for F. – The derived abundances with respect to H2are1010107 for MF and DE, 10121010for F, and1011109 for EC. For all species we ﬁnd a consistent overall range of linewidths (210km s1) and excitation temperatures (20220K). – We ﬁnd very strong correlations ( r0:92) between the abundances of MF, DE, and EC, spanning 3orders of magnitude in abundance, uniformly covered by our sample.We have compared our results with heterogeneous sources from literature (including low-, intermediate- and high-mass star-forming regions, a protostellar shock region, pre-stellar cores and Galactic centre clouds), which conﬁrmed and expanded the correlations to 4orders of magnitude in abun- dance for all tracers. We also ﬁnd nearly constant molecular ratios with respect to source luminosity across all evolution- ary stages and among different types of sources, indicating that the chemistry of COMs is mainly developed at early stages and then preserved during the evolution, barely altered by the changing local physical conditions. These results may suggest a potential link between MF, DE, and EC, whereas for F, though consistent with correlations ( r>0:9), we can- not draw conclusions due to the poor statistics. In particular, we claim that MF and DE are most likely chemically linked, since they show the strongest correlation in most parame- ters (abundance, FWHM, and source size) and a remarkably constant ratio of1across a wide variety of sources at all evolutionary stages (also including comets), spanning a strik- ing9orders of magnitude in luminosity. The link may consist in a common formation pathway, such as from pre- cursor CH 3Oas predicted by Garrod & Herbst (2006) and Garrod et al. (2008), or in one species being the precursor of the other, as proposed by Balucani et al. (2015) with MF forming from DE. MF-EC and DE-EC may share CH 3as common precursor instead (see e.g. Beuther et al. 2007). Although observational correlations alone are not enough to prove a chemical link, this work shows that they are tighter among molecules whose chemistry is expected to be related (e.g. MF and DE). – We have also evaluated the variation of molecular abun- dances with the evolutionary stage of the source (traced by the luminosity/mass ratio) ﬁnding a clear increasing trend for all species over up to 6 orders of magnitude in L=M, ranging from pre-stellar cores and hot corinos to UCHIIs. – Based on correlations, molecular ratios and evolutionary trend, we propose a general scenario for the formation and evolution of COMs, which involves a prevalent formation at low temperatures in the earliest phases of star forma- tion (likely mainly on frozen dust grains) followed by a growing desorption powered by the progressive thermal and shock-induced heating of the core with evolution. This would explain the increasing observed gas-phase abundances and number of molecular detections. Moreover, these results sug- gest that O- and N-bearing COMs might have a similar behaviour in star-forming regions at all stages. Interestingly, this analysis also points out that molecular abundances might serve as evolutionary tracers within the whole star formation process. In conclusion, we stress that the physical parameters derived in our sample represent average values across the whole clumps, and could therefore include also contributions from outside the cores. Relevant improvements to this work will come from high angular resolution observations, able to resolve the inner struc- ture of these regions and hence to better locate the molecular emission, allowing to more accurately identify spatial correla- tions between COMs. In particular, interferometric observations of a large sample of star-forming regions in different evolution- ary stages, like the one studied in this work, will be able to conﬁrm and improve the proposed scenario for the formation and evolution of COMs. Acknowledgements. We thank the IRAM-30m staff for the precious help during the different observing runs. V.M.R. has received funding from the European A54, page 14 of 25	10.1051_0004-6361_202038212	page_0013
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A. Coletta et al.: Evolution of COMs in star-forming regions Appendix A: Sources without detections In this appendix we list the 19 sources of the initial sample of 39 (see Sect. 2) which did not report detections of the COMs analysed in this work (MF, DE, F, and EC). Table A.1. Observed sources of the original sample without detections of any of the COMs studied in this work (see Sect. 2). Source (J2000) (J2000) d Classiﬁcation References (h : m : s) (:0:00) (kpc) I00117-MM1 00 : 14 : 26.1 +64 : 28 : 44 1.8 HMPO (1, 2, 3, 4, 5, 6, 7, 8) I00117-MM2 00 : 14 : 26.3 +64 : 28 : 28 1.8 HMSC (1, 2, 3, 4, 5, 6, 7) AFGL5142-EC 05 : 30 : 48.7 +33 : 47 : 53 1.8 HMSC (1, 3, 4, 5, 6, 7, 9, 15) 05358-mm1 05 : 39 : 13.1 +35 : 45 : 51 1.8 HMPO (1, 3, 4, 5, 6, 7) 18264-1152M1 18 : 29 : 14.6 11 : 50 : 22 3.5 HMPO (8, 10, 11) G028-C3(MM11) 18 : 42 : 44.0 04 : 01 : 54 5.0 HMSC (3, 7) G028-C1(MM9) 18 : 42 : 46.9 04 : 04 : 08 5.0 HMSC (1, 2, 3, 7) G034-F2(MM7) 18 : 53 : 16.5 +01 : 26 : 10 3.7 HMSC (1, 2, 3, 7) G034-F1(MM8) 18 : 53 : 19.1 +01 : 26 : 53 3.7 HMSC (1, 2, 3, 7) G034-G2(MM2) 18 : 56 : 50.0 +01 : 23 : 08 2.9 HMSC (1, 2, 3, 7) I19035-VLA1 19 : 06 : 01.5 +06 : 46 : 35 2.2 UCHII (1, 2, 3, 4, 5, 6, 7) I20293-WC 20 : 31 : 10.7 +40 : 03 : 28 2.0 HMSC (1, 2, 3, 4, 5, 6, 7) I21307 21 : 32 : 30.6 +51 : 02 : 16 3.2 HMPO (1, 2, 3, 4, 5, 6, 7) I22134-B 22 : 15 : 05.8 +58 : 48 : 59 2.6 HMSC (1, 2, 3, 4, 5, 6, 7) I22134-VLA1 22 : 15 : 09.2 +58 : 49 : 08 2.6 UCHII (1, 2, 3, 4, 5, 6, 7) I22134-G 22 : 15 : 10.5 +58 : 48 : 59 2.6 HMSC (1, 2, 3, 4, 5, 6, 7) 22198+6336 22 : 21 : 26.8 +63 : 51 : 37 0.7 HMPO (8, 12, 13, 14) 23033+5951 23 : 05 : 24.6 +60 : 08 : 09 3.5 UCHII (1, 2, 3, 4, 5, 6, 7, 8) NGC7538-IRS9 23 : 14 : 01.8 +61 : 27 : 20 2.8 UCHII (1, 2, 3, 4, 5, 6, 7, 8, 15) References.(1)Fontani et al. (2011);(2)Fontani et al. (2014);(3)Fontani et al. (2015a);(4)Fontani et al. (2015b);(5)Fontani et al. (2016);(6)Fontani et al. (2018);(7)Colzi et al. (2018a);(8)Colzi et al. (2018b);(9)Mininni et al. (2018);(10)Fazal et al. (2008);(11)Leurini et al. (2007);(12)Jin et al. (2016); (13)Sánchez-Monge et al. (2010);(14)Fujisawa et al. (2014);(15)Fontani et al. (2019). A54, page 17 of 25	10.1051_0004-6361_202038212	page_0016
A&A 641, A54 (2020) Appendix B: Selected ﬁts In this appendix we show, for each molecule, selected transitions detected with the molecular line ﬁtting procedure (see Sect. 4) performed in different wavebands and sources. Fig. B.1. Selected transitions of MF detected in different wavebands and sources: ( a)0:9mm waveband, source 18089-1732; ( b)2mm, G31.41+0.31; ( c)3mm, G31.41+0.31. The LTE synthetic spectrum obtained in the line ﬁtting procedure with MADCUBA (see Sect. 4) is overplotted in red. See Tables D.1–D.3 for a list of the brightest lines detected for each molecule in each waveband and their spectroscopic parameters. Fig. B.2. Same as Fig. B.1, but for DE: ( a)0:9mm waveband, source W51; ( b)2mm, G10.47+0.03; ( c)3mm, G31.41+0.31. A54, page 18 of 25	10.1051_0004-6361_202038212	page_0017
A. Coletta et al.: Evolution of COMs in star-forming regions Fig. B.3. Same as Figs. B.1–B.2, but for F: ( a)0:9mm waveband, source W51; ( b)2mm, G31.41+0.31; ( c)3mm, G31.41+0.31. Fig. B.4. Same as Figs. B.1–B.3, but for EC: ( a)0:9mm waveband, source G10.47+0.03; ( b)2mm, G29.96-0.02; ( c)3mm, G31.41+0.31. Appendix C: Other physical parameters obtained from the ﬁts In this appendix we report the results for the physical parameters derived from the molecular line ﬁtting procedure (see Sect. 4) not included in Sect. 5. A54, page 19 of 25	10.1051_0004-6361_202038212	page_0018
A&A 641, A54 (2020) C.1. Excitation temperatures Table C.1 shows the excitation temperatures ( Tex, Sect. 5.3) obtained for each molecule in the different wavebands assuming LTE conditions.C.2. Systemic velocities Table C.2 reports the best-ﬁt LSR source velocities ( VLSR) obtained for each molecule in the 2mm waveband. Table C.1. Excitation temperatures of MF, DE, F, and EC, obtained from the ﬁts (see Sects. 4 and 5.3) in the three observed wavebands: T1(0:9mm), T2(2mm), and T3(3mm). Source Tex(MF) (K) Tex(DE) (K) Tex(F) (K) Tex(EC) (K) T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 05358-mm3 105 42 AFGL5142-MM 29 11 99 30 6713 18182-1433M1 88 13 18517+0437 85 21 18447 116 16 11320 I20293-MM1 134 19 I23385 83 40 18089-1732 191 8 16210 12838 8111 1073 674 10041 73 179 10 8730 G24.78+0.08 114 44 22024 17349 11014 895 7510 97 82 27 87 72 13 9429 G31.41+0.31 177 12 1658 15511 1064 972 845 93 11519 14333 12475 20316 22338 G75-core 28 5 63 29 3415 20126+4104M1 88 34 W3(OH) 188 47 1004 107 7 708 175 193 19 G5.89-0.39 87 14 29 4 G10.47+0.03 84 11 19512 159 9 14214 65 19 1376 G14.33-0.65 89 54 10053 122 8 81 42 G29.96-0.02 121 23 111 50 16821 131 13 G35.20-0.74 162 33 95 13 W51 133 5 1374 120 2 1113 882 83 44 1624 19410+2336 150 34 ON1 21 11 46 40 11114 Notes. Values without error come from ﬁts performed with the Texparameter ﬁxed. Here and in the following table, the horizontal black lines subdivide the sources according to their evolutionary classiﬁcation (see Table 1). Table C.2. LSR source velocities ( VLSR) obtained for each molecule in the 2mm waveband. Source VLSR(km s1) MF DE F EC AFGL5142-MM 2:50:12:30:1 18182-1433M1 59.1 18517+0437 44:10:4 44.0 I20293-MM1 6:30:3 I23385 49.8 18089-1732 32:60:1 32:70:1 32:40:3 33:70:1 G24.78+0.08 110.8 111:10:1 111:40:2 110.2 G31.41+0.31 97:30:1 97:50:1 97:40:1 97:20:1 20126+4104M1 4.0 G75-core 0:20:10:50:2 W3(OH) 47:90:146:80:2 47:60:1 G5.89-0.39 9:20:2 9 :80:4 G10.47+0.03 66:00:1 67:20:3 66 :80:1 G14.33-0.65 22.6 22:90:2 22.5 G29.96-0.02 97.7 97:60:2 97 :60:1 G35.20-0.74 32.3 32.2 W51 55:90:1 56:40:1 57 :90:1 ON1 13.0 11:90:3 Notes. Values without error come from ﬁts performed with the VLSRparameter ﬁxed. A54, page 20 of 25	10.1051_0004-6361_202038212	page_0019
A. Coletta et al.: Evolution of COMs in star-forming regions Appendix D: Detected molecular transitions In this appendix we list the most intense rotational transitions (considering all the sources) detected with MADCUBA (see Sect. 4) for each molecule in each waveband. Table D.1. Selection of the transitions which were detected for each molecule at 2mm and used for the ﬁts. Frequency Transition logAi j(a)Eup(b)Frequency Transition logAi j(a)Eup(b) (GHz) (s1) (K) (GHz) (s1) (K) MF DE 141.037702 12(2,11)–11(2,10) 4.396 47 141.832255 8(3,5)–8(2,6) 4.853 45 141.044354 12(2,11)–11(2,10) 4.396 47 141.835507 8(3,5)–8(2,6) 4.853 45 141.244026 11(3,8)–10(3,7) 4.412 46 143.020781 3(2,2)–2(1,1) 4.862 11 141.260421 11(3,8)–10(3,7) 4.412 46 143.163002 13(2,12)–13(1,13) 5.010 88 141.652995 11(2,9)–10(2,8) 4.392 43 143.599415 7(3,4)–7(2,5) 4.856 38 141.667012 11(2,9)–10(2,8) 4.392 43 143.602992 7(3,4)–7(2,5) 4.856 38 142.733524 13(1,13)–12(1,12) 4.368 49 143.606236 7(3,4)–7(2,5) 4.856 38 142.735139 13(1,13)–12(1,12) 4.368 49 144.858991 6(3,3)–6(2,4) 4.872 32 142.815476 13(0,13)–12(0,12) 4.368 49 144.862041 6(3,3)–6(2,4) 4.868 32 142.817021 13(0,13)–12(0,12) 4.367 49 145.547165 16(1,15)–16(0,16) 5.024 127 143.234201 12(1,11)–11(1,10) 4.374 47 145.680397 5(3,2)–5(2,3) 4.922 26 143.240505 12(1,11)–11(1,10) 4.374 47 145.682677 5(3,2)–5(2,3) 4.896 26 146.977678 12(3,10)–11(3,9) 4.356 52 146.166246 4(3,1)– 4(2,2) 5.100 22 146.988047 12(3,10)–11(3,9) 4.356 52 146.677951 4(3,2)–4(2,3) 5.096 22 148.028088 12(6,6)–11(6,5) 4.442 70 146.704743 3(2,1)–2(1,2) 4.856 11 148.039433 12(6,7)–11(6,6) 4.441 70 146.872547 5(3,3)–5(2,4) 4.914 26 148.040699 12(6,7)–11(6,6) 4.441 70 147.024902 7(1,7)–6(0,6) 4.719 26 148.045822 12(6,6)–11(6,5) 4.441 70 147.025599 7(1,7)–6(0,6) 4.719 26 148.516039 12(5,8)–11(5,7) 4.395 63 147.206816 6(3,4)–6(2,5) 4.855 32 148.545009 12(5,8)–11(5,7) 4.412 63 147.21074 6(3,4)–6(2,5) 4.852 32 148.614838 12(5,7)–11(5,6) 4.411 63 147.731365 7(3,5)–7(2,6) 4.828 38 148.664523 12(5,7)–11(5,6) 4.394 63 147.734969 7(3,5)–7(2,6) 4.827 38 148.79779 12(4,9)–11(4,8) 4.362 57 148.497096 8(3,6)–8(2,7) 4.807 45 148.805941 12(4,9)–11(4,8) 4.361 57 148.500397 8(3,6)–8(2,7) 4.807 45 151.950079 13(2,12)–12(2,11) 4.297 55 148.503843 8(3,6)–8(2,7) 4.807 45 151.956625 13(2,12)–12(2,11) 4.296 55 EC 153.350475 14(1,14)–13(1,13) 4.273 57 142.34633 16(2,15)–15(2,14) 3.624 63 153.352035 14(1,14)–13(1,13) 4.273 57 143.335284 16(8,8)–15(8,7) 3.733 130 153.397844 14(0,14)–13(0,13) 4.273 57 143.335284 16(8,9)–15(8,8) 3.733 130 153.399352 14(0,14)–13(0,13) 4.273 57 143.33771 16(7,10)–15(7,9) 3.701 113 153.512752 13(1,12)–12(1,11) 4.282 55 143.33771 16(7,9)–15(7,8) 3.701 113 153.518739 13(1,12)–12(1,11) 4.282 55 143.343925 16(9,7)–15(9,6) 3.774 148 153.553231 12(2,10)–11(2,9) 4.284 51 143.343925 16(9,8)–15(9,7) 3.774 148 153.56692 12(2,10)–11(2,9) 4.284 51 143.357203 16(6,11)–15(6,10) 3.674 98 F 143.357203 16(6,10)–15(6,9) 3.674 98 140.587141 12(1,11)–12(0,12) 5.162 85 143.360378 16(10,6)–15(10,5) 3.823 170 142.701325 7(1,7)–6(1,6) 3.694 30 143.360378 16(10,7)–15(10,6) 3.823 170 146.871475 7(0,7)–6(0,6) 3.649 28 143.382952 16(11,5)–15(11,4) 3.886 193 148.223143 7(2,6)–6(2,5) 3.673 40 143.382952 16(11,6)–15(11,5) 3.886 193 148.555852 7(6,2)–6(6,1) 4.209 136 143.406553 16(5,12)–15(5,11) 3.652 86 148.555852 7(6,1)–6(6,0) 4.209 136 143.407188 16(5,11)–15(5,10) 3.652 86 148.566822 7(5,3)–6(5,2) 3.943 103 143.410796 16(12,4)–15(12,3) 3.967 218 148.566823 7(5,2)–6(5,1) 3.943 103 143.410796 16(12,5)–15(12,4) 3.967 218 148.596177 9(0,9)–8(1,8) 5.114 45 143.443012 16(13,3)–15(13,2) 4.076 246 148.59897 7(4,4)–6(4,3) 3.804 76 143.443012 16(13,4)–15(13,3) 4.076 246 148.599354 7(4,3)–6(4,2) 3.804 76 143.50697 16(4,13)–15(4,12) 3.635 76 148.667301 7(3,5)–6(3,4) 3.720 55 143.5292 16(3,14)–15(3,13) 3.622 69 148.709018 7(3,4)–6(3,3) 3.720 55 143.53529 16(4,12)–15(4,11) 3.635 76 153.432176 7(1,6)–6(1,5) 3.600 32 144.10474 16(3,13)–15(3,12) 3.617 69 145.41801 16(1,15)–15(1,14) 3.592 61 146.12004 16(2,14)–15(2,13) 3.590 64 146.894524 17(1,17)–16(1,16) 3.578 65 147.756711 17(0,17)–16(0,16) 3.570 65 Notes. The spectral parameters are taken from the JPL catalogue for MF lines and the CDMS catalogue for DE, F, and EC lines. We show transitions with TMB>0:1K (for MF and EC) and TMB>0:3K (for DE); every detected transition of F is present instead.(a)Logarithmic Einstein coefﬁcient;(b)rotational upper level energy. A54, page 21 of 25	10.1051_0004-6361_202038212	page_0020
compounds that decrease AR expression have been reported to decrease AR by inhibiting its synthesis ( 76, 77). As a parallel strategy to rationally design a SARD, we appended the adamantyl moiety to the AR agonist RU59063 ( 70). Gratifyingly, we found that this addition switched the agonist into a pure antagonist capable of degrading AR protein (half-maximal degradation at 1 μM; maximal degradation of 95%). Moreover, this SARD was also able to inhibit proliferation of a model castration-resistant prostate cancer cell line resistant to enzalutamide. FUSION-BASED DEGRON TECHNOLOGIES SERDs, IMiDs, and specific PROTACs are interesting compounds for their ability to degrade disease-relevant proteins, but these tools must be custom designed and synthesized for each target protein. Thus, for a particular protein to be degraded, a sizeable synthetic effort must be made [e.g., the conjugation of the VHL ligand to a ligand of the targeted POI (if such a ligand is available)]. This latter issue is particularly problematic because many attractive proteins to be targeted using degradation strategies are currently undruggable targets for which no ligand exists. Fortunately, to address this need for model systems for controlling intracellular protein levels, the chemical biology community has developed more generalizable approaches to controlling protein levels using small molecules. Here, we review several techniques in which a small molecule recruits a ligand-mediated degron to an E3 ligase, summarized in Table 1. Auxin-Inducible Degron In plants, small-molecule auxin hormones control many different growth and cell cycle functions. The mysteries of this system were elucidated in the past 15 years when it was shown that auxins act as a molecular glue, similar to IMiDs, between CRL1Tir1 and indole-3-acetic acid (IAA)/auxin transcription factors ( 78, 79). This natural protein degradation system has been exploited to recruit exogenous substrates to an E3 ligase for ubiquitination and subsequent degradation. For example, the auxin-binding domain [a.k.a. auxin-inducible degron (AID)] has been fused to GFP and other POIs to induce the efficient and rapid degradation proteins from yeast and mammalian cells ( 80, 81). Although the system requires genetic engineering, the fusion proteins and small molecules used are bioorthogonal and interface well with endogenous machinery. The minimal AID domain must be fused to the POI but is small (44 amino acids) ( 82). Additionally, the Tir1 F- Box protein must also be expressed exogenously but interfaces with the endogenous CRL1 complex within cells. This is unlikely to cause inhibition of the endogenous degradation machinery. Moreover, the small molecule IAA is also thought to be inert in eukaryotic cells, although possible metabolic byproducts may be toxic. Overall, this system is bioorthogonal and enables rapid ( t1/2 = 20 min) depletion of the POI. Several studies have highlighted the usefulness of this system in addressing biological questions, such as in the study of centrosome formation in human cells ( 83, 84) and Bondeson and Crews Page 9 Annu Rev Pharmacol Toxicol . Author manuscript; available in PMC 2017 September 06. Author Manuscript Author Manuscript Author Manuscript Author Manuscript	cfa9da281941a329299bc535ce3bb85c2ee70727	page_0008
calcineurin function in the malarial parasite Plasmodium berghei (85). The use of CRISPR/ Cas9 to introduce AID into the endogenous genomic locus of the gene of interest for conditional protein depletion in Caenorhabditis elegans has proved to be particularly useful (86). Zhang et al. ( 86) first confirmed that the AID system is active in all development stages of the worm and that degradation is rapid: A 20-min half-life in the presence of auxin was observed. Furthermore, the authors compared RNAi-and auxin-mediated depletion and found that whereas RNAi demonstrated a particular phenotype (e.g., 2% progeny arrested in development), the auxin was capable of a much more pronounced phenotype (100% arrest). This indicates that despite RNAi’s limited ability to affect a phenotype owing to tissue distribution, cell penetrance or incomplete knockdown, the chemical genetic tool enables a more robust phenotype. Finally, the authors show the use of the AID system in the germ– line, a tissue understudied because of a complete lack of conditional protein degradation tools. The authors clearly demonstrate complete protein knockdown within 45 min and show that within 6 h of knockdown, meiotic nuclei are disfigured ( 86). These data more closely match knockout rather than knockdown phenotypes, an important corroboration of the chemical and genetic tools. PROTAC-Recruiting Degrons Our lab has used the modified bacterial dehalogenase HaloTag as a model system for induced protein degradation for several years, given the ease of generating chloroalkane- based HaloTag ligands synthetically. The hydrophobic tagging approach was first developed using the HaloTag system and was shown to induce efficiently the degradation of cytosolic as well as transmembrane HaloTag fusion proteins in cell culture and in vivo ( 87–89). The ability to induce selectively the degradation of a single HaloTag fusion protein proved valuable in the study of the unfolded protein response (UPR) of the endoplasmic reticulum. In this case, an endoplasmic reticulum–localized HaloTag fusion protein was unfolded upon addition of a hydrophobic tag, thus inducing the UPR ( 90). Moreover, because this unfolded protein represented only a small portion of total protein within the endoplasmic reticulum, the cell was able to resolve and adapt to the stressor. Thus, more specific transcriptional changes could be studied using the destabilized HaloTag compared to other globally acting agents typically used to study the UPR ( 90). We next sought to develop potent PROTACs able to degrade the more-stabilized HaloTag7. These compounds are similar to the VHL-based PROTACs presented above: The heterobifunctional molecule binds to the E3 Ligase VHL, while the chloroalkane simultaneously forms a covalent bond with the HaloTag receptor protein ( 91). From a panel of different compounds, we found that HaloPROTAC3 was the most efficacious. Interestingly, extending the linker by one PEG unit led to an approximately 20% reduction in activity, and shortening the linker by one PEG unit abrogated any activity completely. A similar HaloTag-based degradation system has also been presented that uses the E3 ligase IAP instead of VHL ( 92). Although this system is not as efficient, the authors show that nuclear proteins can also be degraded. To aid in the use of HaloTag, GeneCopoeia has made available 20,000 human and 15,000 mouse open reading frames fused to HaloTag7 ( http:// www.genecopoeia.com/tech/halo-tag/ ).Bondeson and Crews Page 10 Annu Rev Pharmacol Toxicol . Author manuscript; available in PMC 2017 September 06. Author Manuscript Author Manuscript Author Manuscript Author Manuscript	cfa9da281941a329299bc535ce3bb85c2ee70727	page_0009
