Following analysis n = 23 studies were included. Data extraction details included; sample size, age, gender, injury, the exoskeleton used, intervention duration, weekly sessions, total sessions, session duration and outcome measures. Results indicated that exoskeleton gait training elevated energy expenditure greater than wheelchair propulsion and improved gait function. Patients exercised at a moderate-intensity. Powered exoskeletons may increase energy expenditure to a similar level as non-exoskeleton walking, which may improve cardiovascular function more effectively than wheelchair propulsion alone. Patients with neurological impairments often experience physical deconditioning, resulting in reduced fitness and health. Powered exoskeleton training may be a successful method to combat physical deconditioning and its comorbidities, providing patients with a valuable and novel experience. This systematic review aimed to conduct a search of relevant literature, to examine the effects of powered exoskeleton training on cardiovascular function and gait performance. Two electronic database searches were performed (2 April 2020 to 12 February 2021) and manual reference list searches of relevant manuscripts were completed. Studies meeting the inclusion criteria were systematically reviewed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.  Walking8 km·h−1 . For ablectively  such as,ectively . Anton eectively .Due to a reduction in gait and mobility, individuals with neurological impairments such as multiple sclerosis (MS) , stroke However, robotic exoskeletons enable wheelchair users with little or no walking ability to walk over ground, with maximal external support . ProlongThis systematic review was designed to include and evaluate research on the effects of powered exoskeleton training on cardiovascular function and gait performance.PRISMA guidelines were followed to conduct a search of current literature surrounding the topic area . Specifin = 63 relevant titles with two further relevant titles identified through references list searches. Following abstract analysis (n = 65), n = 37 studies were excluded as they did not meet the inclusion criteria. n = 28 entire manuscripts were examined and a total of n = 23 studies were included  calculated various objective and subjective measurements of energy expenditure associated with powered exoskeleton training. Volume of oxygen (2); which was reported in relative and absolute terms including, millilitres per kilogram of body mass per minute (mL·kg−1·min−1), millilitres per kilogram of body mass per kilometre (mL·kg−1·km−1), litres per minute (L·min−1) or millilitres (mL). Heart rate , was measured. Rate of perceived exertion (RPE) was self-reported using a Borg scale and the metabolic equivalent of task (METs) was measured to assess exercise intensity. Physiological cost index , respiratory exchange ratio (RER), the number of calories expended per minute  and blood pressure  were also recorded (All included studies (recorded . n = 21) also measured an element of gait performance. The authors assessed variables such as; 25-foot walk test (25FWT) which recorded time over a 25-foot walk, six-minute walk test (6 MWT) that recorded total distance covered over six minutes of walking. Time up and go test (TUG), which measured the time needed to stand up, balance, walk three metres and back, and sit down. Ten-metre walk test (10 MWT) which recorded the time over a ten-metre walk distance. Two-minute walk test (2 MWT) which measured distance covered over a two-minute walking duration. The 30-min walk test (30 MWT) which recorded distance over a walking time of 30 min. Total steps, overall distance and walking speed were also recorded in some studies (Some included studies ( studies .n = 7) [n = 1) [n = 1) [n = 1) [n = 1) [n = 1) [n = 2) [n = 1) [n = 1) [n = 5) [n = 1) [n = 1) [n = 13) [n = 2) [n = 2) [n = 2) [n = 2) [n = 1) [n = 236). Across the included studies (n = 23), the participants’ ranged from 18 to 67 years of age. The gender of participants was not disclosed by one study, the remaining studies (n = 22) were comprised of 151 males and 65 females (n = 19 studies [n = 23) used a powered exoskeleton (The design of the selected studies included case series/reports (n = 7) ,42,44,47 [n = 1) , prospec [n = 1) , self-co [n = 1) , longitu [n = 1) , prospec [n = 1) , randomi [n = 2) ,43, open [n = 1) , prospec [n = 1) , experim [n = 5) ,32,33,45 [n = 1)  and comp [n = 1)  studies.[n = 13) ,42,44,45 [n = 2) ,31, Cana [n = 2) ,38, Kore [n = 2) ,43, Ital [n = 1) ; one stu [n = 1) , collect females . The pardies n =  were com studies , another studies  and one  studies . All selskeleton .n = 6) [n = 5) [n = 6) [n = 2) [n = 1) [n = 1) [n = 1) [n = 1) [n = 1) [The powered exoskeleton devices used throughout the selected studies included the EKSO (n = 6) ,40,41,44 [n = 5) ,33,42,45 [n = 6) ,39,43,46 [n = 2) ,34, Powe [n = 1) , Angeleg [n = 1) , Hybrid  [n = 1) , SMA Exo [n = 1)  and REX  [n = 1) . The intn = 16 studies recorded2 associated with powered exoskeleton assisted walking , standing (4.8 ± 0.45 mL·kg−1·min−1) and walking (11.3 ± 1.30 mL·kg−1·min−1) element among an able-bodied control group [−1·min−1) and walking (3.91 ± 0.93 mL·kg−1·min−1) element was also examined by Corbianco et al. using a lokomat, which produced significantly lower (p < 0.001) mean 2 recordings when compared to the Ekso GT [2 when resting (0.27 L·min−1) when standing (0.4 L·min−1), and when walking (0.55 L·min−1) with powered exoskeleton assistance after a 12-week walking intervention [−1·min−1) to post (9.38–32.9 mL·kg−1·min−1) intervention [2 results from pre (1208.1 mL) to mid (1077.9 mL) and from mid to post (901.3 mL) intervention [2 from pre (3.7 ± 0.7 mL·kg−1·min−1) to post (5.3 ± 1.9 mL·kg−1·min−1) intervention among patients with neurological impairments during a five-minute walking bout [−1·min−1) [2-peak during the 6 MWT and 25FWT at both a self-selected (SS) speed and fast speed (FS) at pre-and-post-intervention [2-peak is described as the difference between 2 peak when walking and standing. Results demonstrated a slight increase in N2-peak from pre-intervention (5.76 ± 1.3 N2) to post-intervention (5.91 ± 1.2 N2) during the 6 MWT. N2-peak results during the 25FWT reduced slightly from pre-intervention at a SS speed (2.83 ± 1.1 N2) and FS (3.60 ± 1.3 N2) to post-intervention at a SS speed (2.01 ± 1.0 N2) and FS (3.31 ± 1.9 N2) [2 improvement of 34.92 ± 14.84 mL·kg-1·km−1 during the 6 MWT and a peak 2 improvement of 0.08 ± 0.04 mL·kg−1·km−1 during the graded treadmill endurance test were made, in comparison to unpowered assistance [−1·min−1 [2 of non-exoskeleton assisted walking among able-bodied participants which was reported as 11.7 ± 2.0 mL·kg-1·min−1 [−1·min−1) among SCI patients [In total,  walking . Oxygen pulation ,21,33,45rvention . Oxygen rvention ,41. Jangrvention , whereasing bout ; which w1·min−1) . Afzal ervention ; NV˙O2-p9 NV˙O2) . When wasistance . Oxygen −1·min−1 ,43,47. Cn = 12 studies, using a range of polar HR monitors (p < 0.001) than walking with the lokomat (84 ± 9 bpm) [HR data was collected in a total of monitors . HR was ± 9 bpm) . Accordi± 9 bpm)  (Table 3p = 0.001) from somewhat hard (13 ± 5.95) to very, very light (7 ± 3.52) [As illustrated in  ± 3.52) .p < 0.001) than walking with the lokomat (1.58 ± 0.44 METs) within an SCI population [A total of six studies calculated METs during powered exoskeleton assisted walking. Chang et al. examined the mean METs of walking with (6.5 ± 1.0 METs) and without (3.4 ± 0.6 METs) exoskeleton assistance among able-bodied individuals . Evans epulation , whereaspulation .PCI during exoskeleton assisted walking was calculated in four studies. The combined PCI findings across the four studies ranged from 0.92 ± 0.38 to 1.60 ± 0.84 bpm during powered exoskeleton assisted walking among SCI and able-bodied participants ,32,34,38−1), CHO utilisation was assessed when seated (0.10–0.37 g·min−1), standing (0.08–0.30 g·min−1) and when walking (0.16–0.85 g·min−1) with maximal exoskeleton assistance [−1), when standing (0.01–0.14 g·min-1) and when walking (0–0.11 g·min−1) with maximal support [RER was assessed during powered exoskeleton assisted walking in a total of four studies. During a seated, standing and walking element, mean RER ranged from 0.8 to 1.0 . Carbohysistance . Fat oxi support . Kressle support . Maher e support ; and amo support .−1) during a seated, standing and walking period within an SCI population [−1) to standing  and a further increase when walking , within a single patient [−1), standing  and walking with exoskeleton assistance , within SCI patients [−1), standing  and walking period  [−1 [−1), after nine sessions  and post-intervention after 18 sessions  [−1) and 30 MWT  using a powered exoskeleton, which was slightly less than using unpowered assistance during the 6 MWT  and 30 MWT  among SCI patients [Gorgey et al. and Maher et al. examined energy expenditure  [−1 . Energy l·min−1) . Kwon etpatients .Lester and Gorgey, recorded BP pre-and-post two exoskeleton training sessions, BP changed from 111/77 mmHg to 117/74 mmHg after session one, and increased from 88/58 mmHg to 122/82 mmHg after session two . Gorgey −1, 25.2 ± 6.3 steps·min−1) when compared to the exoskeleton  [As well as assessing energy expenditure, eight studies also employed the 6 MWT as a measurement of gait performance . The 6 Ms·min−1) .