A final system based on PROTACs and a fusion protein-binding domain was highlighted alongside the cereblon-based PROTACs against BRD4 (see above). This system uses a FK506 binding protein 12 (FKBP12) fusion protein and its high-affinity ligand conjugated to Thalidomide. This compound was also able to degrade free FKBP12 with high potency (63). Small-Molecule Modulation of Protein Activity Two other techniques requiring genetic manipulation allow control over protein levels but are more nuanced in their mechanism of action. The first, small molecule–assisted shutoff (SMASH), produces native protein in the absence of compound ( 93). Upon compound binding, however, a tag is prevented from self-excising, which exposes a hydrophobic degron, thus causing the protein to be degraded quickly. Therefore, compound addition does not degrade the protein but inhibits its processing into a functional protein. This limits the technique to instances in which a protein is short-lived (and so protein produced prior to compound addition does not interfere with the experiment) or in which removal of drug and accumulation of protein can suffice. Another report used this technology to boost the statistical power of a dual-reporter screen by tuning the expression of one of those reporters using SMASH ( 94). The second system is a modification of the popular Shield-1 (Shld-1) drug-on technology. This system makes used of a destabilized mutant of FKBP12 that is stabilized in the presence of the compound Shld-1 and has proved useful in controlling fusion protein stability in parasites ( 95), worms ( 96), and medaka ( 97) as well as to potassium channel biology ( 98) and the cytosolic UPR in mammalian cells ( 99). Several technological advancements are worth noting: By combining Shld-1 stabilization with induced dimerization of a split-ubiquitin system, release of the native protein is achieved in response to a small molecule ( 100). In addition, this system can be further combined with Tet-On transcriptional control to produce a 130-fold dynamic range of POI levels ( 101). OUTLOOK Comparison of the Technologies: What Tool Should One Use? As outlined in this review, there are several options for controlling intracellular protein levels using a small-molecule approach, each with its own advantages and disadvantages. The choice of a particular protein degradation system depends on the specific biological problem to be addressed and the properties of the desired protein degrader compound. Currently, PROTACs provide the best means of designing and generating a modular compound that can degrade a POI directly without fusing it to a degron. Although a powerful protein degradation system, PROTAC generation requires a known ligand for a POI. As discussed above, important considerations such as linker geometry, attachment point, and choice of the particular E3 ligase engaged must be considered. Typically, a panel of different PROTACs should be developed and then assayed for their ability to degrade the POI selectively and potently.Bondeson and Crews Page 11 Annu Rev Pharmacol Toxicol . Author manuscript; available in PMC 2017 September 06. Author Manuscript Author Manuscript Author Manuscript Author Manuscript	cfa9da281941a329299bc535ce3bb85c2ee70727	page_0010
If no known ligand for the POI exists, then small molecule–based protein degradation systems using protein fusions are available. Because the POI is likely available as a fusion to the HaloTag protein, HaloPROTACs can provide a robust tool for specific degradation. Alternatively, AID also provides very rapid degradation of the POI, allowing for more granular temporal questions to be answered, and has been demonstrated to be efficacious in vivo. In those cases for which genetic manipulation of the POI is unacceptable and no known ligand is available, modern screening technologies can help develop a high-affinity ligand for the POI. Differential scanning fluorimetry (a.k.a. the thermal shift assay) ( 102), NMR- based fragment screens ( 103), competitive binding screens when a weak ligand is available (69), and affinity-based selection are all popular techniques ( 104). Other techniques to identify cryptic binding sites may also identify novel pockets on protein surfaces that could be targeted by degradation technologies ( 105, 106). What Do the Next Several Years Hold for Targeted Protein Degradation? The past two years have seen an exciting number of novel strategies to target a particular protein for degradation. Importantly, these technologies have expanded beyond the academic chemical biology arena: IMiDs are already FDA approved, and PROTAC-based clinical candidates are currently being developed. Given the fast pace of this field, the next several years will surely be an exciting time for those interested in small-molecule control of intracellular protein levels. The IMiD class of degraders offers many opportunities for new discoveries in this field; undoubtedly, novel protein substrates that can be degraded through structurally diverse IMiDs will be identified. Furthermore, given the recent discovery of the binding motif of CK1α and Ikaros to the cereblon-IMiD complex, it will be exciting to see whether rational substrate design can be performed to either maximize the efficacy of existing IMiDs or create new IMiDs with novel activities. However, given the challenges of stabilizing protein- protein interactions ( 107), this rational approach to IMiD design may prove difficult. The PROTAC strategy has the advantages of modularity, potency, and in vivo efficacy and will likely be the system of choice for those interested in exploring the undruggable proteome via small-molecule modulation of protein levels. Given the hundreds of E3 ligases in the cell, as ligands to ligases are identified, they will likely be incorporated into novel PROTAC degraders, thus expanding the rapidly growing number of known E3 ligases that can be hijacked to induce targeted protein degradation ( 108–112). Overall, targeted protein degradation offers strategies for asking and answering new biological questions as well as targeting functions of hitherto undruggable proteins. Acknowledgments C.M.C. gratefully acknowledges the US National Institutes of Health for their support (R35-CA197589). Glossary POI protein of interestBondeson and Crews Page 12 Annu Rev Pharmacol Toxicol . Author manuscript; available in PMC 2017 September 06. Author Manuscript Author Manuscript Author Manuscript Author Manuscript	cfa9da281941a329299bc535ce3bb85c2ee70727	page_0011
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Figure 1. Compounds that degrade their respective targets without requiring any genetic manipulation. The substrate binding portion is highlighted in yellow, whereas purple dictates parts of the compound that induce target protein degradation. ( a) ARN-810, a bioavailable SERD that causes degradation upon rearrangement of hydrophobic portions of ER α by the vinyl carboxylic acid. Minor changes at the carboxylic acid position can create ER α agonists. ( b) Lenalidomide, an immunomodulatory compound that causes degradation of Ikaros and CK1α by binding to the E3 ligase cereblon and creating a novel surface for their interaction. Minor structural changes in the compound abrogate CK1 α binding but maintain Ikaros binding. ( c) A PROTAC induces degradation of RIPK2 by recruiting it to the E3 ligase VHL. The two binding motifs are separated by the linker, allowing enhanced modularity. Abbreviations: CK1 α, casein kinase 1 α; ERα, estrogen receptor α; PROTAC, proteolysis targeting chimera; RIPK2, receptor interacting serine/threonine kinase 2; SERD, selective estrogen receptor downregulator; SERM, selective estrogen receptor modulator; VHL, von Hippel–Lindau.Bondeson and Crews Page 20 Annu Rev Pharmacol Toxicol . Author manuscript; available in PMC 2017 September 06. Author Manuscript Author Manuscript Author Manuscript Author Manuscript	cfa9da281941a329299bc535ce3bb85c2ee70727	page_0019
Author Manuscript Author Manuscript Author Manuscript Author ManuscriptBondeson and Crews Page 21Table 1 Summary of small-molecule-inducible degrons Fusion tag Compounds N or C terminal? Mass of tag DC50aDMaxbt1/2cReference(s) HaloTag HaloPROTAC3 Both 37 kDa 19 nM 90% 4–8 h 91 HaloTag Bestatin 1b Both 37 kDa 1 μM 39% 1 h 92 SMASHd Asunaprevir C terminal NA 1 nM 100% NA 93–94 AIDe IAA Both 5 kDa 50 μM 100% 20 min 80–86 FKBP12 dFKBP12 Both 13 kDa 10 nM 95% ND 63 Abbreviations: AID, auxin-inducible degron; FKBP12, FK506 binding protein 12; IAA, indole-3-acetic acid; NA, not applicable; ND, not determined; SMASH, small molecule–assisted shutoff.aDC50, concentration to achieve half-maximal degradation; does not correspond to 50% protein remaining. bDMax , maximal percentage of protein degraded; 100% indicates no protein remaining. ct1/2, half-life of degradation; time at which half-maximal degradation is observed. dSMASH is autocleaving and thus not present in the expressed protein.eAID also requires coexpression of the Tir1 F-Box protein. Annu Rev Pharmacol Toxicol . Author manuscript; available in PMC 2017 September 06.	cfa9da281941a329299bc535ce3bb85c2ee70727	page_0020
letters to nature 970 NATURE \|VOL 406 \|31 AUGUST 2000 \|www.nature.comindication that blinking does not occur on the timescales of interest for photon antibunching. Prior measurements on single QD photoluminescence and absorption have demonstrated the existence of discrete QD resonances8. However, just as the observation of discrete absorption lines in an atomic vapour cannot be taken as an evidence that the observed system consists of a single atom, these experiments, at least in principle, cannot rule out the existence of several QDs. In contrast, photon correlation measurements, such as the one reported here, provide a reliable method for deciding whether or not the observed system is a single anharmonic quantum emitter. In addition, owing to interactions with the lattice, a nanostructure that acts like an anharmonic emitter at cryogenic temperatures can be indistinguishable from a higher-dimensional system at room tem- perature. We have demonstrated that a CdSe quantum dot behaves as an anharmonic emitter even at room temperature. Our results constitute a ®rst step in the study of quantum optical phenomena in semiconductor QDs. Demonstration of quantum dynamics in semiconductors at room temperature could lead to applications in quantum information processing and computation. M Methods Sample preparation The CdSe/ZnS (core/shell) quantum dots were synthesized following high-temperature organometallic methods described in the literature11±13. The resulting nanoparticles were capped with the organic ligand trioctylphosphine oxide (TOPO) and had a distributionwith an average diameter of 4.1 nm and a r.m.s. (root mean square) width of 0.33 nm (8% size distribution). The single QD samples were prepared by spin-casting 30 ml of a 0.5 nM solution of the QDs dissolved in hexanes onto a bare glass coverslip, which resulted in a mean separation of the QDs of approximately 1 mm. Experimental set-up Optical pumping was performed using circularly polarized light of the 488-nm line of a continuous-wave Ar+laser, generating electron±hole pairs in the excited states of the CdSe quantum dots. The exciting light was focused by a high numerical aperture (NA is 1.3)oil-immersion objective to a near-diffraction-limited (full-width at half-maximum (FWHM)) spot approximately 300 nm in diameter at the glass±sample interface. In order to minimize the generation of two electron±hole pairs simultaneously we used a lowexcitation intensity of about 250 W cm -2. Typically, a QD was excited approximately every 1±10 ms (ref. 14), whereas the photoluminescence decay time is of the order of 30 ns. Thus the probability of generating two electron±hole pairs was small. The photoluminescence from the QD was collected by the same objective and ®rst passed through the excitation laser beam splitter and then through a holographic notch ®lter to block scattered laserlight. The light was then split with a 50/50 non-polarizing beam splitter and the resulting two photon beams were focused onto the active areas of two single-photon-counting avalanche photodiodes (SPAD). Measurement Photoluminescence images of the nanocrystals were obtained by scanning the QD-covered glass plate through the laser focus and recording the number of counts with one of the SPADs. To measure the photon statistics of a selected QD (or a cluster of QDs), the glassplate was positioned where a single bright spot of typically 300 nm in diameter (resolution limited) was observed in the photoluminescence image. The number of pairs of photons n(t) with arrival-time separations of twas measured using the two SPADs for t<t max200 ns. The pulses from the two SPADs were used to start and stop a time-to- amplitude converter (TAC) where the time delay between the start and stop pulses (to within tres) was converted to a voltage amplitude. The SPADs exhibited the same countingrate. An electronic delay (53 ns) was introduced in the stop channel in order to check the symmetry of the n(t) signal and to avoid the effect of noise for small voltages in the TAC. The output pulses were fed into a multichannel analyser. To reduce the background contribution and therefore decrease the amount of uncorrelated light in the n(t) measurement, the multichannel analyser was enabled only during the `on' periods, that is, when the signal level was above a certain threshold. Received 17 April; accepted 3 July 2000. 1. Walls, D. F. & Milburn, G. J. Quantum Optics (Springer, Berlin, 1994). 2. Hanbury-Brown, R. & Twiss, R. Q. Correlation between photons in two coherent beams of light. Nature 177, 27±29 (1956). 3. Kimble, H. J., Dagenais, M. & Mandel, L. Photon antibunching in resonance ¯uorescence. Phys. Rev. Lett. 39,691±694 (1977). 4. Diedrich, F. & Walther, H. Nonclassical radiation of a single stored ion. Phys. Rev. Lett. 58,203±206 (1987). 5. Basche Â, Th., Moerner, W. E., Orrit, M. & Talon, H. Photon antibunching in the ¯uorescence of a single dye molecule trapped in a solid. Phys. Rev. Lett. 69,1516±1519 (1992). 6. Fleury, L., Segura, J.-M., Zumofen, G., Hecht, B., Wild, U. P . Nonclassical photon statistics in single- molecule ¯uorescence at room temperature. Phys. Rev. Lett. 84,1148±1151 (2000). 7. Nirmal, M. et al. Fluorescence intermittency in single cadmium selenide nanocrystals. Nature 383, 802±804 (1996). 8. Empedocles, S. & Bawendi, M. G. Spectroscopy of single CdSe nanocrystallites. Acc. Chem. Res. 32, 389±396 (1999). 9. Klimov, V . I. & McBranch, D. W. Femtosecond 1P-to-1S electron relaxation in strongly con®ned semiconductor nanocrystals. Phys. Rev. Lett. 98,4028±4031 (1998). 10. Efros, Al. L. & Rosen, M. Random telegraph signal in the photoluminescence intensity of a single quantum dot. Phys. Rev. Lett. 78,1110±1113 (1997). 11. Murray, C. B., Norris, D. J. & Bawendi, M. G. Synthesis and characterization of nearly monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites. J. Am. Chem. Soc. 115, 8706±8715 (1993). 12. Hines, M. A. & Guyot-Sionnest, P . Synthesis and characterization of strongly luminescing ZnS-capped CdSe nanocrystals. J. Phys. Chem. 100, 468±571 (1996). 13. Dabbousi, B. O. et al. (CdSe)ZuS core shell quantum dots: synthesis and characterization of a size series of highly luminescent nanocrystallites. J. Phys. Chem. 101, 9463±9475 (1997). 14. Nirmal, M. & Brus, L. Luminescence photophysics in semiconductor nanocrystals. Acc. Chem. Res. 32, 407±414 (1999). Acknowledgements This work is supported by the David and Lucile Packard Fellowship (A.I. and S.K.B.). P .M. acknowledges the ®nancial support of the Max Kade Foundation. Correspondence and requests for materials should be addressed to A.I. (e-mail: atac@ece.ucsb.edu).01234567891.01.11.21.31.41.51.6CdSe/ZnS single quantum dotIntensity autocorrelation G(2) (t) Time, t (s) Figure 4 Intensity autocorrelation G(2)(t) obtained for the time trace shown in Fig. 3. ................................................................. Organoplatinum crystals for gas-triggered switches Martin Albrecht , Martin Lutz ², Anthony L. Spek ²& Gerard van Koten ³ *Debye Institute, Department of Metal-Mediated Synthesis & ²Bijvoet Center for Biomolecular Research, Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands ............................................................................................................................... ............... Considerable effort is being devoted to the fabrication of nano- scale devices1. Molecular machines, motors and switches have been made, generally operating in solution2±7, but for most device applications (such as electronics and opto-electronics), a maximal degree of order and regularity is required8. Crystalline materials would be excellent systems for these purposes, as crystals com- prise a vast number of self-assembled molecules, with a perfectly ordered three-dimensional structure9. In non-porous crystals, however, the molecules are densely packed and any change in them (due, for example, to a reaction) is likely to destroy the crystal and its properties. Here we report the controlled and fully reversible crystalline-state reaction of gaseous SO 2with non- porous crystalline materials consisting of organoplatinum mole- cules. This process, including repetitive expansion±reduction sequences (on gas uptake and release) of the crystal lattice,modi®es the structures of these molecules without affecting their crystallinity. The process is based on the incorporation of SO 2into the colourless crystals and its subsequent liberation from © 2000 Macmillan Ma gazines Ltd	5a62ad4f00e46cc072cc6f5b2c0e14fa1207aba4	page_0000
letters to nature NATURE \|VOL 406 \|31 AUGUST 2000 \|www.nature.com 971the orange adducts by reversible bond formation and cleavage10. W e therefore expect that these crystalline materials will ®nd applications for gas storage devices and as opto-electronic switches11,12. Chemical transformations that occur in crystalline material, so- called `crystalline-state reactions'13, are very rare because most chemical reactions cause considerable stress and intermolecular reorganization. Hence, the loss of crystallinity is a typical event. Among the best explored crystalline-state reactions are (reversible) photochemical isomerization processes, that is, unimolecular, light- induced transformations in the crystal14±16. Processes in the crystal- line phase that involve substrate binding and release, and thus a change in the overall atom content within the unit cell, have beenOH 12Me2NPt NMe 2Cl + SO 2 SO2 OHMe2NPt NMe 2ClSO2 Figure 1 Reversible adsorption of SO 2by organoplatinum( II) species containing the N,C,N9 terdentate coordinating, monoanionic `pincer' ligand. Figure 2 Molecular structures and hydrogen bonding motifs of organoplatinum complexes 1and2.a,b, The molecular structures were determined by single crystal X-ray diffraction and demonstrate the change in the geometry around platinum upon coordination of SO 2: distorted square-planar in 1(a) versus square-pyramidal in the adduct 2(b).c,d, Thea-type supramolecular connectivity pattern, that is, the Pt±Cl ¼H±O hydrogen bonding, is similarly present in 1(c) and in 2(d). Theb-type network mediated by Pt ¼S¼Cl interactions is only found in 2. All hydrogen atoms except the phenolic O±H hydrogen have been omitted for clarity. Elements are colour-coded: platinum (purple), nitrogen (blue), chlorine (green), oxygen (red), sulphur (yellow). X-ray crystallographic details for the single crystal structure determination for 1: intensities were measured at 150 K using graphite-monochromated Mo K aradiation ( l= 0.71073 A Ê). 2,936 independent re¯ections (6,631 total measured) were analysed by using automated Patterson methods. Structure re®nement on F2structure factors converged with a discrepancy factor R1= 0.0376 and a weighted discrepancy factor w R2= 0.0793(goodness of ®t = 1.024). The oxygen-bound hydrogen was located in the difference Fourier map and allowed to re®ne freely. Residual electron density was only found within 0.84 and -1.08 e A Ê-3. An identical experimental set-up was used for the analysis of the adduct 2. The structure was solved by using automated Patterson methods on 2,166 independent re¯ections (11,626 total measured). Structure re®nement on F2converged atR1= 0.0350 and w R2= 0.0558 (goodness of ®t = 1.054). The oxygen-bound hydrogen was located in the difference Fourier map and re®ned with a rotating model. Residual electron density was only found within 0.97 and -1.01 e A Ê-3(for further details, see refs 10 and 24, respectively). Selected bond distances (in A Ê) and angles (in degrees) around platinum for 1: Pt±C, 1.915(9); Pt±Cl, 2.434(2); Pt±N1, 2.094(8); Pt±N2, 2.082(8); N1±Pt±N2, 163.9(3); C±Pt±Cl, 177.4(4); and for 2: Pt±C, 1.923(10); Pt±Cl, 2.423(3); Pt±N1, 2.106(8); Pt±N2, 2.096(8); N1±Pt±N2, 160.5(3); C±Pt±Cl, 173.1(3). (N1 and N2 are the respective nitrogen atoms, coloured blue). Table 1 Selected crystallographic parameters of 1and2 Complex Space group (number)Unit cell dimensionsDensity, r (g cm-3)Packing index (%) a(AÊ) b(AÊ) c(AÊ) V(AÊ3)............................................................................................................................... ............................................................................................................................... ..................................................................................................... 1(ref. 24) Pna2 1(33) 24.2238(14) 10.1986(8) 5.4483(14) 1,346.0(4) 2.1606(6) 72.2 2(ref. 10) Pna2 1(33) 16.837(3) 10.2189(16) 9.0231(10) 1,552.5(4) 2.1473(6) 70.9............................................................................................................................... ............................................................................................................................... ..................................................................................................... © 2000 Macmillan Ma gazines Ltd	5a62ad4f00e46cc072cc6f5b2c0e14fa1207aba4	page_0001
letters to nature 972 NATURE \|VOL 406 \|31 AUGUST 2000 \|www.nature.comrestricted to enzymes and other materials with an `open' (that is, porous)17structure with large cavities (for example, zeolites)18±20.A versatile way of overcoming this problem relies on crystal engineering21, whose intention is the design and processing of supramolecular properties in solids22. Our approach towards the property-directed synthesis of new and well-de®ned materials involves the use of the N,C,N 9 terdentate coordinating, monoanionic `pincer' ligand system[C 6H2(CH 2NMe 2)2-2,6-R-4], abbreviated as NCN-R, where R is a functional group23. When complexed to four-coordinated metal centres (for example, Pt( II)), the fourth site is forced to occupy a trans position to the metal±carbon bond, due to the chelating binding mode of the pincer ligand (Fig. 1). In addition, modi®ca- tion of the aryl ring of the ligand allows for the introduction of functional groups such as an acceptor or donor site. Using this method, the organoplatinum complex [PtCl(NCN-OH)] ( 1) was prepared recently24; it contains a metal-bound chloride as a hydro- gen bond acceptor and a phenolic hydroxide group on the pincer ligand as a hydrogen donor. With this supramolecular synthon25, the organoplatinum complex self-assembles in the solid state to form an a-type network via intermolecular Pt±Cl ¼H±O (hydrogen) bonds (Fig. 2). When crystalline 1is exposed to an atmosphere of SO 2, adsorption of this gas by the organoplatinum sites is indicated by a dramatic colour change of the material from colourless to deep orange, a colour change previously also observed in solution10,26. This adduct formation modi®es the geometry around platinum and was therefore expected to destroy the crystallinity. Surprisingly however, exposure of crystalline 1to SO 2gas (about 1 minute exposure time) gives the correspond- ing adduct [PtCl(NCN-OH)(SO 2)], 2(Fig. 1), in a quantita- tive, crystalline-state reaction. Unequivocal evidence for this process has been obtained by X-ray powder diffraction andsolid-state infrared spectroscopy. Similarly, the reverse reaction in an SO 2-free environment leads to the complete regeneration of crystalline 1. The structural features of 1and 2are known from single crystal analyses taken on crystals that were grown from saturated solutions (Fig. 2a, b)10,24. On absorption of SO 2, the molecular structure is changed predominantly around the platinum atom. The metal centre is no longer of square-planar but of approximate square-pyramidal geometry. This has an in¯uence on the packing index and the density, which decrease, and on the unit cell volume, which is expanded by more than 15% after absorption of SO 2(Table 1). Intermolecular Pt±Cl ¼H±O hydrogen bonding in 1results in the formation of an a-type network (Fig. 2c). Tight ®xation of SO 2in2 occurs by a Pt ¼S(O) 2¼Cl bond formation perpendicular to the hydrogen bond network27, which results in a unusual b-type net- work (Fig. 2d). The distances between hydrogen bond donors and acceptors (H ¼Cl is 2.33(9) A Êin2and 2.32(13) A Êin1;O¼Cl is 3.127(8) A Êin2and 3.126(8) A Êin1) as well as the bond angles (O±H ¼Cl is 165(8) 8in 2and 161(15) 8in 1)a r e statistically identical. When SO 2-free organoplatinum crystals are subjected to an environment of SO 2for a few seconds, only partial binding of the substrate is observed and a mixture of the two crystalline com- pounds 1and 2is obtained. The precise composition of this mixture is strongly dependent on the exposure time and the sample preparation. However, the powder diffraction diagram of the mixture can be used to access the ratio of 1and 2because their patterns are very different. Therefore, the comparison of the measured powder diagram with those simulated from the single crystal structures affords a good estimation of the ratio between 1 and 2. The reversibility and kinetics of these crystalline-state reactions have been elucidated by time-resolved X-ray powder diffraction28 and infrared spectroscopy. By overlapping of the time-resolvedinfrared spectra, various isosbestic points have been identi®ed which strongly suggest a direct transformation of 1into 2and vice versa. We examined the stretching vibrations of bound SO 2 (ns= 1,072 and nas= 1,236 cm-1). The (dis)appearance of these 1.0 0.8 0.6 0.4 0.2 50 0 100 1501.0 0.8 0.6 0.4 0.2 51 5 2 0Time (h) Time (s)off on off on off on 10Relative absorbance Figure 3 `On/off' switching between crystalline 1and2. Switching was monitored by infrared spectroscopy in an environmental DRIFT (diffuse re¯ectance infrared Fourier transform) chamber. The vibration of platinum-bound SO 2(ns= 1,072 cm-1) is diagnostic for the position of the switch. Spectra were recorded at 293 K every 20 seconds (average of 4 scans) under a continuous ¯ow of SO 2(crystalline-state reaction giving 2) and N 2 (giving 1), respectively. Repetitive switching `on' and `off' does not reduce the amplitude.8 1 01 21 41 61 82 02 250100150200250 2q (degrees) Intensity at 2q = 13.35° Time (min)Intensity0 200 4002060100 Figure 4 Time-resolved X-ray powder diffraction analysis of the transformation of crystalline 2to1in an atmosphere of air. 2was mounted in an open glass capillary on an Nonius Kappa charge-coupled device (CCD) diffractometer with Cu K aradiation, graphite monochromator, l= 1.5418 A Êat a temperature of 293 K. Scans were performed every hour with a scan time of 6 minutes. After background subtraction, the characteristic intensities at 2 v= 11.33 (diagnostic for 1) and 13.35 (for 2) have been related to the reaction time. Inset, correlation exceeds 0.996 for a linear approximation. © 2000 Macmillan Ma gazines Ltd	5a62ad4f00e46cc072cc6f5b2c0e14fa1207aba4	page_0002
letters to nature NATURE \|VOL 406 \|31 AUGUST 2000 \|www.nature.com 973signals is dependent on the atmospheric constitution and thus demonstrates the potential of this material as a crystalline switch (Fig. 3). The gas ®xation process of crystalline 1(`off' position) in a steady atmosphere of SO 2is best approximated by a pseudo ®rst- order rate law in organoplatinum species (that is, rate = k[Pt] where [Pt] is the concentration of 1; Fig. 3), whereas a linear decrease of the characteristic absorption bands is noted when 2(`on' position) is exposed to an inert gas. (In fact, kis (to our knowledge) the ®rst reliable rate constant measured for the diffusion of a gas through a crystalline material. It is strongly temperature dependent: at a given particle size, the gas release is accelerated approximately ®vefold when the temperature is raised from ambient (293 K) to 333 K and enormously retarded at low temperature (253 K, about 50 times slower). Preliminary measurements suggest that the gas desorption mechanism at higher temperatures may be different from that operating at ambient temperatures.) This suggests that in the rate-determining step of substrate binding, single platinum sites are involved. Although absorption of SO 2on molecules which are located at the surface of the crystalline material is assumed to be facile, the transport of the gas into the interior of the crystal to neighbouring molecules is probably less favoured. This can be explained by considering competitive release of SO 2back to the atmosphere. More importantly, incorporation of the substrate into the inner sphere is considerably complicated as an expansion of the crystal lattice occurs, involving, for example, reorganization of the relative position of the a-type networks (Fig. 2). This implies that building blocks containing bound SO 2must in¯uence their sur- rounding substrate-free neighbours in such a way that space is available for the gas molecule to move inside the densely packed supermolecule. Using powder diffraction techniques, gradual transformation of 2to1without formation of an intermediate has been observed, and the decrease of the intensity peaks of crystalline adduct 2is accompanied by a proportional increase of the intensities assigned to1(see above, Fig. 4). Analysis of the characteristic intensities at low 2vvalues strongly suggests a linear relationship between the change of the intensities and the elapsed time (see inset to Fig. 4), thus corroborating the results obtained from infrared spectroscopy (see above). Hence, a zero-order rate law is deduced for the release of SO 2gas from crystalline 2. The exact reaction rate is strongly dependent on the surface area of the sample (for example, particlesize). Moreover, in a thin capillary, the desorption reaction of SO 2 from 2proceeds considerably more slowly than in an open cup, but is still linear with time. This result emphasises the importance of area and the properties of the surface with respect to exchange withthe local environment. Additionally, single crystals of 2lose SO 2 primarily at the solid±gas interphase, resulting in the observation that a colourless zone `grows' from the outside to the inside of the crystal block (Fig. 5). The desorption of SO 2at the surface is assumed to be relatively fast and therefore the rate-determining step is presumably the diffusion-controlled transportation of the gaseous molecule from one metal centre to another, that is, the transfer of SO 2from the core to the surface of the crystal. The full reversibility of the crystalline-state absorption and desorption reaction is demonstrated by exposing a sample of regenerated 1to atmospheres of air and SO 2alternately. No loss in signal intensities, for example, owing to the formation of amorphous powder, is observed even after several repeated cycles. When single crystals of 2are exposed to air, 1is obtained as a colourless crystalline powder that is no longer suitable for a full single crystal X-ray analysis. These results provide access to the preparation and engineering of ef®cient crystalline switches with unusual properties. The `on/off' position of this switch may be de®ned, for example, by the crystal size and properties, or by the diagnostic colour of these materials. Most importantly, such switches are triggered primarily by their gaseous environment and may also be regulated by temperature. Owing to the insensitivity of the present organoplatinum systems towards light or oxidation13±16, these materials have high potential for applications as gas sensors29and as photochemical switches in opto-electronic devices in, for example, data processing or non- linear optical technology. M Received 19 April; accepted 21 June 2000. 1. Fox, M. A. Fundamentals in the design of molecular electronic devices: long-range charge carrier transport and electronic coupling. Acc. Chem. Res. 32,201±207 (1999). 2. Kelly, T. R., De Silva, H. & Silva, R. A. Unidirectional rotary motion in a molecular system. Nature 401, 150±152 (1999). 3. Koumura, N., Zijlstra, R. W. J., van Delden, R. A., Harada, N. & Feringa, B. L. Light-driven monodirectional molecular rotor. Nature 401, 152±155 (1999). 4. Balzani, V ., Go Âmez-Lo Âpez, M. & Stoddart, J. F. Molecular machines. Acc. Chem. Res. 31,405±414 (1998). 5. Sauvage, J. P . Transition metal-containing rotaxanes and catenanes in motion: toward molecular machines and motors. Acc. Chem. Res. 31,611±619 (1998). 6. Fabbrizzi, L., Licchelli, M. & Pallavicini, P . Transition metals as switches. Acc. Chem. Res. 32,846±853 (1999). 7. Collier, C. P . et al. Electronically con®gurable molecular-based logic gates. Science 285, 391±394 (1999). 8. Liu, C. -Y. & Bard, A. J. Optoelectronic properties and memories based on organic single-crystal thin ®lms. Acc. Chem. Res. 32,235±245 (1999). 9. Dunitz, J. D. in Perspectives in Supramolecular Chemistry. The Crystal as a Supramolecular Entity (ed. Desiraju, G. R.) 7 (Wiley, Chichester, 1996). 10. Albrecht, M., Gossage, R. A., Lutz, M., Spek, A. L. & van Koten, G. Diagnostic organometallic and metallodendritic materials for SO 2gas detection: reversible binding of sulfur dioxide to aryl platinum(II) complexes. Chem. Eur. J. 6,1431±1445 (2000). 11. Chen, P ., Wu, X., Liu, J. & Tan, K. L. High H 2uptake by alkali-doped carbon nanotubes under ambient pressure and moderate temperatures. Science 285, 91±93 (1999). 12. Dillon, A. C. et al. Storage of hydrogen in single-walled carbon nanotubes. Nature 386, 377±379 (1997). 13. Ohashi, Y., Y anagi, K., Kurihara, T., Sasada, Y. & Ohgo, Y. Crystalline-state reaction of cobaloxime complexes by X-ray exposure. An order-to-order racemization in the crystal of [( S)-1-cya- noethyl](pyridine)-bis(dimethylglyoximato)cobalt(III). J. Am. Chem. Soc. 104, 6353±6359 (1982). 14. Novak, K., Enkelmann, V ., Wegner, G. & Wagener, K. B. Crystallographic study of a single crystal to single crystal photodimerization and its thermal reverse reaction. Angew. Chem. Int. Edn Engl. 32, 1614±1616 (1993). 15. Kobatake, S., Y amada, T., Uchida, K., Kato, N. & Irie, M. Photochromism of 1,2-bis(2,5-dimethyl-3- thienyl)per¯uorocyclopentene in a single crystalline phase. J. Am. Chem. Soc. 121, 2380±2386 (1999). 16. Scheffer, J. R. & Pokkuluri, P . R. in Photochemistry in Organized & Constrained Media (ed. Ramamurthy, V .) 185 (VCH, New York, 1990). 17. Langeley, P . J. & Hulliger, J. Nanoporous and mesoporous organic structures: new openings for materials research. Chem. Soc. Rev. 28,279±291 (1999). 18. Estermann, M., McCusker, L. B., Baerlocher, C., Merouche, A. & Kessler, H. A synthetic gallo phosphate molecular sieve with a 20-tetrahedral-atom pore opening. Nature 352, 320±323 (1991). 19. Hajdu, J. et al. Millisecond X-ray diffraction and the ®rst electron density map from Laue photographs of a protein crystal. Nature 329, 178±181 (1987). 20. Buss, C. E. et al. Structural investigations of vapochromic behavior. X-ray single-crystal and powder diffraction studies of [Pt(CN- iso-C3H7)4][M(CN) 4] for M = Pt or Pd. J. Am. Chem. Soc. 120, 7783± 7790 (1998). 21. Desiraju, G. R. Crystal Engineering; The Design of Organic Solids (Elsevier, Amsterdam, 1989). 22. Lehn, J.-M. Supramolecular Chemistry: Concepts and Perspectives (VCH, Weinheim, 1995). 23. van Koten, G. Tuning the reactivity of metals held in a rigid ligand environment. Pure Appl. Chem. 61, 1681±1694 (1989). Figure 5 Formation of crystalline 1from a single crystal of 2. Shown after about 14 h in air, showing the colourless zone at the periphery and the orange colour in the core of the crystal. © 2000 Macmillan Ma gazines Ltd	5a62ad4f00e46cc072cc6f5b2c0e14fa1207aba4	page_0003
letters to nature 974 NATURE \|VOL 406 \|31 AUGUST 2000 \|www.nature.com24. Davies, P . J. et al. Organoplatinum building blocks for one-dimensional hydrogen-bonded polymeric structures. Angew. Chem. Int. Edn Engl. 35,1959±1961 (1996). 25. Braga, D., Grepioni, F. & Desiraju, G. R. Crystal engineering and organometallic architecture. Chem. Rev. 98,1375±1405 (1998). 26. Albrecht, M. & van Koten, G. Gas sensor materials based on metallodendrimers. Adv. Mater. 11,171± 174 (1999). 27. Darensbourg, M. Y., Tuntulani, T. & Reibenspies, J. H. Structure/function relationships in ligand- based SO 2/O2conversion to sulfate as promoted by nickel and palladium thiolates. Inorg. Chem. 34, 6287±6290 (1995). 28. Moffat, K. Time-resolved crystallography. Acta Crystallogr. A 54,833±841 (1998). 29. Kong, J. et al. Nanotube molecular wires as chemical sensors. Science 287, 622±625 (2000). Acknowledgements We thank E. T. H. Lutz and A. M. M. Schreurs for technical assistance during the measurements and R. A. Gossage for discussions. This work was partially supported by the Council for Chemical Sciences from the Dutch Organization for Scienti®c Research (CW±NWO). Correspondence and requests for material should be addressed to G.v.K. (e-mail: g.vankoten@chem.uu.nl). ................................................................. Automatic design and manufacture of robotic lifeforms Hod Lipson & Jordan B. Pollack Computer Science Department, Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454, USA ............................................................................................................................... ............... Biological life is in control of its own means of reproduction, which generally involves complex, autocatalysing chemical reac- tions. But this autonomy of design and manufacture has not yet been realized arti®cially1. Robots are still laboriously designed and constructed by teams of human engineers, usually at con- siderable expense. Few robots are available because these costs must be absorbed through mass production, which is justi®ed only for toys, weapons and industrial systems such as automatic teller machines. Here we report the results of a combined computational and experimental approach in which simple elec- tromechanical systems are evolved through simulations frombasic building blocks (bars, actuators and arti®cial neurons); the `®ttest' machines (de®ned by their locomotive ability) are then fabricated robotically using rapid manufacturing technol- ogy. W e thus achieve autonomy of design and construction using evolution in a `limited universe' physical simulation 2,3coupled to automatic fabrication. In the ®eld of arti®cial life, `life as it could be' is examined on the basis of understanding the principles, and simulating the mechan- isms, of real biological forms4. Just as aeroplanes use the same principles as birds, but have ®xed wings, arti®cial lifeforms may share the same principles, but not the same implementation in chemistry. Stored energy, autonomous movement, and even animal communication are replicated in toys using batteries, motors and computer chips. Our central claim is that to realize arti®cial life, full autonomy must be attained not only at the level of power and behaviour (the goal of robotics, today5), but also at the levels of design and fabrication. Only then can we expect synthetic creatures to sustain their own evolution. We thus seek automatically designed and constructed physical artefacts that are functional in the real world, diverse in architecture (possibly each slightly different), and auto- matically producible with short turnaround time, at low cost and in large quantities. So far these requirements have not been met. The experiments described here use evolutionary computation for design, and additive fabrication for reproduction. The evo- lutionary process operates on a population of candidate robots,each composed of some repertoire of building blocks. The evolu- tionary process iteratively selects ®tter machines, creates offspring by adding, modifying and removing building blocks using a set of operators, and replaces them into the population (see Methods section). Evolutionary computation has been applied to many engineering problems6. However, studies in the ®eld of evolutionary robotics reported to date involve either entirely virtual worlds2,3,o r , when applied in reality, adaptation of only the control level ofmanually designed and constructed robots 7±9. These robots have a predominantly ®xed architecture, although Lund10evolved partial aspects of the morphology, Thompson11evolved physical electric circuits for control only, and we evolved static Lego structures, but had to manually construct the resultant designs12. Other works involving real robots make use of high-level building blocks com- prising signi®cant pre-programmed knowledge13. Similarly, addi- tive fabrication technology has been developing in terms of materials and mechanical ®delity14but has not been placed under the control of an evolutionary process. Our approach is based on the use of only elementary building blocks and operators in both the design and fabrication process. As building blocks are more elementary, any inductive bias associated with them is minimized, and at the same time architectural ¯exi- bility is maximized. Similarly, use of elementary building blocks in the fabrication process allows the latter to be more systematic and versatile. As a theoretical extreme, if we could use only atoms as building blocks, laws of physics as constraints and nanomanipula- tion for fabrication, the versatility of the manufacturable design space would be maximized. Earlier reported work that used higher- level components and limited architectures (such as only tree structures2,3) resulted in expedited convergence to acceptable solu- tions, but at the expense of truncating the design space. Further- more, these design spaces did not consider manufacturability. Morphology (body)Control (brain) BarLinear actuator Ball jointNeuron Infinite planeSynapse Figure 1 Schematic illustration of an evolvable robot. Bars connect to each other to form arbitrary trusses; by changing the number of bars and the way they connect, the structural behaviour of the truss is modi®edÐsome substructures may become rigid, while others may become articulated. Neurons connect to each other via synapses to form arbitrary recurrent neural networks. By changing the synapse weights and the activation threshold of the neuron, the behaviour of the neuron is modi®ed. By changing the number of neurons and their connectivity, the behaviour of the network is modi®ed. Also, we allow neurons to connect to bars: in the same way that a real neuron governs the contraction of muscle tissue, the arti®cial neuron signal will control the length of the bar by means of a linear actuator. All these changes can be brought about by mutational operators. A sequence of operators will construct a robot and its controller from scratch by adding, modifying and removing building blocks. The sequence at the bottom of the image illustrates an arbitrary progression of operators that create a small bar, elongate it andsplit it. Simultaneously, other operators create a neuron, add another neuron, connect them in a loop, and eventually connect one of the neurons to one of the bars. The bar is now an actuator. Because no sensors were used, these robots can only generate patterns and actions, but cannot directly react to their environment. © 2000 Macmillan Ma gazines Ltd	5a62ad4f00e46cc072cc6f5b2c0e14fa1207aba4	page_0004
University of Groningen Light-Controlled Conductance Switching of Ordered Metal-Molecule-Metal Devices Molen, Sense Jan van der; Liao, Jianhui; Kudernac, Tibor; Agustsson, Jon S.; Bernard, Laetitia; Calame, Michel; Wees, Bart J. van; Feringa, Ben L.; Schönenberger, Christian Published in: Nano Letters DOI: 10.1021/nl802487j IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2009 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Molen, S. J. V. D., Liao, J., Kudernac, T., Agustsson, J. S., Bernard, L., Calame, M., Wees, B. J. V., Feringa, B. L., & Schönenberger, C. (2009). Light-Controlled Conductance Switching of Ordered Metal- Molecule-Metal Devices. Nano Letters , 9(1), 76-80. https://doi.org/10.1021/nl802487j Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 03-04-2023	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0000
1 Supporting Information Light-controlled conductance switching of ordered metal-molecule- metal devices S.J. van der Molen *,1,2,3 , J. Liao 1, T. Kudernac 4, J.S. Agustsson 1, L. Bernard 1, M. Calame 1, B.J. van Wees 2, B.L. Feringa 4, C. Schönenberger1 1: Department of Physics, University of Basel, Klingelbe rgstrasse 82, 4056 Basel, Switzerland 2: Physics of Nanodevices, Zernike Institute for Advanced Materi als, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands 3: Kamerlingh Onnes Laboratorium, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands 4: Stratingh Institute / Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0001
2 1. Details on sample preparation and experimental techniques a) Sample preparation: We synthesize diarylethene molecules with two acetyl-protected thiol endgroups connected to the central switching unit via a metap henyl spacer (Fig. 1a of the manuscript) [27,28]. Two-dimensional, hexagonally ordered arrays of octanemonothiol-covered nanoparticles are fabricated using the me thod described in Refs. 31 and 32. In the present study, two sample geometries are us ed. For the first type (including 'sample 1'), we stamp a large-scale array ( ≅ 4×4 mm), assembled on a water surface, onto a quartz substrate using a flat PDMS stamp. Nex t, we evaporate two gold contacts on top of the network, employing a thin Cu-wire as a sh adow mask (45 nm Au / 5 nm Ti). Thus, the effective array size of 'sample 1' is rel atively large (width: 3.14 mm, length: 200 µm). For the second type (including 'sample 2'), we use patter ned PDMS stamps to transfer the self-assembled nanoparticle array s. These stamps are prepared using standard UV-lithography to define narrow lines. Subsequently , we evaporate gold contacts using a TEM-grid as a shadow mask [31, 32]. The uncovered arr ay on 'sample 2' has a width of 125 µm and a length of 8 µm. Molecular insertion is performed by immersing a sample in a 0.5 mM solution of the 'on'-state switch in tetrahydrofurane, for over 24 hours at 295 K. Finally, a sample is electrically contacte d and inserted in an argon flow cell. b) Experimental techniques: Optical absorption spectroscopy is performed with a halogen lamp and an Andor Shamrock 163i spectrograph connected to the side- port of an Olympus IX71 inverted microscope. For the experiments in Figs . 2 and 3 of the manuscript, a Labview routine is used. This program commences by measuring an I-V (current-voltage) curve (-10<V<10V) in the dark. Then, it opens a shut ter for 5 seconds, during which the sample can be illuminated with the Hg-lamp, usin g different filters (‘UV-filter’: 300< λ<400 nm; ‘Vis-filter’: 590< λ<650 nm, or complete blocking). After the shutter is closed, the cycle is repeated. The conductance is m easured exclusively in the dark to exclude photocurrents or heating effects. One cycle ta kes 26 seconds. Thus, real time in Figs. 2 and 3 of the manuscript is 26/5 times the illumination time.	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0002