−1 and 5 to 15 m, respectively [−1; 8–20 m) intervention [−1, 10–35 m) was also reported [−1) [The 10 MWT was employed in seven studies . Out of ectively . The 10 ectively . Mixed rectively . Benson ectively . Walkingrvention . Post-inrvention . Jang etrvention . The resrvention . An incrreported . Khan etted [−1) . Benson ted [−1) . Mean 10ted [−1) ,34.A total of five studies performed the TUG test to assess gait performance. TUG was assessed at pre-intervention in three studies. The mean pre-intervention TUG results across the three studies were 33.4 ± 12.2 s, 35.0 s, 38.3 s and 76.16 s, respectively ,30,36. T−1) and 25FWT-FS (0.51 ± 0.2 to 0.58 ± 0.3 m·s−1) [−1 [Afzal et al. recorded walking speed during the 25FWT at both a self-selected walking speed (25FWT-SS) and fast walking speed (25FWT-FS) . A sligh3 m·s−1) . Kozlowss−1) [−1 . −1, 22.6 ± 7.6 steps·min−1) in comparison to the powered exoskeleton , slightly greater distance was covered during the 30 MWT using the KAFO (242.9 ± 119.8 m) when compared to the ReWalk (196.2 ± 35.7 m) [Kwon et al. recorded walking distance during the 30 MWT with both the ReWalk powered exoskeleton and unpowered KAFO . Due to  35.7 m) .n = 11 of the included studies recorded walking speed with powered exoskeleton assistance (−1) in two studies; the mean walking speed in both studies was 21.18 ± 1.75 m·min−1 and 13.8 ± 6.0 m·min−1, respectively [p = 0.000) than walking with unpowered assistance  [−1) by Jang et al. which increased from pre (6.8 cm·s−1) to post (10.3 cm·s−1) intervention [−1, the results collectively ranged from 0.02 to 1.5 m·s−1 [−1) was recorded in two studies; Jang et al. showed an improvement from pre (16.4 steps·min−1) to post (24 steps·min−1) intervention [−1 [−1), the combined results ranged from 59 to 2616 steps·session−1 [As illustrated below, sistance . Speed wectively ,45. Arazm·min−1) . Walkingrvention . A furth.5 m·s−1 ,38,40,41.5 m·s−1 ,40,41,45rvention , and Chation [−1 . The remession−1 . Total wession−1 ,45. Therefore, powered exoskeleton assisted walking may be an effective method to help individuals with neurological impairments improve their cardiovascular fitness. The increase in oxygen consumption ,43,45,47n = 23 manuscripts, which included SCI, MS and stroke patients as well as able-bodied participants were reviewed. The energy expenditure variables examined in the selected studies included, 2, HR, RPE, METs, PCI, RER, energy expenditure and BP. The gait performance parameters measured in the selected studies included a variety of assessment protocols, such as, the 6 MWT, 10 MWT, TUG, 25 FWT, 2 MWT, 30 MWT, total steps, distance and walking speed. The primary aim of the current study was to systematically review current literature on the effects of exoskeleton assisted gait training on cardiovascular function. Cardiovascular disease is one of the leading causes of mortality among individuals who suffer from paraplegia [The purpose of this review was to systematically search and analyse existing publications that examined energy expenditure and gait performance during powered exoskeleton training. A total of raplegia . Thereforaplegia . In turn2 from seated to standing, and a further increase from standing to walking with powered exoskeleton assistance. Mean 2 decreased slightly from pre-to-post-intervention across two studies [2 was also examined among able-bodied participants during non-exoskeleton assisted walking in two studies and results were compared to powered exoskeleton assisted walking. The results reported by Chang et al. highlighted that powered exoskeleton assisted walking produced double the 2 compared to non-exoskeleton assisted walking among able-bodied individuals [2 during exoskeleton assisted walking among SCI patients was slightly less than the 2 during non-exoskeleton assisted walking within an able-bodied control group [−1) and without (1.3 ± 0.2 m·s−1) exoskeleton assistance [−1) than the SCI group (13.8 ± 6.0 m·min−1) [2 within the SCI group [2 results, whereas when walking with exoskeleton assistance the group used by Chang et al. walked over five times faster and consumed over double the amount of oxygen than the SCI group in Maher et al. study [The included studies displayed an increase in  studies ,41. V˙O2ividuals ; whereasol group . Chang esistance , whereassistance . The ablm·min−1) . When coCI group . When wal. study ,45. Furtl. study . A simill. study ,33,40,47l. study . The RPEl. study . −1 produces 3.4 METs [−1) [p < 0.001) [Powered exoskeleton assisted walking produced