3 2. Optical spectra of diarylethene molecules in solution In Fig. S1, we show optical absorption spectra for thiolated me taphenyl-substituted diarylethene molecules in dry toluene. Figure S1a, displays the spectra for molecules in which the thiol group is ‘protected‘ by an acetyl group. The cl osed, ‘on’-state isomer (dashed red line) shows a broad absorption for 420 < λ < 630 nm. Illumination at these wavelengths induces ‘on’ to ‘off’ switching. The open, ‘off’ isome r (solid black line) has only got absorption peaks in the UV region. Illumination with UV-lig ht yields ‘off’ to ‘on’ switching. In Fig. S1b, spectra are shown for thiolated metaphenyl-substi tuted diarylethene molecules bound (at one side) to small gold nanoparticles (2 nm in di ameter) in dry toluene. Mie scattering on the nanoparticles clearly influences the absorption curves. Most notably, a small surface plasmon peak is present, as se en in the spectrum for the open molecules (black solid line, cf. Fig. S1a). However, the abso rption peak of the closed diarylethene molecules (red, dashed line) still dominates t he plasmon peak due to the small particle diameter. The ring-closure process is less ef ficient for molecules bound to nanoparticles (quantum yield: 7%) than for free molecules (qua ntum yield: 40%) (R. Hania et al. in preparation). We note that for the 10-nm gold particles we use in 2D-arrays, the surface plasmon peak is strongly enhanced, as compared to the 2-nm ones in Fig. S1b. There fore, the surface plasmon peak constitutes the main absorption peak around 600 nm. It dominat es the typical absorption peak of the closed form molecules. Figure S1 \| a, Optical absorption spectra of thiolated metaphenyl-substit uted diarylethene molecules in dry toluene . Black, solid line: open, ‘off’- state molecule; red, dashed l ine: closed, ‘on’-state molecule; note the broad absorption peak around 550 nm. b, Optical absorption spectra for the same molecules, after coupling to small gold nanoparti cles. The nanoparticles induce Mie scattering. The surface plasmon peak is relatively weak, due to the sma ll particle diameter (2 nm), so that the ‘on’-state absorption peak dominates the spectrum of the closed molecules. For larger particles, as in the arrays, the surface plasmon peak dominates t he spectrum. 300 350 400 450 500 550 600 650 700 0.0 0.5 1.0 Optical density λ (nm) a b 400 600 800 1000 0.0 0.5 1.0 1.5 Optical density λ (nm)	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0003
4 3. Experiments in ambient conditions Experiments in air at room temperature show reversible swit ching. However, switching is less pronounced than in an argon flow cell and the devices deteriorat e faster. After 1 to 2 switching cycles the samples break down. An example is shown i n Fig. S2, for a device of similar dimensions as ‘sample 1’ in Fig. 3 of the main manuscr ipt (3.1x4.1 mm). We relate this to enhanced photodegradation effects during UV irradiation in air. Figure S2 \| Conductance and optical absorption experiments in ambient conditions . The device is similar to ‘sample 1’, length: 3.1 mm, width, 4.1 mm. Fi rst, at t<0, we take absorption spectrum 1 (see inset, using a halogen lamp). At t=0, we star t irradiating the sample with visible light (see coloured bar; same 100 W Hg-lamp and filters as i n Figs. 2 and 3 of the manuscript). This induces a conductance decrease, as seen in the main panel. We stop illumination for a short time around t=560 s, after which we proceed with visible illuminat ion. Next, we stop illumination again and take spectrum 2 (inset). Clearly, the surface plasm on peak is blue shifted with respect to spectrum 1, demonstrating molecular ‘on’-to-‘off’ conversion. Sub sequently, we illuminate with UV-light (t=980 s). This results in a conductance increase . We stop UV-illumination and measure spectrum 3, which is very similar to spectrum 1. Fi nally, we irradiate with visible light again for an extended period. After this, we measure spect rum 4. The plasmon peak has moved to the red again, although the effect is limited now. 0 2000 4000 1.6 1.7 1.8 1.9 2.0 640 680 0.52 0.54 0.56 G (nS) Illumination time (s) Vis Vis UV Vis 1 Absorption λ (nm) 3 24 1 2 3 4	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0004
5 4. Photodegradation due to long UV-illumination in Ar flow cell In Figure S3, we show an illumination experiment on a nanoparticl e network with ‘on’- state diarylethene bridges. The sample is similar to ‘samp le 1’ in Figure 3 of the manuscript (length: 0.25 mm, width: 6.3 mm). The experiment is done i n an argon flow cell, at room temperature. Visible illumination is started at t=0, resul ting in a conductance decrease due to ‘on’ to ‘off’ switching. At t=245 s, we begin UV- irradiation. In contrast to Figures 2 and 3 of the manuscript, we use an extended UV-illuminat ion time, which allows us to observe degradation effects. Just after UV-illumina tion has started, there is a steep conductance increase, which we relate to molecular ‘on’ to ‘ off’ conversion. Some time later, the slope of the conductance curve decreases. We re late this effect to a competition of ‘on’ to ‘off’ conversion and irreversible reactions. Inde ed, photodegradation is most pronounced for fully conjugated, ‘on’ state mol ecules, which are increasingly present during UV-irradiation. In this case, the extended UV- illumination led to failure of the sample. As shown in Fig. S3, the sample could not be switched a second time by illumination with visible light. Interestingly, a similar change of slope during UV-irradiati on can be seen in Figure 2 of the manuscript, although it is much less pronounced. By restricting UV irradiation, and hence limiting irreversible reactions, we were able to obt ain 8 full switching cycles for ‘sample 2’. Figure S3 \| Illumination experiment on a network with ‘on ’-state bridges. At t=0, illumination with visible light is started. At t=245 s, long-term UV irradiation begins (see coloured bar at top). This leads to a competition of ‘on’- to-‘off’ conversion and photodegradation effects. Subsequent illumination with visible light has no effect on the conductance. -200 0 200 400 600 29 30 31 32 33 34 35 36 37 38 G (nS) Illumination time (s) Dark Vis UV Vis	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0005
6 5. Illumination of octanemonothiol-covered nanoparticle networks We performed reference experiments on octanemonothiol-covered nanopartic le networks. These are samples as prepared before molecular bridging, of similar dimensions as ‘sample 2’ considered in Figure 2 of the main manuscript (lengt h: 10 µm, width: 230 µm). Figure S4a shows the influence of illumination on the conductance of such a sample (Argon flow cell, room temperature). The conductance G(t) has be en normalized to its initial, low value G(t=0)=0.496 nS. This allows for a comparison with ‘ sample 2’ and Fig. 2 of the main manuscript (replotted in Fig. S4b, after norma lization). Visible light is found to have little effect on the octanemonothiol device: conductance cha nges are small and can go either way. UV illumination, on the other hand, induces a sma ll conductance decrease in the first run. We relate this to photodegradation effects, pos sibly of the Au-S bond in the monothiol molecules. Figure S4 \| a, Conductance evolution during illumination for a net work of octanemonothiol- covered nanoparticles. The conductance is normalized to G(t=0) = 0.496 nS . For t<0, the sample is in the dark. Illumination with visible light is sta rted at t=0, followed by a period in the dark, by UV-illumination etc. b, First five switching events for ‘sample 2’ (see Fig. 2, main manuscript). Also here we normalized the conductance. -500 0 500 1000 1500 2000 0.6 0.8 1.0 UV Vis UV Vis G/G(t=0) Illumination time (s) 0 500 1000 1500 2000 0.6 0.8 1.0 Vis UV G/G(t=0) illumination time (s) Vis UV Vis UV Vis UV Vis UV Vis Vis UV Vis UV a b	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0006
7 6. Illumination of nanoparticle networks bridged by OPV-dithiol molecu les We performed reference experiments on nanoparticle networks, i n which (oligo(=3)- phenylenevinylene-dithiol molecules (OPV) have formed bridges bet ween neighbouring nanoparticles (see Fig. S5a). These samples are of similar dimensions as ‘ sample 2’ considered in Figure 2 of the main manuscript (length: 10 µm, width: 150 µm). Figure S5b shows the evolution of the conductance during illumination (Argon flow ce ll, room temperature). The conductance values have been normalized to the initial conductance G(t=0)=50.6 nS. This allows for a comparison to ‘sample 2’ and Fig. 2 of the main manuscript (replotted in Fig. S5c, after normalization). Indeed, for the OPV sample, the overall conductance variations are small. In the dark, the sample c onductance increases somewhat, giving rise to a small background slope. Visible lig ht is found to have virtually no effect on the device. UV illumination gives rise t o a slight decrease of the conductance. This is consistent with photodegradation effects, taking pl ace under UV irradiation. Note the pronounced difference with the data on the dia rylethene switches, Figs. 2 and S5c, where UV irradiation gives rise to a large conductance increase . Figure S5 \| Conductance evolution (b) during illumination for a network of nanoparticles, bridged by OPV-dithiol molecules (drawn in a) . The conductance is normalized to G(t=0) = 50.6 nS . For t<0, the sample is in the dark. Illumination with visib le light is started at t=0, followed by a period in the dark, by UV-illumination etc. c, First five switching events for ‘sample 2’ (see Fig. 2, main manuscript). Also here we normalized the conduc tance. HS SH 0 500 1000 1500 2000 0.6 0.8 1.0 Vis G/G(t=0) Illumination time (s) UV Vis UV Vis 0 500 1000 1500 2000 0.6 0.8 1.0 Vis UV G/G(t=0) illumination time (s) Vis UV Vis UV Vis UV Vis UV Vis Vis UV Vis UV a b c	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0007
8 7. Scanning electron microscopy In Fig. S6, we show a scanning electron microscopy (SEM) ima ge of ‘sample 1’ taken after the experiment shown in Figure 3 of the manuscript. The nano particle structure is still intact. Moreover, neither visible nor UV-illumination has c hanged the nanoparticle lattice. Hence, structural changes during illumination cannot explain the data in Figure 3. Figure S6 \| Scanning electron micrograph of ‘sample 1’ . The image was taken after the experiment displayed in Figure 3 of the manuscript. The nanopart icle lattice structure has not been influenced by visible nor by UV illumination.	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0008
9 8. Percolation Here, we employ percolation theory to model the conductance for ‘s ample 2’ after sequential visible and UV illumination (see Fig. 2 of manuscript ). Our calculation is based on the general effective medium equation, which has proven to give a successful relation between conductance, G, and the percentage of neighbouring nanoparticle pairs that are bridged by closed, ‘on’-state molecules, p [36, 37]. For a 2D-system, it r eads: 0)())(1 ( 4 / 3 4 / 3 14 / 34 / 3 1 4 / 3 4 / 3 04 / 34 / 3 0=+−++− − GAGGGp GAGGGp c c (1) where G 1 is the conductance of the sample if all nanoparticle pairs a re bridged by closed, ‘on’-state molecules, i.e., G 1=G(p=1). Equivalently, G 0 is the conductance of the sample if all nanoparticle pairs are bridged by open, ‘off’-sta te molecules, i.e., G 0=G(p=0). We take G 1 = 25 G0. The constant A c=(1-pc)/p c=1.88 is related to the percolation threshold, pc=2sin(π/18)=0.35, for a triangular, 2D network. We assume that at t=0 all nanoparticle pairs are bridged b y closed, ‘on’-state switches, i.e. p(t=0)=1 so that G 1=3.2 nS (see Fig. 2). After the first illumination with visible light (defined as t=1/2, i.e., half an illumination period), a fraction x of t he closed molecules will have switched open, i.e., p(t=1/2)=1-x. After subsequent UV il lumination (defining t=1), a fraction y of the open molecules will have switched back, etc. F igure S7 shows the conductance for the 2D-network after each illumination step, calculat ed using eq. (1). To fit the first points of Fig. 2 in the manuscript, we need x=0.2 and y=0.7. As seen in Fig. S7, two ‘photostationary states’ come about, partly explaining the decay in Fig. 2. The additional decay observed in Fig. 2 is therefore related to photodegradation effe cts. Figure S7 \| Calculation of sample conductance using a percolat ion model. The initial conductance is based on ‘sample 2’. With time, two photostationary states appear, partly explaining the decay in Fig. 2 of the manuscript. Note that t he time axis is defined in periods. After each full period of visible and UV illumination, t=n is integer. Af ter visible illumination, t = n+1/2 0 1 2 3 4 5 6 7 81.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 G (nS) Illumination period	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0009
10 References (numbering is the same as in the main manuscript) [27] Kudernac, T., de Jong, J.J., van Esch, J., Feringa, B.L., Dulic, D., van der Molen, S.J., van Wees, B.J. Mol. Cryst Liq. Cryst . 430 , 205-210 (2005); [28] Kudernac, T., van der Molen, S.J., van Wees, B.J., Feringa, B.L. Chem. Comm. 3597-3599 (2006) [31] Liao, J., Bernard, L., Langer, M., Schönenberger, C.; Calame, M. Adv. Mater. 18 , 2444-2447 (2006) [32] Bernard, L., Kamdzhilov, Y., Calame, M., van der Molen, S.J., Liao, J., Schönenberger, C. J. Phys. Chem. C 111 , 18445-18450 (2007) [36] Kirkpatrick, S., Rev. Mod. Phys. 45 , 574-588 (1973) Additional Reference: [37] Wu, J.; McLachlan, D.S. Phys. Rev. B 56 , 1236-1248 (1997)	184873c219ecbb27e85e30a1aeee4a42757b1e52	page_0010
BioMed Central Page 1 of 1 (page number not for citation purposes)BMC Systems Biology Open Access Poster presentation Molecules for memory: modelling CaMKII Melanie Stefan* and Nicolas Le Novere Address: EMBL – European Bioinformatics Inst itute (EBI), Hinxton, Cambridge, CB10 1SD, UK Email: Melanie Stefan* - mstefan@ebi.ac.uk * Corresponding author Introduction Long-term modifications of synaptic strength, such as long-term potentiation (LTP) or long-term depression (LTD) are thought to underlie some forms of learning and memory. At the excitatory glutamate synapse, LTP is dependent on calcium influx through the N-methyl-D- aspartate (NMDA) receptor and subsequent activation of calcium/calmodulin-dependent protein kinase II (CaM- KII). The NMDA receptor, CaMKII, and its activator cal- modulin are all embedded in a complex hyperstructure consisting of more than 180 molecules [1] that acts as as a "synaptic plasticity nanomachine". Our current work aims at exploring CaMKII function in the context of the NMDA receptor complex Materials and methods We used StochSim [2] to carry out stochastic single-parti- cle simulations. Molecules react with each other according to probabilities computed from kinetic constants. An important feature of StochSim is that different states (e.g. open/closed, phosphorylated/unphosphorylated at a spe- cific site) or combinations of different states can be repre- sented. Reactions can modify the state of a molecule, and likewise, the state of a molecule can have an influence on its propensity to participate in a given reaction. Results A series of models of CaMKII has been created to explore the interplay between phosphorylation, interaction with other proteins, conformational change and kinase activ- ity. Simulations have been carried out using the stochasticsimulator StochSim [2]. So far, the model was successful in explaining the effect of calmodulin binding and Thr286 autophosphorylation on CaMKII conformation. Further- more, it suggests functional implications for CaMKII binding to the NR2B subunit of the NMDA receptor. Conclusion A series of models have been created to explore CaMKII function in the context of the NMDA receptor complex. First simulations using StochSim confirm mechanisms described in the experimental literature and suggest fur- ther predictions about the system. Future work includes the extension of the model to include a larger number of processes and interaction partners. References 1. Collins MO, Yu L, Coba MP, Husi H, Campuzano I, Blackstock W, Choudhary JS, Grant SGN: Molecular charac terization and comparison of the components and multiprote in complexes in the postsynaptic proteome. J Neurochem 2006, 97:16-23. 2. Morton-Firth CJ: Stochastic Simulation of Cell Signalling Path- ways. In PhD thesis University of Cambridge ; 1998. from BioSysBio 2007: Sy stems Biology, Bioinformatics and Synthetic Biology Manchester, UK. 11–13 January 2007 Published: 8 May 2007 BMC Systems Biology 2007, 1(Suppl 1):P40 doi:10.1 186/1752-0509-1-S1-P40 <supplement> <title> <p>BioSysBio 2007: Systems Biology, Bioinformatics, Synthetic Biology</p> </title> <editor>John Cumbers, X u Gu, Jong Sze Wong</editor> <note>Meeting abstracts – A single PDF containing all abstracts in this Supplement is available <a href="www.biomedcentral.com/content/files/pdf/1752-0509-1-S1-full.pdf">here</a>.</note> <url>http://www.biomedcentral.com/cont ent/pdf/1752-0509-1-S1-info.pdf</url> </supplement> This abstract is availabl e from: http://www.biomedcentral.com/1752-0509/1?issue=S1 © 2007 Stefan and Le Novere; li censee BioMed Central Ltd..	b3c74a438f16a267df78ff34834a8c465b29db3b	page_0000
molecules Review Continuous Flow Photochemistry for the Preparation of Bioactive Molecules Mara Di Filippo, Cormac Bracken and Marcus Baumann * School of Chemistry, University College Dublin, Science Centre South, Belﬁeld, Dublin 4, Ireland; mara.diﬁlippo@ucdconnect.ie (M.D.F.); cormac.bracken@ucdconnect.ie (C.B.) *Correspondence: marcus.baumann@ucd.ie; Tel.: +353-01-617-2117 Academic Editors: Gioiello Antimo and Cerra Bruno Received: 28 December 2019; Accepted: 10 January 2020; Published: 15 January 2020 /gid00030/gid00035/gid00032/gid00030/gid00038/gid00001/gid00033/gid00042/gid00045 /gid00001 /gid00048/gid00043/gid00031/gid00028/gid00047/gid00032/gid00046 Abstract: The last decade has witnessed a remarkable development towards improved and new photochemical transformations in response to greener and more sustainable chemical synthesis needs. Additionally, the availability of modern continuous ﬂow reactors has enabled widespread applications in view of more streamlined and custom designed ﬂow processes. In this focused review article, we wish to evaluate the standing of the ﬁeld of continuous ﬂow photochemistry with a speciﬁc emphasis on the generation of bioactive entities, including natural products, drugs and their precursors. To this end we highlight key developments in this ﬁeld that have contributed to the progress achieved to date. Dedicated sections present the variety of suitable reactor designs and set-ups available; a short discussion on the relevance of greener and more sustainable approaches; and selected key applications in the area of bioactive structures. A ﬁnal section outlines remaining challenges and areas that will beneﬁt from further developments in this fast-moving area. It is hoped that this report provides a valuable update on this important ﬁeld of synthetic chemistry which may fuel developments in the future. Keywords: continuous ﬂow chemistry; photochemistry; photocatalysis; bioactive molecules; medicinal chemistry; process development; enabling technologies 1. Introduction The last decade has witnessed a much-welcomed renaissance and subsequent exploitation of photochemical transformations within the chemistry community. This renewed interest in utilizing light to bring about chemical reactions is largely fueled by a desire to realize more sustainable approaches for target molecule synthesis, along with signiﬁcant advances in the ﬁeld of photoredox catalysis where both transition metals and organic dyes have been exploited. The availability of suitable light sources, ranging from light-emitting diodes (LED) to continuous ﬂuorescent lamps (CFL) and powerful UV lamps (e.g., medium-pressure mercury lamp), furthermore, has driven this development. Consequently, a plethora of known and newly developed photoreactions has been reported in the literature, rendering facile access to diverse chemical entities through selective transformations that oftentimes o er orthogonal approaches to the synthetic chemist [1–6]. In addition, continuous ﬂow technology [ 7–13] has had a major impact on popularizing photochemical reactions, as it not only provides the tools to e ectively perform photochemical reactions, but moreover helps overcoming limitations that are inherent to photochemistry. As such, the short path lengths provided by narrow, light-permeable tubing ensure that light can easily penetrate the substrate solution, thereby mitigating limitations commonly arising from the Beer–Lambert law. Furthermore, by placing the light source near the reactor coil or microchip of the ﬂow set-up, very ecient and uniform radiation of the substrate can be achieved. Oftentimes low-energy, tunable light sources are favored in combination with suitable cooling mechanisms to e ectively control Molecules 2020 ,25, 356; doi:10.3390 /molecules25020356 www.mdpi.com /journal /molecules	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0000
Molecules 2020 ,25, 356 2 of 13 both temperature and energy input. Finally, continuous ﬂow processing allows for simple reaction scale-up of the resulting photoreaction without alteration of reaction parameters and the short residence times within the photoreactor reduce the likelihood of decomposition of substrate or product due to over-radiation. As a consequence of the successful union of ﬂow reactor technology with modern photochemical applications, a growing body of literature has emerged, including topical reviews by leading experts in the ﬁeld [ 14–17]. As the ﬁeld of continuous ﬂow photosynthesis is moving forward at fast pace, we wish to provide a focused review on its impact on generating bioactive molecules [ 18–20]. This appears both timely and paramount, as the last ten years have provided the foundations to now apply continuous photochemistry to the syntheses of various biologically active entities—drugs, natural products and their precursors. In addition, we wished to evaluate whether there had been signiﬁcant uptake of this technology by chemists outside traditional academic laboratories and whether ﬂow photochemistry had advanced to be a viable option to generating bioactive molecules in industrial settings. To accomplish this, we ﬁrst review the design of various reactor types and outline innovative aspects that aid in overcoming traditional bottlenecks. The second section investigates progress on continuous photochemical approaches targeting greener and more sustainable synthesis means. That is then followed by a dedicated section reviewing recent applications of photochemical transformations to enable the formation of bioactive species, before an assessment of the feasibility of readily integrating photochemical transformations into advanced multistep sequences in ﬂow mode. 2. Reactor Design and Technology The diminishing penetration depth of light into solutions of substrates is one of the major challenges that has prevented e ective exploitation of photochemistry in research settings. This phenomenon, described by the Beer–Lambert law, means that photochemical batch processes that typically su er from poor mass transfer cannot be scaled e ectively. Consequently, most batch photo-reactors (e.g., Rayonet reactor) use a set of lamps closely arranged around the reaction vessel to maximize irradiation. Alternatively, a single light source may be placed at the center of a double jacketed reaction vessel to irradiate the solution in an inverse arrangement. In both cases, appropriate cooling mechanisms are vital to ensure e ective dissipation of the heat generated by the lamps. The superior performance of continuous ﬂow reactors over batch systems arises from miniaturization of mixing and reactor elements that results in excellent mass and heat transfer. Therefore, chemists and chemical engineers have taken inspiration from ﬂow reactor designs to address the aforementioned challenges regarding photochemical reactors. Consequently, several diverse and e ective ﬂow reactor designs have been reported over the last decade and applied to di erent photochemical transformations [21]. An example by Oelgemöller and coworkers [ 22] demonstrates a falling ﬁlm reactor set-up to bring about the e ective photooxygenation of -terpinene in the presence of Rose Bengal to generate ascaridole (Figure 1). More recently, the Corning reactor system that is based on irradiated reactor plates has gained popularity as it allows one to e ectively perform continuous photochemical reactions at various scales. In this system, panels of LEDs are arranged next to mesoﬂuidic reactor plates, allowing for uniform radiation of the substrate solution. Published examples include the photooxidation of methionine, -terpinene or citronellol in the presence of organic dyes (e.g., Rose Bengal, methylene blue or tetrakis(4-carboxyphenyl)porphyrin) [23,24].	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0001
Molecules 2020 ,25, 356 3 of 13 Figure 1. Images of a falling ﬁlm reactor plate (reproduced with permission from [ 25]) and the Corning G3 photoreactor [26]. Another approach to realize such photooxygenation reactions in a continuous manner was reported by Pergantis and Vassilikogiannakis, who developed a nebulizer-based photo-reactor [ 27]. Therein, an aerosol is generated and sprayed into an irradiated reaction chamber by mixing a stream of air or oxygen with the substrate solution. Low-energy LED strips are mounted around this chamber providing uniform radiation of the aerosol, which upon condensation is collected at the outlet of this set-up (Figure 2). Figure 2. Image of the nebulizer-based photo-reactor (reproduced with permission from [27]). Related applications that also exploit thin ﬁlms were recently reported by Poliako and George who used a modiﬁed rotary evaporator set up (Figure 3). In this case substrate solutions are delivered into the rotating ﬂask via the side port of the set-up. LEDs or UV–Vis lamps e ectively irradiate the ﬁlm generated rendering a system that can be run either continuously or semi-continuously based on low budget components that are readily available in research laboratories [28]. Figure 3. Image of the photochemical reactor set up (reproduced with permission from [28]). Raston and Stubbs recently reported on a vortex ﬂuidic device that comprises a temperature-controlled reactor zone into which the substrate solution can be delivered continuously (Figure 4). The resulting thin ﬁlm again provides a high surface-to-volume ratio, and thus enables eective and uniform irradiation. This was e ectively demonstrated for the generation of new C–C bonds via photo-redox transformations [29].	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0002
Molecules 2020 ,25, 356 4 of 13 Figure 4. Representations of the vortex ﬂuidic device. ( A) A schematic of the vortex ﬂuidic device; ( B) A photograph of the vortex ﬂuidic device; (reproduced with permission from [30]). In order to enable large scale generation of desired photo-products, Booker-Milburn and co-workers recently reported on a new photo-ﬂow reactor known as Fireﬂy (Figure 5). In this set-up, several parallel tubes are interconnected and arranged around a powerful light source (e.g., 400 W Hg-lamp). Equipped with an e ective cooling mechanism, this reactor provides an internal volume of 120 mL and can deliver multigram quantities of photo-adducts per minute, which can be scaled to kilogram quantities per day [31]. Figure 5. Representations of the ﬁreﬂy photoreactor. ( a) Diagram of reactor; ( b) Fireﬂy reactor in operation; (reproduced with permission from [31]) . In addition to the above photo-ﬂow reactors that display innovative engineering to uniquely resolve limitations known from batch applications, a variety of photochemical reactors exist that are readily integrated with commercial ﬂow modules. These are typically based on reactor coils made of various ﬂuorinated polymers that are combined with suitable light sources ranging from LEDs to medium pressure Hg lamps. Examples of these systems include Vapourtec’s UV150 and high-power LED reactor (e.g., [ 32–34]) system and Uniqsis’s PhotoSyn (e.g., [ 35,36]), as depicted in Figure 6. Due to their modular nature, these systems are easy to use in everyday lab applications, and can be complemented with broad-band ﬁlters or dedicated photo-spectrometers. The popularity and eectiveness of these continuous ﬂow photoreactors can be seen in many applications that have been reported over the years.	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0003
Molecules 2020 ,25, 356 5 of 13 Figure 6. Images of the Vapourtec high-power LED reactor and Uniqsis’s PhotoSyn reactor. As can be seen from this compilation of continuous photo-reactors, numerous reactor types have been developed, and their successful application to di erent types of chemical reactions (i.e., cycloadditions, photooxygenations, etc.) demonstrates how limitations inherent to batch photochemistry can be overcome by appropriately engineered devices. All these systems have short pathlengths in common that result from thin ﬁlms or small tubing diameters, and thus enable the eective and uniform irradiation of molecules. In addition, continuous ﬂow processing ensures homogeneous irradiation proﬁles without over-irradiating substrates that might otherwise lead to undesired side reactions and accompanying discoloration of reaction products. 3. Greener and More Sustainable Approaches Photochemistry is recognized as a valuable means for making new molecules, not only because it complements traditional thermal reactions, but also as it utilizes photons as cheap and readily available reagent equivalents. As such, photons may be viewed as traceless inputs, whose energy can be tuned based on their wavelength via Planck’s equation. Together with the notion that continuous ﬂow chemistry may o er a more sustainable technology for chemical synthesis compared to traditional batch chemistry [ 37–40], this suggests that ﬂow photochemistry is a green and almost ideal approach towards the synthesis of target molecules. In fact, photochemical transformations share this feature with electrochemical syntheses, as discussed in a recent comparative study [ 41]. While several articles have reported on the greener credentials of ﬂow chemistry, and photochemistry potentially provides a cleaner means to making molecules, this section attempts to assess the remaining limitations of continuous ﬂow photochemistry in this context. One key to successfully performing photochemical reactions is the exploitation of appropriate light sources that emit photons of suitable wavelength to bring about a desired transformation. While traditional applications have been based on UV light, modern photochemical transformations such as those based on photoredox catalysis, commonly exploit LEDs. This change is desirable not only as it allows one to use lower energy light sources ( 10 W for LED versus 100 W for Hg lamp) that provide better selectivity, but also as considerably less heat is generated by LEDs. Consequently, using LEDs oftentimes avoids the need for high-power cooling systems, and concurrently minimizes the formation of thermal side-products. Arguably, the availability of LEDs delivering light of speciﬁc wavelengths contributes to more energy e cient chemical synthesis. In this vein, Ryu and co-workers reported in an early study on the energy e ciency of various light sources used in a typical [2 +2]-cycloaddition reaction [ 42]. This comparative study evaluated a Hg lamp (300 W), a black light source (15 W) and an UV LED (1.7 W), and found that the LED not only gave the highest yield, but with a signiﬁcantly higher energy e ciency. Noël and co-workers [ 43,44] have recently described the exploitation of solar light in continuous ﬂow settings. The use of solar light is highly attractive [ 45]—and in fact the ﬁrst photochemical transformations were studied using sunlight [ 46,47] (many of which are associated with the pioneering	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0004
Molecules 2020 ,25, 356 6 of 13 works of Cannizzaro, Paterno and Ciamician); however, the heterogeneity of solar light ( 5% UV , 43% visible, 52% IR), together with inconsistent photon ﬂux (seasons, cloud coverage, day /night pattern and geographical impact), make this approach challenging. In order to mitigate these challenges, luminescent solar concentrators (LSC, Figure 7) can be used to down-convert solar radiation to low-energy visible light that matches the absorption of speciﬁc photo-redox catalysts (e.g., methylene blue) which would then enable a given transformation. In such set-ups most of the incoming solar light is thus down-converted by ﬂuorescent dyes embedded within the LSC, and transported within the device to microchannels where it is absorbed by a speciﬁc photocatalyst that triggers a chemical reaction. Although this technology is attractive, inevitable ﬂuctuations in solar light input may require this approach to be coupled with conventional light sources to achieve a consistent photochemical process. Figure 7. Exploiting solar light for scaled luminescent solar concentrators (LSC) applications (reproduced with permission from [48]). Besides the availability and use of suitable and energy-e cient light sources, a further challenge impacting the sustainability of photochemical reactions is the excessive use of solvents. An inherent problem in photochemistry is the requirement for high dilutions to favor productive interactions between photons and substrate (or catalyst) molecules. Therefore, most photochemical reactions are run at or below concentrations of 20 mM, which in turn results in the generation of copious amounts of solvent waste. It is therefore paramount to develop e ective photochemical transformations based on non-hazardous solvents such as water, alcohols, acetone and ethyl acetate. Additionally, future continuous photoreactions should aim to incorporate solvent recycling systems whilst striving for processes that are tolerant to higher e ective concentrations. Recent progress in this area has already highlighted the feasibility of performing multi-phasic photoreactions in slug-ﬂow where suspensions of substrate and /or photocatalyst can be processed through microchannels. Micro-mixing can be achieved by Taylor ﬂow in between immiscible phases (liquid /liquid, liquid /gas), which enhances the performance of such systems ([49]). 4. Synthesis of Bioactives: Drugs and Natural Products As can be seen from the previous sections, several continuous photochemical approaches have been developed to overcome some of the intrinsic restrictions associated with light-driven reactions and subsequently applied to the synthesis of various compounds. Whilst this was in many cases directed towards establishing new or improved synthetic methods, this section will highlight some recent applications that have generated bioactive target compounds. This will showcase a selection of natural products, drugs and drug-like entities prepared in both academic and industrial laboratories. Early applications of continuous photochemical approaches oftentimes demonstrate the complexity of structures that can be realized in an e ective manner. Several prominent examples, such as the generation of the anti-malaria drug artemisinin and its derivatives [ 50,51] and the preparation of vitamin D 3[52–54], outline the power of photochemistry when applied to the construction of unique targets. Further examples include the synthesis of neostenine [ 55] and goniofufurone [ 56], in which key bonds were realized photochemically, providing rapid access to advanced bioactive structures (Figure 8).	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0005
Molecules 2020 ,25, 356 7 of 13 Figure 8. Structures of complex bioactive molecules featuring ﬂow photochemistry key steps. Other examples include the photochemical preparation of the anti-inﬂammatory drug ibuprofen by a photo-Favorskii rearrangement, which highlights an attractive and atom-economical continuous approach to this important target [57]. More recent examples describe routes towards various poly(ADP-ribose) polymerase inhibitors aided by an intramolecular photocyclization reaction [ 58], unnatural aza-rocaglates via an excited state intramolecular proton transfer (ESIPT)-mediated (3 +2) photocycloaddition [ 59] or the preparation of new derivatives of clausine A via an azide-mediated carbazole formation followed by an arylation reaction [60] (Figure 9). Figure 9. Structures of poly(ADP-ribose) polymerase inhibitors and natural product analogues prepared in ﬂow. Pyocyanin, a small naturally occurring virulence factor, was recently prepared in an e ective and scalable multi-step approach featuring a photochemical oxygenation reaction (Figure 10). This process was, furthermore, coupled with immobilized reagents to facilitate the puriﬁcation and isolation of the target product [61].	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0006
Molecules 2020 ,25, 356 8 of 13 Figure 10. Continuous photosynthesis of pyocyanin (reproduced with permission from [61]). In addition to natural products and their analogs, several recent reports detail the use of continuous photochemical methods for the generation of drug-like heterocyclic sca olds that hold interest in medicinal chemistry programs. 2,4-Methanopyrrolidines are an important heterocyclic class of compounds that display higher hydrophilicity than regular pyrrolidines. Several applications demonstrate that their continuous photochemical synthesis based on an intramolecular [2 +2] cycloaddition (Figure 11A) can be scaled to kilogram quantities in order to facilitate further derivatization [ 31,62,63]. The synthesis of 3-hydroxyazetidines via a continuous Norrish–Young photocyclization reaction (Figure 11B) represents a further demonstration of e ectively creating drug-like structures in a simple and atom-economical fashion, and a recent study reports on the versatility of this transformation [ 64]. A ﬁnal example outlines the combination of photochemical with thermal processes in a reaction sequence rendering several isoindolin-1-one derivatives in a continuous manner, in which a photobenzylation of substituted phthalimides features as key step [ 65] (Figure 11C). Figure 11. Structures of drug-like heterocyclic entities prepared photochemically and their precursors. (A) synthesis of 2,4-Methanopyrrolidines; ( B) synthesis of 3-hydroxyazetidines; ( C) photobenzylation of substituted phthalimides.	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0007
Molecules 2020 ,25, 356 9 of 13 5. Remaining Challenges for E ectively Integrating Photoreactions within Multistep Sequences The previous section outlined the breath of applications in which continuous photochemical transformations were e ectively exploited to bring about the generation of bioactives in the form of drugs, natural products or their analogues. A variety of chemical transformations features in these syntheses ranging from cycloadditions and rearrangements to di erent radical-mediated sequences. In addition, photochemical ﬂow approaches have been developed with great success for functionalization reactions, such as halogenations [ 66–69], triﬂuoromethylations [ 70–72], oxygenations [ 73–75] and C-H activations [ 76–78]. All these applications showcase how continuous set-ups help to overcome limitations commonly encountered with classical photochemistry, and it is apparent that a variety of light sources as well as reactor conﬁgurations are exploited to best serve the intended chemical route. At this stage it appears that most of the highlighted studies were exploring the feasibility of photochemistry to achieve the improved ﬂow synthesis of molecules of interest, which in many cases also addressed scalability to gram or even kilogram quantities based on tailored reactor designs. However, it is currently less common that such continuous photochemical syntheses are embedded within telescoped ﬂow sequences in the same manner; this has been achieved for non-photochemical reactions [ 10]. Based on the advances presented in this review, it will take place; however, some bottlenecks will need to be overcome. As such, many photochemical reactions are still prone to generate multiple impurities, albeit in signiﬁcantly lower amounts than in most batch applications. Currently, the eective separation of these impurities is typically achieved by o -line column chromatography rather than e ective in-line puriﬁcation tools. In addition, most cases reported in this review still rely on relatively high dilutions in order to realize e ective photochemical transformations. While this will prevent formation of precipitates that can be detrimental to ﬂow reactions, these high dilutions are unattractive, as they result in larger amounts of solvent waste. Furthermore, as the e ective telescoping of additional downstream reactions requires matched concentrations of reactive species, various in-line analysis tools will be vital in achieving e ective multi-step sequences [ 13,79]. Additional tools for unit operations that help with recycling solvents and concentrating streams in situ may be required both to improve the sustainability (E-factor and process mass index) and ensure the kinetics of bimolecular reactions are not hampered by exceedingly high dilutions. Additional advances can be expected based on substituting high-power UV lamps with high-e ciency LEDs and utilizing solar light to bring about the photochemical synthesis of target molecules in a much more sustainable manner. 6. Conclusions and Outlook Based on the applications highlighted in this short review, continuous photochemistry has indeed enjoyed a very successful period with numerous reports detailing its various beneﬁts. From this, it is evident that the best solutions are obtained when innovative reactor designs are developed and applied to a given synthetic challenge, thereby exemplifying the synergy that can be gained from the union of synthetic chemistry and chemical engineering. At this point it appears that most applications of continuous ﬂow photochemistry target synthetic methodology based on photo-cycloadditions and photo-rearrangement reactions. However, several reports demonstrate the applicability of these methods to the generation of more complex structures, such as natural products and their analogs, which in many cases is complemented by more scalable processes to deliver gram or even kilogram quantities of material. A further positive development in this ﬁeld concerns the transition from non-selective UV lamps to more energy-e cient, high-power LEDs, and in some cases even solar light. This directly impacts the sustainability of the resulting processes and will be a key element in achieving greener chemical synthesis. Whilst these developments were typically led by academic researchers, it is understood that all major pharmaceutical companies are developing their own continuous photochemical transformations.	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0008
Molecules 2020 ,25, 356 10 of 13 In addition, many publications outline collaborative e orts between academics and their industry-based colleagues, again highlighting the synergy of such joint projects. It can be anticipated that this development will render further applications of continuous light-driven reactions, enabling the synthesis of bioactive compounds, and as a consequence, will compensate for the relative scarcity of drug-based applications at this point through innovative and powerful applications in both medicinal chemistry settings and the scaled production of drugs. Author Contributions: Conceptualization, writing, editing and reviewing—M.D.F., C.B. and M.B. All authors have read and agreed to the published version of the manuscript Funding: This research received no external funding. Acknowledgments: We would like to acknowledge support of our research in this area from the School of Chemistry (PhD demonstratorships to M.D.F. and C.B.) and University College Dublin (SF1606 and SF1609) as well as SSPC through PharM5. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Bach, T.; Hehn, J.P . Photochemical Reactions as Key Steps in Natural Product Synthesis. Angew. Chem. Int. Ed.2011 ,50, 1000–1045. [CrossRef] 2. Homann, N. Photochemical Reactions as Key Steps in Organic Synthesis. Chem. Rev. 2008 ,108, 1052–1103. [CrossRef] [PubMed] 3. Kärkäs, M.D.; Porco, J.A., Jr.; Stephenson, C.R.J. Photochemical Approaches to Complex Chemotypes: Applications in Natural Product Synthesis. Chem. Rev. 2016 ,116, 9683–9747. 4. Ravelli, D.; Protti, S.; Fagnoni, M. Carbon-Carbon Bond Forming Reactions via Photogenerated Intermediates. Chem. Rev. 2016 ,116, 9850–9913. [CrossRef] [PubMed] 5. Romero, N.A.; Nicewicz, D.A. Organic Photoredox Catalysis. Chem. Rev. 2016 ,116, 10075–10166. [CrossRef] [PubMed] 6. Skubi, K.L.; Blum, T.R.; Yoon, T.P . Dual Catalysis Strategies in Photochemical Synthesis. Chem. Rev. 2016 , 116, 10035–10074. [CrossRef] [PubMed] 7. Gutmann, B.; Cantillo, D.; Kappe, C.O. Continuous-Flow Technology—A Tool for the Safe Manufacturing of Active Pharmaceutical Ingredients. Angew. Chem. Int. Ed. 2015 ,54, 6688–6729. [CrossRef] 8. Movsisyan, M.; Delbeke, E.I.P .; Berton, J.K.E.T.; Battilocchio, C.; Ley, S.V .; Stevens, C.V . Taming hazardous chemistry by continuous ﬂow technology. Chem. Soc. Rev. 2016 ,45, 4892–4928. [CrossRef] 9. Baumann, M.; Baxendale, I.R. The synthesis of active pharmaceutical ingredients (APIs) using continuous ﬂow chemistry. Beilstein J. Org. Chem. 2015 ,11, 1194–1219. [CrossRef] 10. Britton, J.; Raston, C.L. Multi-step continuous-ﬂow synthesis. Chem. Soc. Rev. 2017 ,46, 1250–1271. [CrossRef] 11. Jensen, K.F. Flow chemistry—Microreaction technology comes of age. AIChE J. 2017 ,63, 858–869. [CrossRef] 12. Fitzpatrick, D.E.; Ley, S.V . Engineering chemistry for the future of organic synthesis. Tetrahedron 2018 ,74, 3087–3100. [CrossRef] 13. Baumann, M. Integrating continuous ﬂow synthesis with in-line analysis and data generation. Org. Biomol. Chem. 2018 ,16, 5946–5954. [CrossRef] [PubMed] 14. Cambie, D.; Bottecchia, C.; Straathof, N.J.W.; Hessel, V .; Noël, T. Applications of Continuous-Flow Photochemistry in Organic Synthesis, Material Science, and Water Treatment. Chem. Rev. 2016 ,116, 10276–10341. [CrossRef] [PubMed] 15. Sambiago, C.; Noël, T. Flow Photochemistry: Shine Some Light on Those Tubes! Trends Chem. 2019 . [CrossRef] 16. Knowles, J.P .; Elliott, L.D.; Booker-Milburn, K.I. Flow photochemistry: Old light through new windows. Beilstein J. Org. Chem. 2012 ,8, 2025–2052. [CrossRef] 17. Elliott, L.D.; Knowles, J.P .; Koovits, P .J.; Maskil, K.G.; Ralph, M.J.; Lejeune, G.; Edwards, L.J.; Robinson, R.I.; Clemens, I.R.; Cox, B.; et al. Batch versus Flow Photochemistry: A Revealing Comparison of Yield and Productivity. Chem. Eur. J. 2014 ,20, 15226–15232. [CrossRef] 18. Politano, F.; Oksdath-Mansilla, G. Light on the Horizon: Current Research and Future Perspectives in Flow Photochemistry. Org. Process Res. Dev. 2018 ,22, 1045–1062. [CrossRef]	e3b3828c6c32f511c9b5fa9c4f08188c1746e1a9	page_0009
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IAGG 2017 World Congressa Chinese city. Frailty was assessed using the FRAIL Scale. A healthy lifestyle accumulation index was constructed using measures on physical activity, daily living habits and social participation. Data on ADL, IADL, depression, cognition and socio-demographics were also collected. The prevalence of frailty was 3.9%, higher in women (4.7%) than in men (2.5%). Women adopted more healthy lifestyles (mean 9.8) than did men (mean 8.9). Increasing age, female, ADL and IADL impairments, and depression were associated with an incremental risk of frailty. Healthy lifestyle was an independ - ent protective factor of frailty (OR = 0.89, 95% CI = 0.82 - 0.97) after adjusting for socio-demographics and general health status, significantly in men (OR = 0.84, 95% CI = 0.73 - 0.97), but not in women (OR = 0.91, 95% CI = 0.82–1.00). Healthy lifestyle accumulation attenuates the risk of frailty in Chinese community-dwelling older adults. This suggests that it is relevant for elders, especially for aged males, to adopt healthy lifesyle in order to prevent or delay the onset of frailty. PHYSICAL FRAITY AND ONE-YEAR READMISSIONS AMONG GERIATRIC TRAUMA PATIENTS C. Maxwell1, M.S. Dietrich1, R.S. Miller2, 1. Vanderbilt University School Of Nursing, Nashville, Tennessee, 2. Vanderbilt University, Nashville, Tennessee Background: An understanding of acute care readmissions among older adults informs quality improvement efforts and guides policy initiatives. Among geriatric trauma patients (GTPs), studies examining one-year readmissions have been confined to retrospective analyses of trauma registry data and/or large state-wide discharge data. We conducted a pro - spective cohort study among GTPs who remained alive at four time points over one year, and examined all causes of readmissions, repeat readmissions, and pre-injury frailty sta - tus. We hypothesized that pre-injury physical frailty would predict readmissions among GTPs. Methods: Sample: 188 adults >/= age 65 admitted to a level one trauma center over a six month period (Oct2013- Mar2014) with a primary injury. Pre-injury physical frailty measures were obtained on admission from surrogate respondents. Procedure: After hospitalization, home phone calls were made to patients or surrogates at 30, 90, 180 and 360 days. Data Analysis: Frequency distributions, logistic regression models. Results: Over a 1-year period, 46 patients (25%) died and 55 patients (40%) still living at 4 time points experienced one or more readmissions, for a total of 125 readmissions. Seventeen patients (31%) had > 1 readmission prior to death or within 1-year. Reasons for readmission included: follow- up surgeries (30%), medical complications (28%), medical comorbidities (22%), and repeat falls (9%). After controlling for age, comorbidites, injury severity, pre-injury frailty and cognitive impairment, pre-injury frailty status was the only statistically significant predictor of at least 1 readmission </= one-year post injury (O.R.=1.52, p=0.039). Conclusion: Our findings extend our understanding of the influence of frailty on GTP outcomes, and highlight the importance of frailty screening upon hospital admission to facilitate patient-centered care and shared decision-making.PATTERNS OF MULTIPLE HEALTH-RELATED BEHA VIORS IN NON-DISABLED OLDER ADULTS WITH FRAILTY W. Chang , 1. National Health Research Institutes, Taipei City, Taiwan, 2. Ministry of Health and Welfare, Taipei City, Taiwan Previous research has found the protective effects of health-related behaviors such as social engagement and using preventive services on the onset of disability for older adults with frailty. This study aimed to identify heterogeneous per - formance types based on multiple health-related behaviors, and to investigate the factors associated with the behavio - ral patterns. Data was drawn from the 2011 survey data of Taiwan Longitudinal Study on Aging (TLSA) with a sample of 1685 individuals aged 58 years or older who had prefrailty or frailty and no ADL disability. Health-related behaviors including smoking, harmful alcohol use, betel quid chewing, leisure-time physical activity, Qui-gong or transcendental meditation, preventive services utilization, and volunteer - ing or community activity were analyzed to identify latent behavioral patterns by applying latent class analysis (LCA) with covariates using a multinomial logistic regression frame - work. Four behavioral patterns were identified: Healthy Life Style with High Social Engagement (prevalence of 25.4%), Physical Activity (19.0%), Inactive Life Style (37.7%), and Risky Life Style (17.9%). Younger age, being male, lower socioeconomic status (SES), lower number of chronic dis - eases, and being more frail increased the risk of being in the Inactive and Risky classes. Compared with the Healthy and Social Engagement class, the Physical Activity class tended to have lower SES. Intervention programs improving healthy behavioral patterns to prevent the development of disability for frail older adults could be designed according to the iden - tified related factors. Policies on aging health should focus more on disadvantaged people and those who still have no or few chronic diseases. FRAILTY TRAJECTORIES OF MEXICAN ORIGIN OLDER ADULTS B.T. Howrey , S. Al Snih , K.J. Ottenbacher , Family Medicine, University of Texas Medical Branch at Galveston, Galveston, Texas Progressive physical frailty in older adults is associated with increased risk of falls, disability, institutionalization and mortality; however, there is considerable heterogeneity in frailty trajectories over time. We identify heterogeneous frailty trajectory groups and examine the specific contribu - tion of health conditions to frailty trajectories among older Mexican origin adults. We use a sample from the Hispanic Established Population for the Epidemiological Study of the Elderly (HEPESE) with a count of frailty criteria in 1995: slow gait, weak hand grip strength, exhaustion, and unex - plained weight loss (n=2040). Using group based trajec - tory models we identified three frailty groups: low stable (n=96), moderate progressive (n=1456), and high progressive (n=488). The small stable group was significantly younger and had fewer baseline health conditions than either the moderate or high frailty groups, while the high frailty group had comparatively higher rates of stroke and hip fracture Innovation in Aging , 2017 , Vol. 1, No. S1 817 Copyedited by: OUP	d92c345558961e8290e78a7ae37fe235ab540a8a	page_0000
IIAGG 2017 World Congressat baseline than the other groups. Poisson regression mod - els found that frailty counts increased with diabetes (IRR 1.19, 95%CI 1.09–1.29 and IRR 1.17, 95% CI 1.10–1.24 respectively) and pain (IRR 1.19, 95% CI1.09-1.29 and IRR 1.18, 95% CI 1.16–1.41 respectively) and decreased with church attendance (IRR 0.88, 95% CI 0.82–0.95 and IRR 0.88, 95% CI respectively) in the moderate and high groups. Covariates were not associated with changes in frailty in the stable group. These results suggest that while frailty increases for the majority, it does so at different rates. The presence of a small, low frailty group over time warrants further study. THE LINK BETWEEN DEPRESSION AND FRAILTY AMONG OLDER VETERANS FROM A VA GERIATRIC CLINIC C.J. Burant2,1, P.A. Higgins2,1, H. Kim3, D.H. Canaday2,1, T. Hornick2,1, 1. Case Western Reserve University, Bedford, Ohio, 2. Louis Stokes VA Medical Center, Cleveland, Ohio, 3. Mayo Clinic, Rochester, Minnesota Frailty is described as the vulnerability to declining health that could contribute to increased morbidity and mortal - ity. Exhaustion and overall weakness are key components of frailty and may have links to depression. Depression and frailty in many ways may not be distinct from one another and may be bidirectional in nature. As older adults’ health becomes compromised, patients may be at higher risk for frailty and depression. The purpose of this prospective pilot study of older veterans (n= 146) mean age = 83.7 (sd=6.1) was to identify the prevalence and associated factors with depression and two measures of frailty (Fried and Gill). In this study of a VA outpatient geriatric clinic, patients were screened for depression using the GDS-15 and for frailty using the Fried Frailty Criteria (weight loss, exhaustion, physical activity, walk time, grip strength) the Gill Frailty Instrument (walk time, sit to stand). In the current study, 25 (17.1%) patients reported some signs of depression (GDS 5 or greater). Regarding frailty, using the Fried criteria 77(52.7%) had some frailty, while 48 (32.9%) were frail. For the Gill criteria, 31 (21.2%) had some frailty and 49 (33.6%) were frail. Depression was correlated with Fried Frailty (r=.30) and its components including: difficulty in walking (r=.22); exhaustion [lack of effort (r.=33) and could not get going (r=.41)]. Depression was not related to Gill frailty, except for its component of difficulty in walking (r=.22). More in-depth studies are needed to fully understand the cyclical nature of the relationship between depression and frailty. SESSION 3625 (POSTER) GENDER ISSUES IN AGING GENDER DIFFERENCES IN CARDIOVASCULAR DISEASE: COMORBID POST-TRAUMATIC STRESS DISORDER AND DIABETES C.J. Gibson1,2, Y. Li1,3, S. Inslicht1,2, A.L. Byers1,2, 1. San Francisco VA Medical Center, San Francisco, California, 2. University of California, San Francisco, San Francisco, California, 3. NCIRE-The Veterans Health Research Institute, San Francisco, CaliforniaPost-traumatic stress disorder (PTSD), an often chronic and debilitating condition with health impacts across the lifespan, is associated with increased risk for both diabe - tes mellitus and cardiovascular disease. Depression comor - bid with diabetes is known to contribute more strongly to cardiovascular disease and mortality than either condition alone, and this risk may be particularly pronounced among women. Similar patterns linking comorbid PTSD and dia - betes with cardiovascular risk may be expected, but have not been established. These associations were examined in the Department of Veterans Affairs National Patient Care Database (n=163,184, 4% female), a retrospective cohort study of Veterans 55 years and older. PTSD and diabetes mel - litus at baseline (2000–2003) were related to risk for inci - dent cardiovascular disease (myocardial infarction, stroke, or transient ischemic attack) over a 10 year follow-up period (2004–2014) using Cox proportional hazard models. After adjusting for demographic variables, medical conditions, and psychiatric diagnoses, there was a significant three-way interaction between gender, diabetes, and PTSD predicting incident cardiovascular disease (p=0.03). Risk for incident cardiovascular disease was over three times higher for women Veterans with comorbid diabetes and PTSD compared to PTSD alone (HR 3.18, 95% CI 1.10–9.24), but comorbid diabetes did not increase risk for male Veterans with late-life PTSD (HR 0.96, 95% CI 0.86–1.07). These findings high - light the need for research to identify mechanisms contrib - uting to increased risk for morbidity and mortality among women affected by these commonly co-occurring conditions, and the importance of examining gender differences in PTSD and its health-related comorbidities. MEN AND AGING: NEGOTIATING MASCULINITIY M. Charpentier , 1. Social Work, UQAM-University of Quebec in Montreal, Montreal, Quebec, Canada, 2. Research chair on Aging and Citizen Diversity, Montréal, Quebec, Canada This poster presents the findings of a qualitative study on men’s experience of aging that was conducted in Québec with 24 men aged 65 to 92. Their experience was examined from a subjective and reflexive (relationship to the self, body, others) point of view and based on the conduct of actors in their daily environment and the public arena (Dubet 1994). We will show that older men experience grief associated with aging, especially losses linked to the body in terms of physical aptitudes/performance, sexuality and sex appeal. They see themselves as increasingly unable to meet expecta - tions associated with the hegemonic model of masculinity, in other words, the dominant standards and values concern - ing masculinity (Thompson and Wearthy 2004), which can be translated into the traditionally male qualities of emo - tional control, strength, and competitiveness (Roy 2008). Paradoxically, their susceptibility to the hegemonic stand - ards of masculinity, which causes them to experience aging in terms of loss, is also what inspires these men to (re)act, and exercise what could be called their power to act. Faced with the loss of power over their body, older men develop various strategies, which change over time, to negotiate the effects of aging. Of these, the principal ones are: recreating a significant Innovation in Aging , 2 01 7, Vol. 1, No. S1 818 Copyedited by: OUP	d92c345558961e8290e78a7ae37fe235ab540a8a	page_0001
Creation of a low-entropy quantum gas of polar molecules in an optical lattice Steven A. Moses, Jacob P. Covey, Matthew T. Miecnikowski, Bo Yan,Bryce Gadway,yJun Yez, and Deborah S. Jinz JILA, National Institute of Standards and Technology and the University of Colorado, and the Department of Physics, University of Colorado, Boulder, CO 80309, USA (Dated: October 1, 2018) Ultracold polar molecules, with their long-range electric dipolar interactions, oer a unique plat- form for studying correlated quantum many-body phenomena such as quantum magnetism. How- ever, realizing a highly degenerate quantum gas of molecules with a low entropy per particle has been an outstanding experimental challenge. In this paper, we report the synthesis of a low entropy molecular quantum gas by creating molecules at individual sites of a three-dimensional optical lat- tice that is initially loaded from a low entropy mixture of K and Rb quantum gases. We make use of the quantum statistics and interactions of the initial atom gases to load into the optical lattice, simultaneously and with good spatial overlap, a Mott insulator of bosonic Rb atoms and a single-band insulator of fermionic K atoms. Then, using magneto-association and optical state transfer, we eciently produce ground-state molecules in the lattice at those sites that contained one Rb and one K atom. The achieved lling fraction of 25% indicates an entropy as low as 2 :2kB per molecule. This low-entropy molecular quantum gas opens the door to novel studies of transport and entanglement propagation in a many-body system with long-range dipolar interactions. Polar molecules are an ideal candidate system for studying spin physics and emulating quantum mag- netism [1{4]. However, low temperatures and long life- times are required. Ultracold fermionic KRb molecules have been created at a temperature, T, close to the Fermi temperature, TF[5], but cooling the trapped gas deeply into quantum degeneracy has yet to be demonstrated. The largest obstacle arises from the fact that two KRb molecules can undergo a chemical reaction and this lim- its the lifetime of the trapped gas [6]. Furthermore, the chemical reaction rate increases in an applied electric eld because of the attractive part of the dipole-dipole interactions [6]. A solution to this problem is to conne the molecules in a deep optical lattice in order to restrict collisions [7{9]. In particular, the lifetime of ground- state molecules in a deep three-dimensional (3D) lattice was demonstrated to be longer than 20 s and limited by o-resonant scattering of the lattice light [9]. With the chemical reactions mitigated, the remaining challenge is to create a low entropy system, which in the lattice corre- sponds to increasing the lling fraction. In this paper we report the realization of a high-lling, low-entropy quan- tum gas of ground-state molecules in a deep 3D lattice using a quantum synthesis approach. Simulating quantum many-body physics with lattice- conned atoms requires a lling near unity and corre- spondingly low entropy [10]. This condition can be sig- nicantly relaxed with polar molecules thanks to their long-range dipolar interactions, which allow for a decou- pling of spin (rotational state of the molecules) and mo- tion so that only the spin entropy, which can be pre- Current Address: Department of Physics, Zhejiang University, Hangzhou, China 310027. yCurrent Address: Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. zTo whom correspondence should be addressed: ye@jila.colorado.edu, jin@jilau1.colorado.edupared to be near zero, is relevant [11]. This was recently demonstrated in Refs. [12, 13], where a spin-1/2 system was realized by encoding spin in the rotational degree of freedom of the KRb molecules. At dilute lattice ll- ings, spin exchange via dipolar interactions was observed in the density-dependent decay of, and oscillations in, the spin coherence. In order to go beyond the obser- vation of dipolar spin-exchange interactions and explore new scientic frontiers, such as studying the spin-1 =2 Hamiltonian for quantum magnetism [14{20], the propa- gation of excitations and the growth of entanglement and correlations [21, 22], many-body localization [23], exotic quantum phases [24{28], and spin-orbit coupling with molecules [29], higher lattice llings will be essential. De- termining what constitutes high lattice lling depends on the specic experiment in question; however, for dynam- ics studies, one benchmark is the percolation threshold, which for an innite simple cubic lattice with nearest neighbor interactions corresponds to a lling 0:3 [30]. Because of the molecules' long-range interactions and the nite system size, a lling near this percolation threshold is sucient for exploring dynamics such as the propaga- tion of excitations. The original success in realizing a nearly quantum de- generate gas of polar molecules [5] relied on devising tech- niques to make ground-state molecules from an ultracold atom gas rather than directly cooling the molecules. Con- tinuing in this general approach and to sidestep the di- culty in direct cooling of molecules, our strategy for real- izing higher lattice llings for polar molecules is to take advantage of the precise experimental control available for manipulating the initial atomic quantum gas mixture in a 3D lattice. While this basic approach has been pro- posed in a number of papers [31, 32], it is very challeng- ing to realize experimentally. Specically, one needs to prepare a low entropy state of two atomic species in the lattice and combine this with ecient molecule produc- tion. Our molecule production uses magneto-associationarXiv:1507.02377v1 [cond-mat.quant-gas] 9 Jul 2015	bf565c21c1851f395772ed9d29d23bb4c5a53360	page_0000
2 FIG. 1. Quantum synthesis for creating polar molecules. Left: strategy for realizing high lling of molecules in a 3D lattice. We load K (blue) and Rb (red) atoms into a 3D optical lat- tice, with many more K atoms than Rb atoms. In the center of the lattice where the two atom clouds overlap, we realize a Rb Mott insulator and a K single-band insulator, each with near unity lling. Right: Zoom-in showing molecule produc- tion. Sites with one Rb and one K have a high probability of producing molecules, while sites with multiple Rb or with only a single atomic species do not yield molecules. to rst create very weakly bound Feshbach molecules fol- lowed by optical transfer to the molecular ro-vibrational ground state. In previous work, we showed that the con- version eciency from atoms to Feshbach molecules is high (8713%) for lattice sites containing exactly one Rb atom and one K atom [9]. In addition, previous mea- surements of inelastic collisional loss rates for Feshbach molecules with K or Rb atoms [6, 33] suggest that hav- ing an extra atom on a lattice site will be detrimental to molecule production at that site. The basic scheme is illustrated in Fig. 1. By loading a nearly pure Bose-Einstein condensate (BEC) of Rb atoms into a 3D optical lattice, we can achieve a Mott insulator (MI) state. Here, repulsive interactions between the Rb atoms drive a transition to a state that has an integer number of particles per site [34], and the lattice depth is subsequently increased to pin the Rb atoms. For making molecules, the initial BEC density should be suciently low to avoid having multiply occupied sites. For spin- polarized fermionic K atoms, Pauli blocking will prevent any site from having more than one K atom if the atoms are all prepared in the lowest band. The optimum case is a K band insulator [35, 36] of one atom per site, which requires starting with a relatively large initial K den- sity. While a MI of Rb and a band insulator of K are relatively straightforward to achieve separately, creating both simultaneously is very challenging. The densities of both the Rb and K gases should be (=2)3prior to loading the lattice, where =2 is the lattice spacing. When loading both species into a common optical lattice, we thus need to work with a Rb BEC with small atom number and a degenerate Fermi gas with large atom num- ber. The Rb MI must be well spatially overlapped with the center of the much larger K distribution. We also need to preserve the high lling of each atomic species in the presence of the other. For this, control over the interspecies interactions is an essential tool. Finally, anyexcess atoms should be removed from the lattice after the molecule production. To prepare the atomic quantum gases, we evaporate Rb in thej1;1istate and sympathetically cool K in the j9=2;9=2istate in a crossed-beam optical dipole trap with a wavelength = 1064 nm. Here, the atomic hyper- ne states are denoted by jF;mFi, whereFis the total atomic spin and mFis its projection. The evaporation is performed at a magnetic eld, B, of 540 Gauss, where the interspecies scattering length, a, is100a0, wherea0is the Bohr radius. This eld provides for modest interac- tions between the two atomic species while being close to an interspecies Feshbach resonance [37] at B0= 546:6 G that is used for tuning of the interactions as well as for the molecule creation. The nal optical trap is cylindri- cally symmetric with a typical axial trap frequency of !z= 2180 Hz (in the vertical direction) and a radial trap frequency of !r= 225 Hz for Rb. The measured trap frequencies for K are 2 260 Hz and 2 30 Hz. The larger vertical trap frequency helps prevent separa- tion of the Rb and K clouds due to gravitational sag. Immediately after the evaporation, we turn o the in- terspecies interactions by ramping Bto 543.6 G where a= 0. At this point, we have a Fermi gas of between 1105and 2105K atoms and a nearly pure Rb BEC with 103to 104atoms. Once the Rb BEC forms, Rb no longer thermalizes eciently with K, and as a result the temperature of the K gas is limited to T=TF0:3. We then smoothly turn on, in 150 ms, three retro-re ected beams with = 1064 nm that form a cubic optical lat- tice. Two of the lattice beams are in the horizontal plane, while the third beam is at an angle of 6from vertical. The nal lattice depth is between 20 and 25 ERb R, where ERb R=~2k2 2mis the recoil energy for Rb, k=2 , andm is the mass of the Rb atom. We image the atom clouds, either in situ in the lattice or after a time-of- ight (TOF) expansion, using resonant absorption imaging with a probe beam that propagates along the vertical direction. Figure 2A shows an example of TOF images of the Rb gas that show the disappear- ance of coherent matter wave interference as the lattice depth is increased beyond the super uid-Mott insulator transition. For these images, the number of Rb atoms is 8104and the nal lattice depth in units of ERb R is 12, 17, and 22, from left to right. Figure 2B shows an image of 1 :8105K atoms after expansion from the lattice, where the lattice was turned o more slowly for band-mapping [38]. A trace through this image along the direction of one of the horizontal lattice beams, which is rotated by roughly 45with respect to the camera axes, is shown in Fig. 2C. This trace, which is averaged along the other horizontal lattice direction, shows that most of the atoms are in the lowest band. The spatial coordi- nate for the expanded gas image has been converted to quasimomentum in units of ~k. Figures 2D and 2E show in situ images of Rb and K, re- spectively. Note that the Rb cloud is signicantly smaller than the K cloud. To verify that the clouds are spa-	bf565c21c1851f395772ed9d29d23bb4c5a53360	page_0001
3 A B C F E D−2 −1 0 1 200.10.20.30.4 Quasimomentum (¯ hk)OD FIG. 2. (A)The super uid-Mott insulator transition for Rb. The three images of Rb were taken after 8 ms of expansion from the lattice, where the nal lattice depth is 12, 17, and 22 ERb Rfrom left to right. The optical depth (OD) for each image is indicated by the colorbar to the right of the image. (B) Band-mapping of K, imaged after 11.5 ms of expansion. (C) Cut through the K band-mapping image showing the OD vs. quasimomentum. (D) In situ image of 2104Rb atoms. (E)In situ image of 1:8105K atoms. (F)In situ image of the K cloud after initiating loss due to K-Rb inelastic collisions. The resulting hole in the K cloud demonstrates that the initial spatial overlap with the Rb cloud was good in all three directions. tially overlapped, we use an RF pulse to transfer the Rb atoms to thej2;2istate in order to induce spin-changing collisions that result in loss of K and Rb atoms on the same lattice site. The resultant hole in the K distribu- tion (Fig. 2F) clearly demonstrates that the clouds are overlapped in the trap. We determine the peak lling fraction from ts to the measured atomic distributions. The K Fermi gas is de- scribed by a Fermi-Dirac distribution, which can be ap- proximated by a Gaussian. In this case the peak lling is: fGauss =N(=2)3 (2)3=2xyz; (1) whereNis the number of atoms and x,y, andzare the Gaussian rms widths. For the Rb MI, the distribution is better described by a Thomas-Fermi (TF) distribution[39]. In this case the peak lling is: fTF=15N(=2)3 8RxRyRz;: (2) whereRx,Ry, andRzare the Thomas-Fermi radii. We image the gas along z, so we determine the radial size. The vertical size is smaller by a factor of A= 6:4(1), which is measured for a thermal gas of Rb in the com- bined potential of the optical trap and lattice. Figure 3A shows the measured peak lling ( fTF) for Rb. For a comparison to the data, we calculate the T= 0 MI distribution for our trap, convolve this distribution with a Gaussian lter to account for the nite imaging resolution, bin the data into pixels, and then t with a TF distribution [40]. For very small samples, the size of the cloud is about twice the imaging resolution. The data matches well with the T= 0 calculation, and from this comparison, we infer that the N= 1 Rb MI occurs	bf565c21c1851f395772ed9d29d23bb4c5a53360	page_0002
4 00.20.40.60.81fwith K/fRb −300−200−100 0100 20000.20.40.60.81 Scattering length ( a0)BEC fractionA BC 00.20.40.60.81Peak ﬁlling 0.20.30.40.50.60.7 T/TF 0.20.30.40.50.60.7 0 10 20 3000.20.40.60.81 K number (104)12 51020 50100012345 Rb number (103)Peak ﬁlling FIG. 3. (A)Measured Rb MI peak lling fTF(blue points) vs. Rb number. The cloud size was extracted from a TF t to the in situ image, while the number was either extracted from the in situ image or a Gaussian t to the cloud after a few ms of free expansion. In either case the lling was computed according to Eq. (2) in the text. The green staircase displays the calculated peak occupancy for a T= 0 distribution [40]. The total harmonic connement (including the lattice light) is represented by !r= 2(382) Hz and!z= (6:40:1)!r. The orange band shows a t to the calculated density distribution, accounting for nite imaging resolution and pixelation present in the experiment. (B)Peak lling fGauss of K for a lattice depth of 9 EK R (blue points), indicating the onset of a K band insulator for NK>105. The red points show the measured T=TFof the initial K gas before loading the lattice. (C)Top: peak lling of Rb in the lattice in the presence of 1 :4105K as a function of the interspecies scattering length, normalized to the lling of Rb without K. Bottom: the initial BEC fraction in the optical trap under the same condition. Here, the number of Rb atoms is between 2 :8103and 5:2103. The background (non-resonant) scattering length is indicated by the dashed red line. The red shaded bar indicates the BEC fraction of Rb without K. for less than 5000 atoms in our trap. Figure 3B shows the measured peak lling of K ( fGauss ) in the lattice. Here, the lattice depth is 23 ERb R; however, given the dierent mass and ac polarizability for K, this is equivalent to only 9 EK R, whereEK Ris the recoil energy for K atoms. We nd that the measured fGauss rises with increasing K atom number (blue points), and saturates around 80% for K numbers 1105. For this data, T=TFdecreases with increasing K number (red points). The saturation of the lattice peak lling is consistent with the onset of a band insulator in the center of the lattice. The data in Figs. 3A and 3B show that to achieve op- timal molecule production, the initial BEC should have less than 5000 atoms for a MI with mostly one atom per site, while the Fermi gas should have more than 105 atoms to reach the band insulating limit. When loading both atom species simultaneously, K can aect the lling of Rb, and we nd empirically that turning o interac- tions by going to a= 0 is optimum. To illustrate this eect, Fig. 3C shows both the measured peak lling of Rb in the lattice and the initial BEC fraction in the opti- cal trap in the presence of 1 :5105K atoms as a functionofaat the end of the evaporation. We observe a clear dependence, with the highest Rb lling and BEC frac- tion achieved near a= 0. Furthermore, we have checked that the Rb lling at a= 0 is unaected by the K for Rb numbers between 2 103and 105. While the interac- tions between K and Rb atoms during the lattice loading could aect the MI [32, 41], in our data the dominant ef- fect appears to be a higher initial Rb temperature (lower BEC fraction), which results in a poor MI. This higher initial temperature comes from the intrinsic diculty of sympathetically cooling a large K gas through thermal contact with a smaller number of Rb atoms. In preparing the gas for molecule creation, we nd an additional issue arising from interspecies interac- tions. The rst step in creating ground-state molecules is magneto-association, which consists of adiabatically sweepingBacross the K-Rb Feshbach resonance from high to low eld. A schematic of the Feshbach resonance and molecule creation process is shown in Fig. 4A. How- ever, starting from a= 0, which occurs for B < B 0, we rst need to jump Bto the high-eld side of the reso- nance. This jump should ideally be diabatic in order to	bf565c21c1851f395772ed9d29d23bb4c5a53360	page_0003
5 540 550 560−600−400−2000200400 \|9/2,−9/2/angbracketright \|9/2,−7/2/angbracketright B (Gauss)Scattering length ( a0) 540 550 560 B (Gauss)540 550 560 B (Gauss)A B C 1 3510 2030508015000.20.40.60.81 Rb number (103)NKRb/NRb FIG. 4. (A) The interspecies Feshbach resonance. The atoms are loaded into the lattice at a= 0. The K atoms are then transferred to a hyperne state ( j9=2;7=2i) that does not participate in the resonance, and Bis swept above the resonance. After transferring K back to the j9=2;9=2istate, magneto-association proceeds by sweeping Bfrom above to below the resonance (middle and right panels). The Feshbach molecules are then transferred to the absolute ground state using STIRAP. (B)Fraction of Rb atoms converted to Feshbach molecules. The orange shaded region shows the expected fraction of Rb that are on a site with exactly one Rb atom and one K atom. (C)In situ images of ground-state KRb molecules in the lattice. The ground-state molecules are held in the lattice for 40 ms before imaging. For low initial Rb number (top image, average of three repeated experiments), we nd a lling fraction of 25(4)%. For higher Rb number (bottom image, average of seven shots), we observe a hole in the center of the molecular distribution, and the lling is much less. avoid promoting atoms to higher lattice bands [38]; how- ever, with the relatively high local atom densities in the lattice, it is dicult to sweep the eld fast enough. To overcome this problem, we use an RF pulse to transfer the K atoms to a spin state, j9=2;7=2i, that does not ex- perience the 546.6 G resonance. After ramping Babove B0, we transfer the K atoms back to the j9=2;9=2istate and then proceed with the Feshbach association process. We nd that applying these RF transitions improves the nal lling of ground-state molecules by 60% compared to the case of not doing these RF transitions. Figure 4B shows the measured fraction of the Rb num- ber,NRb, that is converted to Feshbach molecules (blue circles). Since we operate with many more K atoms than Rb atoms, a large background of K atoms remains in the lattice after making molecules. This presents a chal- lenge for determining the number of Feshbach molecules, NKRb, which we typically measure by dissociating the molecules and imaging K. To selectively count only the molecules, we use an RF pulse to transfer the back-ground K atoms to the j9=2;7=2istate before dissociat- ing the molecules by ramping Bback above the resonance and selectively imaging the K atoms in the j9=2;9=2i state [40]. For comparison with the data, the shaded band in Fig. 4B shows the product of the measured fGauss = 0:80(5) for K, the calculated fraction of Rb atoms of a T= 0 MI that are on singly occupied sites, and the conversion eciency of preformed pairs reported in Ref. [9]. We nd that the trend of the calculation matches the data, with the conversion eciency decreas- ing for higher Rb number. This is consistent with the assumption that molecules are not produced on sites that have more than one Rb atom. The data lie slightly be- low the calculation; possible explanations for this include nite temperature eects on the MI (which could lead to fewer singly occupied sites) or reduced conversion e- ciency for sites with one Rb and one K atom. For small Rb numbers, we nd that NKRb*=NRbis larger than 50%. As a last step, we use stimulated Raman adiabatic passage (STIRAP) to transfer the Feshbach molecules	bf565c21c1851f395772ed9d29d23bb4c5a53360	page_0004
6 to their ro-vibrational ground state [5]. The typical ef- ciency of this transfer is 89(4)%. Once the molecules are in the ground state, we apply resonant light pulses to remove all unpaired atoms from the lattice. This atom removal is essential since the molecule lifetime in the lattice without atom removal is only a few ms, which we attribute to tunneling of the K atoms that en- ables molecule-atom chemical reactions [6]. After holding the ground-state molecules for 40 ms in the lattice, we take in situ images of the molecule distribution by re- versing the STIRAP process, dissociating the Feshbach molecules, and then imaging the K atoms. Figure 4C shows images of the ground-state molecules in the lattice for cases of both high and low conversion (the arrows indicate the regimes for the two images). The top im- age corresponds to starting with 2500 Rb atoms while the bottom image corresponds to starting with roughly 25000 Rb atoms. The bottom image exhibits a central hole in the molecule distribution, which is consistent with the fact that the central lattice sites contained more than one Rb atom and therefore did not produce molecules. For the higher conversion case, we perform a TF t to the ground-state molecular distribution. From the t we nd 7:9(5)102molecules with a TF radius of 12 :0(2) m. This gives fTF= 0:27(2). As an alternative ap- proach, we can determine the lling by comparing the width of the molecular cloud with that of our simulated T= 0 Rb distribution and assuming a uniform conver- sion eciency of Rb into molecules. The molecules are best described by a distribution that corresponds to an initial Rb number of 3 :2(4)103. Taking the ratio of the measured number of molecules to this Rb number, we ndfmol= 0:25(4), which is consistent with fTF. From the product of the previous measurements, namely the Rb lling,NKRb*=NRb, and the STIRAP eciency, one might expect a ground-state molecule lling of 35%. We attribute the lower measured lling to molecular loss caused by the atom removals. Given the ac polarizability [42] and mass of the ground-state molecules, a lattice with a depth of 25ERb Rcorresponds to 62 EKRb R, whereEKRb R is the re- coil energy for a KRb molecule. The tunneling rate formolecules is therefore negligible. In this case, the en- tropy per molecule can be estimated from the lling in the lattice, with some assumption about the shape of the distribution. Our approach of creating molecules from overlapping Rb and K insulators likely leads to a molecular distribution that is much more homogenous than the alternative approach of adiabatically loading a Fermi gas of molecules into the lattice. The K Fermi gas is homogeneous within the connes of the initial Rb single-shell MI, which should result in a relatively uni- form molecular distribution. For an average lling of fmolin a uniform lattice, the entropy per particle is kB fmol[fmolln(fmol) + (1fmol) ln(1fmol)], which is 2:2kBforfmol= 25% [43]. For comparison, to reach this entropy by adiabatically loading a Fermi gas into a lattice would require starting with a quantum degenerate gas of molecules at T=TF= 0:25. We nd that the employment of dual atomic insula- tors has produced ground-state molecules in a 3D lattice with a very low entropy and a lling that is near the percolation threshold. Under this condition, the system of polar molecules in a 3D lattice is well connected and well suited for experiments probing the propagation of spin excitations in a system with long-range dipolar in- teractions. While the overall number of molecules now is lower than in previous work [12], the system realized here is appropriately sized for imaging with recently developed quantum gas microscope techniques [44{46]. More gen- erally, this work elucidates the many challenges in, and extends the experimental toolbox for, synthesizing ultra- cold molecule systems that can realize novel quantum many-body behavior. ACKNOWLEDGMENTS We thank Zhengkun Fu for experimental assistance, and Michael Wall, Arghavan Safavi, Kaden Hazzard, and Ana Maria Rey for many useful discussions. We acknowl- edge funding from NIST, AFOSR-MURI, ARO-MURI, and NSF grant number 1125844. J. P. C. is funded with an NDSEG graduate fellowship. [1] A. Micheli, G. K. Brennen, and P. Zoller, Nat. Phys. 2, 341 (2006). [2] R. Barnett, D. Petrov, M. Lukin, and E. Demler, Phys. Rev. Lett. 96, 190401 (2006). [3] M. L. Wall, K. R. A. Hazzard, and A. M. Rey, arXiv1406.4758v1 (2014). [4] T. Takekoshi, L. Reichs ollner, A. Schindewolf, J. M. Hut- son, C. R. Le Sueur, O. Dulieu, F. Ferlaino, R. Grimm, and H.-C. N agerl, Phys. Rev. Lett. 113, 205301 (2014). [5] K.-K. Ni, S. Ospelkaus, M. H. G. de Miranda, A. Pe'er, B. Neyenhuis, J. J. 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Greiner, Nature 462, 74 (2009). [45] J. F. Sherson, C. Weitenberg, M. Endres, M. Cheneau, I. Bloch, and S. Kuhr, Nature 467, 68 (2010). [46] N. Gemelke, X. Zhang, C.-L. Hung, and C. Chin, Nature 460, 995 (2009). SUPPLEMENTARY MATERIALS Rb MI calculation For a perfect Rb MI, we calculate the distribution at zero temperature and without tunneling, based on Ref. [39]. We numerically nd the relationship between the chemical potential 0and particle number N. The localat lattice site ( i;j;k ) is(i;j;k ) =0V(i;j;k ), whereVis the harmonic connement. In the zero tun- neling approximation, the occupancy on site ( i;j;k ),n, satises (n1)<(i;j;k) Un. The green staircase in Fig. 3A displays the peak T= 0 occupancy. The green shaded areas indicate experimental uncertainty of the harmonic trap frequency !r= 238(2) Hz and aspect ratio A= 6:4(1). To make a closer comparison to the experiment, we sum the number of atoms along the zdirection (following the experimental geometry where the probe beam integrates along z), and convolve the re- sulting 2D distribution with a Gaussian lter with rms width 4.5(5) lattice sites to simulate the eect of nite imaging resolution. We also account for pixelation by mapping arrays of 6 6 lattice sites onto single pixels. This gives us a convolved, pixelated 2D distribution that we then t with a 2D TF surface to extract fTF, shown as the orange shaded band in Fig. 3A. The width of the band again accounts for the uncertainties in the trap.	bf565c21c1851f395772ed9d29d23bb4c5a53360	page_0006
8 Molecule production and detection Similar to previous work [5, 8, 9], we create weakly bound Feshbach molecules by magneto-association, in which the magnetic eld is swept from above the reso- nance to below the resonance. In the experiments re- ported here, the sweep takes 5 ms, starts at 563 G, and ends at 545.6 G. We then perform STIRAP to trans- fer the Feshbach molecules to the ro-vibrational ground state. The two STIRAP lasers, at 968 nm and 689 nm, are frequency stabilized to a common high-nesse opti- cal cavity. After STIRAP, we remove the unpaired Katoms with a pulse of resonant light and we remove the Rb atoms with a series of microwave adiabatic rapid pas- sages (ARPs) to transfer the atoms to the j2;2istate fol- lowed with pulses of resonant light. We nd that these removals are required in order for the molecules to have a long lifetime in the lattice. To detect the ground-state molecules, we reverse the STIRAP process to transfer the ground-state molecules back to the Feshbach molecule state, sweep the magnetic eld back to 563 G in 1 ms to dissociate the Feshbach molecules, and then image the resulting K atoms. The molecules can also be detected by measuring the resulting Rb atoms, and the numbers agree within the experimental uncertainty.	bf565c21c1851f395772ed9d29d23bb4c5a53360	page_0007
INFECTION ANDIMMUNITY, Aug.1982,p.601-608 Vol.37,No.2 0019-9567/821080601-08$02.00/0 Alteration ofCell-Mediated Immunity toListeria monocytogenes inProtein-Malnourished MiceTreated with Thymosin Fraction Vt THOMAS M.PETRO,* GRACECHIEN, ANDRONALD R.WATSON Department ofFoodsandNutrition, PurdueUniversity, WestLafayette, Indiana47907 Received 14December 1981/Accepted 30April1982 Cell-mediated immunereactivity, measured bylymphocyte responsiveness to phytohemagglutinin, washigherinbothyoungoragedmicefeda4%caseindiet compared withage-matched controls. Treatment invivowithbovinethymosin fraction Vdecreased theresponsiveness tophytohemagglutinin oflymphocytes frommicefedeitherthecontrol ormoderately protein-deficient dietswhen compared withmicetreated invivowithsaline.Resistance againstListeria monocytogenes, knowntobeacell-mediated immunefunction, wasimpaired in youngandagedmicewhichwerefedthelow-protein diet.Treatment with thymosin wasabletosignificantly improve thecell-mediated immune resistance toL.monocytogenes ofmoderately protein-malnourished mice.Thymosin treat- mentimpaired theresistance toL.monocytogenes ofyoungoragedmicefedthe controldiet.Thesplenicnaturalkillercellcytotoxicity ofprotein-malnourished micewasimpaired compared withthatofmicefedthecontroldiet.Treatment withthymosin didnotrestorethenaturalkillercellcytotoxic activityinprotein- malnourished mice,butdidenhance thatactivityincontrolmice. Malnutrition inhumans canadversely affect manyaspects ofthymus-derived (T)cellre- sponses(1,4,26,32).Anincreased incidence of infection withpathogenic (26)orevennormal floramicroorganisms (9)hasbeenobserved in protein-malnourished children. Asimilarde- creaseinresistance toawidevarietyofpatho- genshasbeenshowninmalnourished experi- mentalanimals (6,15,24).However, the mechanisms ofaltereddiseaseresistance are oftenunclear, asmoderate proteinmalnutrition inexperimental animalshasbeenshowntoboth enhance (2,5,24,33)anddecrease (18,25,31) immune responses. Sincethedevelopment ofthecell-mediated immune (CMI)systemisdependent uponthe production ofthymichormones (8),itisnot surprising thattheretarded development ofthe thymusgland(34)anddecreased thymichor- moneactivity (4)havebeenassociated with nutritionally relatedimmunological deficiencies inanimals. Although certaininvestigators have shownbeneficial effectsduetotreatment with thymichormones ofimmunodeficient humans (8,33),otherinvestigators havefoundthatthe treatment oflymphocytes withthymosin frac- tionVcanresultinasuppression ofimmune responses. Thelatterresultispresumably dueto theincreased proportion ofsuppressor lympho- tThisreportisPurdueExperiment Stationpaperno.8753.cytes(22,35).Sincemalnourished animals have beenfoundtobeimmunologically deficient with respecttoresistance toinfection, itisofinterest ifthesehostsrespond inafavorable mannerto treattnent withthymosin fraction V.Thepur- poseofthisinvestigation wastotestthethera- peuticvalueofthymosin fraction Vinwell- nourished andprotein-malnourished mice.The effectsofbothmoderate proteindeficiency or thymosin fraction Vtreatment orbothonthe responsiveness oflymphocytes tophytohemag- glutinin (PHA), natural killercellactivity (NKCC), andresistance againstListeria mono- cytogenes wereexamined. MATERIALS ANDMETHODS Animals anddiets.FemaleBALB/c mice,obtained fromHarlanSprague-Dawley (Indianapolis, Ind.), werecagedingroupsoffive.Themicewhichwere6 weeks(young)or11months(aged)ofagewerefedthe experimental dietsfor5or8weeks,respectively. The micewerefedoneoftwoexperimental diets:(i)a controlcontaining 20%casein,or(ii)amoderately protein-deficient (MPD)dietof4%caseinthatwas equalincalories tothecontroldiet(U.S.Biochemical Corp.,Cleveland, Ohio)(Table1).Whilecontinuing onthesediets,themicewereinjectedintraperitoneally with0.5mlofeitherendotoxin-free 100lOgbovine thymosin fraction V(courtesy ofTeresaL.K.Low, GeorgeWashington University) orphosphate-buffered saline(PBS)(pH7.2)everyotherdayfor1or2weeks, yielding atotaltreatment of400or700F±gofthymosin fraction Vforthemiceondiets(i)and(ii),respective- 601	6af22847549485aaa772d087f74d54cd9382aee3	page_0000
602PETRO, CHIEN, ANDWATSON TABLE 1.AINsemipurified rat-mouse dieta %oftotaldietfor:ComponentControl Lowprotein Caseinhighnitrogen 20.0 4.0 DL-Methionine 0.3 0.3 Cornstarch 15.0 15.0 Sucrose 50.0 66.0 Fiber-Celufil 5.0 5.0 Cornoil 5.0 5.0 AINmineralmix 3.5 3.5 AINvitaminmix 1.0 1.0 Cholinebitartrate 0.2 0.2 aU.S.Biochemical Corp.dietno.10662. ly.Thepreparation ofthymosin fraction Vwasprevi- ouslydescribed (17).Briefly,thebovinethymustissue washomogenized andcentrifuged at14,000xg.The supernatant washeatedto80°C,precipitated with acetone, thenprecipitated withammonium sulfate, andpurifiedonaSephadex G-25column. Resistance toinfection. L.monocytogenes wasini- tiallycultured inTrypticase (BBLMicrobiology Sys- tems,Cockeysville, Md.)soybroth(TSB)overnight andstoredat-70°Cin1-mlsamples of20%glycerol- TSB.Cellsfromstocksuspensions weregrownfor16 hinTSBat37°Cbeforeeachinjection. Thecellswere harvested bycentrifugation at10,000xgandwere washedtwiceinsterilePBS.Theconcentration of washedcellswasadjusted spectrophotometrically (at 550nm)toapproximately 3x108cellsperml.The cellswerethenadjusted totheappropriate concentra- tion,1x104perml,neededforinjection. Theexact numberofviablebacterial cellswasdetermined onthe basisofdeveloping colony-forming units(CFU)on Trypticase soyagar(TSA)afterincubation at37°Cfor 24h.Approximately sevenmicepergroupwere injectedwithL.monocytogenes at:(i)day1after receiving fourinjections, (ii)day1afterseveninjec- tions,or(iii)day7afterseveninjections ofthymosin fractionVorPBS.Atday7afterinjection, thelivers ofmicewereextracted andhomogenized inPBS, usingaTeflon-coated pestleglasshomogenizer. Fifty microliters ofseveraldilutions fromanindividual mousehomogenate wasspreadontoTSAplatesin duplicate todetermine thenumberofCFUdueto infection presentinmicefromeachgroup. Preparation oflymphoid cellsuspensions. The spleensofmiceinfected 7dayspreviously orspleens ofuninfected miceweredispersed intoacellular suspension insterilePBSbygentlyrubbingtheorgans through asterilestainless steelscreen.Erythrocytes werelysedbywashing thesuspensions inTris-buff- ered0.15Mammonium chloride(pH7.2).Cellswere washed onceinPBSandonceinRPMI1640cell culturemedium(GIBCO Laboratories, GrandIsland, N.Y.)containing 10oheat-inactivated fetalcalfserum (culturemedium). Theviability oflymphocytes was assessed bytrypanblueexclusion, andcellconcentra- tionswereadjusted to5x106permlintheculture medium. Measurement oflymphocyte mitogenesis. Quadrupli-catesamplesoflymphocytes fromeachmousewere seededintowellsofCostarCluster%platesata concentration of2.5x10Wcellspermlor,whereindicated, at5.0x10'cellsperwell.Mitogenesis was induced intwoofthecultures with0.25,ugofPHA (Burroughs Wellcome Co.,Research Triangle Park, N.C.).Theremaining twocultures, aftertheaddition ofthemedium only,servedascontrols. Thecell suspensions wereincubated at37°Cin5%C02-95% airfor72h.At24hbeforetheendofthatincubation, 1 ,Ciof[methyl-3H]thymidine (45Ci/mmol) (Amersham Corp.,Arlington Heights, Ill.)wasaddedtoeach culture. Thecellsuspensions wereharvested onto glassfiberfiltersandwashedwithabsolute methanol. Theradioactivity present oneachfilterpaperwas measured withaliquidscintillation spectrometer (Packard Instrument Co.,Rockville, Md.). Cytolytic assay.TheNKCCwasmeasured inyoung micefromeachindicated dietary/injection group. Briefly,2.5x10451Cr-labeled L1210mouseleukemia cellswereincubated with5x106spleencellsin1.5ml ofculturemedium at37°Cin5%CO2for16h.After incubation, thecellsuspensions werecentrifuged at 600xgfor5min,and1mlofthesupernatant was evaluated astoradioactivity releasedbymeasurement inagamma counter(Beckman Instruments, Inc., Fullerton, Calif.).Resultswereexpessed astheper- centlysisoftargetcellsaccording totheformula, % lysis=(experimental 51Crrelease -spontaneous 5Cr release)/(maximum 51Crrelease -spontaneous 51Cr release). Statistical analysis. Ananalysisofvariance wasused todetermine thesignificance ofdifferences orinterac- tionsamongthegroupsofmicewithrespecttodiet andtypeoftreatment. Tofacilitate theanalysisofthe datafromtheresistance studies,deadmiceweregiven astandard scoreof8.0log10viableL.monocytogenes cellsperliver.Itwasdetermined thatmoribund ani- malshadapproximately 8.0log10viableL.monocyto- genescellspresentperliver.Thevaluesreported in Fig.1and2donotcontaindatafromdeadanimals; however, thenumberofdeadanimalspergroupwas noted.AnyvalueswithP<0.05wereconsidered significant. RESULTS Therewerenosignificant differences infood consumption between micefedthe20%(2.75g permouseperday)or4%(3.5gpermouseper day)caseindiet.Theyoungmicefedthe20% caseindietweighed 19.63±0.27g,whereas the micefedthe4%caseindietweighedsignificantlyless(13.96±0.76g)attheendofthe5-week feedingperiod. Resistance againstL.monocytogenes. Todeter- minetheeffectofmoderate proteinmalnutrition uponCMIresistance, micefedthecontrol or MPDdietfor3weekswereinfectedintraperito-neallywith1x104L.monocytogenes cells.The numberofviableL.monocytogenes cellspres- entininfected micewasdetermined ondays3 and7afterinfection. Thelevelofinfection on day3isindicative ofthenativepreimmune macrophage phagocytic andbactericidal capaci-tyofthemouse(7),whereas thelevelofinfec- tionbyday7isdependent uponCMIenhance- mentofmacrophage phagocytic andbactericidalINFECT. IMMUN.	6af22847549485aaa772d087f74d54cd9382aee3	page_0001
PROTEIN MALNUTRITION ANDTHYMOSIN TREATMENT 603 TABLE 2.EffectofMPDdietonresistance of youngmicetoinfection withL.monocytogenes Dietary Log1oCFUperliverandspleenatdayprotein afterinfection'level(%) 3b 7c 7d 20 5.29±0.223.38±0.473.38±0.47 45.23±0.194.11±0.624.59±0.72 aSix-week-old BALB/c micewerefedeithera20or 4%caseindietfor3weeks,theninfectedintraperito- neallywith4.0logloofL.monocytogenes cells;the CFUintheliverandspleenwereevaluated ondays3 and7afterinfection. bMean±standard erroronlogloCFUperliverand spleenfromaminimum ofeightmice. cOnlysurviving mice. dSurviving plusdeadmice. activity(18).Theearlyresistance against L. monocytogenes wasnotsignificantly affected in youngmicefedthelow-protein diet(Table2). Byday7,theCMIclearance ofinfecting L. monocytogenes cellswasimpaired inmicefed theMPDdiet,whenCFUfromdeadmicewere included (control, 3.38+0.47versusmalnour- ishedmice,4.59+0.72). Effectofthymosin treatment onCMIresist- ance.Totestthehypothesis thattheimpairment inCMIresistance againstL.monocytogenes observed inmicefedtheMPDdietwasatleast partially theresultoflowthymichormone pro- duction, wetreatedmicefromeachdietary groupofbothageswithaseriesofinjections of bovinethymosin fraction VorPBS.Oneday afteratotaltreatment of400or700,ugofthymosin, micewereinfected with1x104L. monocytogenes cells.Intheyounger mice,the CMIresistance 7daysafterinfection wassignifi- cantlyaffected(F=9.79,P=0.003)bytreat- mentwiththymosin (Fig.1).Thiseffectupon CMIresistance wasdependent onthedietwhich themicereceived duringthetreatment (F= 12.36,P<0.0001).Although theCMIresistance ofmicefedtheMPDdietwasnotalteredbythe thymosin treatment, theCMIresistance ofmice fedthecontroldietwassignificantly suppressed bythethymosin treatment. Asimilarpatternfortheeffectsofthymosin treatment uponCMIresistance wasobserved in agedmice(Fig.2).Again,theeffectthatthymo- sintreatment hadupontheCMIresistance was significantly dependent uponthedietwhichthe micewerefed(F=17.31,P<0.001).The resistance againstL.monocytogenes ofaged micefedtheMPDdietwasenhanced bythe thymosin treatment, beingmostdramatic at7 daysaftertreatment. Theresistance ofmicefed thecontroldietwasimpaired atalltimesafter thymosin treatment. Itshouldbenotedthat,for anunknown reason,thereseemedtobeade-creaseinresistance afterthefirsttreatment point,irrespective ofwhether theanimals were treatedwiththymosin orPBSorwereyoungor aged.Asaresult,therewasanoverallsignifi- canteffectduetotheduration oftreatment inthe young(F=31.24,Ps0.0001)andaged(F= 9.83,P<0.0001)micethatwasnotdependent upondietortypeofinfection. Theresponsiveness ofspleniclymphocytes to thePHAofmiceafterday7ofinfection was significantly affectedbydietinbothyoung(F= 39.06,P<0.001)andaged(F=22.86,P' 0.001)mice.SpleencellsfrommicefedtheMPD diethadsignificantly higherresponsiveness to PHAthandidspleencellsfromcontrolmice (Fig.3and4).Intheyounger mice(Fig.3), thymosin treatment significantly suppressed spleniclymphocyte responsiveness toPHA(F= 12.83,P<0.001)compared withthatofdiet- matched micetreatedwithPBS.Unlikethe youngmice,theeffectofthymosin treatment uponspleencellresponsiveness tothePHA 20%ProteInDet-{ECMiceThymosinInjected 4%ProteinDiet{CCm"MiceThymos Injected (5) 5.0 (4)~~~~~~~~~~~(40 ~3.0 pgThyosinInjctd FIG.1.CFU(standard errorindicated bybars)in liversat7daysafterinfection ofsevenmiceintraperi- toneally with1x104L.monocytogenes cells.The numbers inparentheses indicate deadmicebyday7 afterinfection. Young(6-week-old) micefedeitherthe control orMPDdietweretreatedwithaseriesof injections withPBSoratotalof400or700p.gof thymosin. Micewereinfected 2daysaftertreatment with400or700,ugofthymosin or1weekafter treatment with700p.gofthymosin (*).VOL.37,1982	6af22847549485aaa772d087f74d54cd9382aee3	page_0002
604PETRO, CHIEN, ANDWATSON 6.0 5.0 iI4.0 0 5 93.0 2.0 1.020%ProteinDiet_KIContro nGMMiceThymosinInjected 4%ProteinDiet_CCControlOMiceThymosinInjected pgThymosinInjeced FIG.2.CFU(standard errorindicated bybars)in liversat7daysafterinfection ofsevenmiceintraperi- toneally with1x104L.monocytogenes cells.Num- bersinparentheses indicate deadmicebyday7after infection. Old(11-month) micefedeitherthecontrolor MPDdietweretreatedwithaseriesofinjections with PBSoratotalof400or700,ugofthymosin. Micewere infected 2daysaftertreatment with400or700,ugof thymosin or1weekaftertreatment with700pugof thymosin (). fromtheoldermicewasnotsignificant, irre- spective ofthelevelofdietaryprotein. Spleniclymphocyte responsiveness toPHA.A separate groupof6-week-old micewasfedei- therthecontrol orMPDdietfor5weeks, to determine whether athymosin-induced suppres- sionofspleenlymphocyte responsiveness to PHAwaspresentinuninfected mice.Micewere thentreatedeveryotherdayfor2weekswitha totalof700,ugofthymosin orPBS.Thespleen cellnumbers andresponsiveness toPHAwere evaluated 1dayafterthelastthymosin treat- ment.Thenumberofspleniclymphocytes was reducedsignificantly bythelowdietaryproteinlevels(Table3).Treatment withthymosin did notsignificantly enhance thenumberoflympho- cytesinmicefedtheMPDdiet.However, thymosin treatment significantly increased the lymphocyte numberinmicefedthecontroldiet. Theresponsiveness oflymphocytes toPHAwas significantly enhanced (F=9.53,P=0.004)inmicefedtheMPDdiet.Asintheprevious experiment, thethymosin treatment significant- lylowered thelymphocyte responsiveness to PHA,especially atthehigherconcentration (F =4.66,P=0.038).Again,thesuppressive effect ofthethymosin treatment uponspleniclympho- cyteresponsiveness toPHAwasnotdependent (F=0.161,P=0.691)uponthelevelofdietary proteinfedtothemice.Thethymosin treatment onlyaffected theTlymphocytes; nosignificant effectinthemitogenic responsiveness oflym- phocytes tolipopolysaccharide wasseen(Table 4)(4,693±572and3,908±429cpminmicefed a20%caseindietinPBSorthymosin-treated mice,respectively; 2,979±542and3,641±653 cpminmicefeda4%caseindietinPBSor thymosin-treated mice,respectively). NKCC.The0antigen-bearing lymphocytes, _11 x4,0 62 0. 9 zI- 0 .56 S430.220%ProteinDiet{ymoun Injected 4%ProteinDWt C Control %E MicThymeinInjected 400hI700 700* pgThymosin InjectedI FIG.3.Countsperminute(standard errorindicat-edbybars)of[3H]thymidine in2.5x105PHA- stimulated spleencellsfrommiceinfectedintraperito-neally7daysearlierwith1x104L.monocytogenes cells.Young(6-week-old) micefedeitherthecontrol (20%oprotein) orMPD(4%protein)dietweretreated withaseriesofinjections withPBSoratotalof400or 700,ugofthymosin. Micewereinfected 1dayafter treatment with400or700p.gofthymosin or1week aftertreatment with700,ugofthymosin(*).INFECT. IMMUN. .	6af22847549485aaa772d087f74d54cd9382aee3	page_0003
PROTEIN MALNUTRITION ANDTHYMOSIN TREATMENT 605 ; a% prw.in oi(DMiceThms Injectd g~~~~~4P6o 4#Dist CBOWmoht e DI.K~CMice ThWInjected j5- 3m400 MO0 700 gThyrnosn Injected FIG.4.Counts perminute(standard errorindicat- edbybars)of[3H]thymidine in2.5x105PHA- stimulated spleencellsfrommiceinfected intraperito- neally7daysearlierwith1x104L.monocytogenes cells.Old(11-month) micefedeitherthecontrol(20% protein) orMPD(4%protein) dietweretreatedwitha seriesofinjections withPBSoratotalof400or700Fg ofthymosin. Micewereinfected 1dayaftertreatment with400or700,ugofthymosin or1weekafter treatment with700,ugofthymosin (*). which areresponsive toPHA(12),havefunc- tionswithintheCMIsystemdistinctfromthe0- antigen-negative lymphocytes responsible for NKCCagainst tumorcells(10).Theeffectsof thymosin treatment uponthespleencellpopula- tionresponsible forNKCCactivity wasdeter- minedinyoungmicefedeitherthecontrol or MPDdietinjected everyotherdayfor2weeks withatotalof700,ugofthymosin orPBS.The splenicNKCCactivity against L1210 mouse leukemia cellswasmeasured 1to7daysafter thelastthymosin orPBSinjection (Table5). Unlikespleencellresponsiveness toPHA, NKCCactivity wassignificantly reduced in micefedtheMPDdiet(F=19.42,P<0.001). Nosignificant effect uponNKCCactivity was observed asaresultofthymosin treatment in micefedtheMPDdiet.However, NKCCactivi- tydidincrease 7daysafterthelastthymosin treatment inmicefedthecontrol diet. DISCUSSION Animalmodelstudiesoftheeffectsofmalnu- trition uponimmunity andresistance toinfection haveyieldedresultsthatsometimes differfrom clinicalobservations inhumans(32).Theresults presented inthisreportfurtherdemonstrate this dichotomy, inthatmoderate proteinmalnutri- tioninmiceresultedinhigherresponsiveness of spleencellstoPHA,butlowerCMIresistance againstL.monocytogenes. Themajoracquired resistance mechanism ofmiceagainstL.mono-cytogenes istheCMIsystem(16),whereas the preimmune susceptibility isgoverned bymono- cytephagocytic andbactericidal activity(21).Defective CMIresistance againstL.monocyto- genes, asinmicefedtheMPDdiet,maybethe resultofoneormore"lesions" inthecascadeof eventsthatarepartofCMIresistance, which leadstothekillingofbacteria. Forinstance, one suchlesion maybeintheproduction oflympho-kinesbyimmune Tcells,whichactivate mono- cytesandmacrophages toincrease phagocytic andbactericidal activity(18).EventhoughTcell activityinmicefedtheMPDdietwasenhanced onapercellbasis, astheconcentration ofcells fortheassaywasadjusted toafixedlevel,the totallymphocyte activityinthewholehostmay beless,resulting inlowerCMIresistance. In- deed,spleniclymphocyte concentrations were significantly impaired inmicefedtheMPDdiet. Another suchlesion maybethemacrophage level,thecellresponsible forkillingtheinfecting bacterial cells.However, Cooper etal.(5)have shownthatnativeperitoneal macrophages of moderately protein-malnourished miceactually phagocytize Listeria cellsbetterthanmacro- phagesfromwell-fed mice.Watson etal.(33) havealsoshownthatmacrophages fromprotein- malnourished animals arelargerandhavehigher levelsofsuperoxide dismutase. Peritoneal mac- rophages fromproteincalorie-malnourished rats hadintactbactericidal activity against both gram-positive andgram-negative bacterial cells (13),butanimpairment innon-CMI resistance factors, suchaschemotactic factorsandthe serumopsonins oftheseproteincalorie-mal- nourished animals. Thesetwoeventswould impairtheinvivofunction ofmacrophages. Complement, anon-CMI resistance factor,has beenshown toplayaroleinthedecreasing susceptibility ofmicetoL.monocytogenes (23). Indeed,certaincomplement components have beenshowntobeimpaired byproteinmalnutri- tioninrats(32). TABLE 3.Effectofthymosin fraction V(Thy)on spleniclymphocyte number incontrol ormoderately protein-malnourished BALB/c micea dietary Treatment Spleniclymphocytes (10')protein(%) ype 20 PBS 9.69±0.50"20 Thy 11.23±0.84 4 PBS 5.07±0.81 4 Thy 5.53±0.71 aMicefromeachdietarygroupwereinjected intra- peritoneally with0.5mlofeither100-Lgthymosin fractionVorPBS,oneveryotherdayfor2weeksfora totalof700p.gofthymosin. bMean +standard error.VOL.37,1982 I I	6af22847549485aaa772d087f74d54cd9382aee3	page_0004
606PETRO, CHIEN, ANDWATSON TABLE 4.Effectofthymosin fraction V(Thy)onresponsiveness ofspleencellsfromcontrolormoderately protein-malnourished BALB/c micetoPHA' cpmof[3HJthymidine incorporated" Levelof Treatment 2.5x105cells 5.0x105cells dietary type______________ __________________protein WithPHA Without WithPHA Without WithHA PH WithPHA PHA 20 PBS 29,228±2,916 1,691±364 84,789±5,705 2,014±313 20 Thy 27,804±4,901 634±148 67,144±8,052 1,775±328 4 PBS 51,255±9,208 816±204 100,601 ±6,548 2,319±330 4 Thy 38,514±7,947 888±139 85,572±1,160 1,233±120 Micefromeachdietarygroupwereinjectedintraperitoneally with0.5mlofeither100-Rgthymosin fraction VorPBS,oneveryotherdayfor2weeks,foratotalof700,ugofthymosin. Cellsstimulated with lipopolysaccharide had4,693±572,3,908±429,2,979±542,3,641±653cpminPBS-treated 20%o,Thy-treated 20%o,PBS-treated 4%,Thy-treated 4%mice,respectively. bMean±standard error. SincemicefedtheMPDdietwereimmuno- suppressed withrespect tospleniclymphocyte numbers andCMIresistance againstL.monocy- togenes, whileexhibiting anenhanced respon- siveness toPHA,attempts weremadeinthis studytorestore thatresistance withbovine thymosin fraction V.Thymosin fraction Vcon- tainsatleast30different polypeptides andhas beenusedtohelprestore CMIresponses in humans withimmunodeficiency diseases (8,33) andincrease resistance totumorcellsinwell-fed andmalnourished mice(24a).Inthepresent study,treatment withthymosin hadasuppres- siveeffectuponlymphocyte responsiveness to PHAinanimals fedeitherthecontrol orMPD diet.Wehaveshown, forthefirsttime, a suppressive effectduetothymosin treatment upontheCMIresistance againstL.monocyto- genesinwell-nourished mice.However, thymo- sintreatment enhanced theCMIresistance againstL.monocytogenes inmicefedtheMPD dietcompared withsaline-treated micefedthe MPDdiet.Theseresults wereobserved inboth youngadultandmiddle-aged mice.Itispossible thatthethymosin treatment ofmiceinduced an increase inthesuppressor lymphocyte popula- tion,therefore decreasing theresponsiveness of thetotallymphocyte population toPHA.Thy- mosin a7,oneofthepolypeptides withinfractionV,hasbeenshowntoinduce suppressor Tcells (17).Also,spleniclymphocytes fromthethymo- sin-treated nudemice wereabletoactively suppress theinvitrogeneration ofcytotoxic T cells(22).Otherstudiesinimmunocompetent micehaveshownthatthymosin treatment has noeffect oraslightlysuppressive effect upon spleencellresponsiveness toPHA(26).Wolf (35)hasindicated thatinvitropretreatment of immunocompetent humanlymphocyte popula- tionswiththymosin cansuppress responsive- nesstoPHAandpokeweed mitogen. Interest- ingly,thymosin hasbeenabletosuccessfully reconstitute concanavalin Aresponsiveness in spleencellsfromnudemice.Thismitogen is capable ofinducing suppressor Tcells(28). Previous studieshaveindicated thatthein- creasedresponsiveness oflymphocyte toPHA orsheeperythrocytes inprotein-malnourished miceisrelated toadecrease insuppressor lymphocytes (14,20).Thedatafromthepresent reporthaveshownthatthymosin treatment is abletoinsignificantly decrease theresponsive- nessoflymphocytes frommalnourished miceto PHA.Thiswouldindicate apartialrestoration of thesuppressor lymphocyte population inthese miceasaresultofthethymosin treatment. Otherpolypeptides fromthethymosin prepa- rationhavebeenshown tohaveotherspecific TABLE 5.Effectofthymosin fraction V(Thy)onNKCCagainstL1210leukemia cellsincontrolof moderately protein-malnourished BALB/c micea Levelof Treatment %Cytotoxicity ondayafterlasttreatment: dietaryprotein(%) type 1 7 20 PBS 15.84±1.49b 18.46±2.08 20 Thy 15.16±1.19 24.22±1.60 4 PBS 11.40±1.31 13.33±1.71 4 Thy 12.72±2.68 12.15±2.76 aMicefromeachdietarygroupwereinjectedintraperitoneally with0.5mlofeither100-,ugthymosin fraction VorPBS,oneveryotherdayfor2weeks;thelymphocyte/target cellratiowas200:1. bMean±standarderror.INFECT. IMMUN.	6af22847549485aaa772d087f74d54cd9382aee3	page_0005