higher METs (6.5 ± 1.0 METs) in comparison to non-exoskeleton walking (3.4 ± 0.6 METs) within an able-bodied population . Jang et3.4 METs ; therefoETs [−1) . PCI durETs [−1)  and, on ETs [−1) . ArazpouETs [−1) ,34. Ramp< 0.001) . The RER results collected suggested that CHO utilisation was significantly higher than fat oxidation during powered exoskeleton assisted walking. Energy expenditure increased from seated to standing and further increased from standing to walking in both SCI and able-bodied control groups ,45. Enern = 21) also assessed gait performance parameters associated with powered exoskeleton assisted walking. Notable improvements were present from pre/mid-to-post-intervention across the 6 MWT [−1) and less walking distance was covered during the 6 MWT (5.0 ± 1.4 m) [As well as measuring an element of energy expenditure, some included studies  , therefoThe results demonstrated that powered exoskeleton assisted walking enabled participants to exercise at a moderate-intensity, mainly by elevating oxygen consumption and HR . The eleAs well as improving cardiovascular function and gait performance, corresponding research has suggested that powered exoskeleton assisted training can potentially improve secondary health conditions ,54,55,56Powered exoskeleton devices have been shown to be safe to use with patients who suffer from neurological impairments, with no falls, no serious adverse events , no fracn = 8) were all made up sample sizes ranging from (1–10) participants. Although the findings reported by the studies which consisted of smaller sample sizes (n = 1–10) were consistent with the results of studies made up of larger sample sizes (n > 12); having a larger sample size may allow results to be generalised to a larger population. It may be suggested that funding is another major restriction for therapists as a powered exoskeleton device is expensive therapeutic tool. Study limitations include the sample sizes of some of the selected studies. Research in this area is sparse, albeit as illustrated below publications surrounding energy expenditure associated with powered exoskeletons has increased over the past decade . As a reFurther research is necessary with larger sample sizes in order to support the findings of current literature. Future research should include control groups for further comparison of results. Further research is also necessary to explore the longitudinal effects of exoskeleton gait training on cardiovascular function, walking ability and secondary health conditions as the longest study duration was 24 weeks. In conclusion, the studies included within this systematic review examined the effects of exoskeleton gait training on cardiovascular function and gait performance. The included studies indicated that powered exoskeleton assisted training may increase oxygen consumption to a similar level to non-exoskeleton walking and elevate HR to a greater level than non-exoskeleton waking. The metabolic equivelant and the physiological cost index of exoskeleton assisted walking were shown to be three to four times greater than non-exoskeleton walking and wheelchair propulsion. Respiratory exchange ratio results highlighted that carbohydrate utilisation was higher during exoskeleton walking in comparison to non-exoskeleton walking. Energy expenditure was slightly less than non-exoskeleton walking and rate of percieved exertion reported during exoskeleton assisted walking was equal to moderate-intensity. The studies demonstrated an improvement in gait performance parameters from pre-to-post-interventions with patients improving across the six-minute walk test, ten-metre walk test, time up and go test and 25 foot walk test, as well as increasing walking speed, distance and total number of steps. Therefore, powered exoskeleton gait training may be an effective way to improve cardiovascular function and walking ability within spinal cord injury, multiple sclerosis and stroke patients. This enabled patients to walk faster and further for a longer duration, and as a result may enhance quality of life. An exoskeleton training programme has been shown to diminish secondary health conditions such as, pain and spasticity, improving bowel and bladder function, increasing bone mineral density ,54,55,56


In the structures of the title salts, two independent cations  are situated on a crystallographic twofold axis and on a center of inversion, respectively. Mutual intermolecular hydrogen bonding between the conjugate 3,5-dinitopyrazole NH-donor and 3,5-dinitropyrazolate N-acceptor sites of the anions governs the self-assembly of the translation-related anions in a predictable fashion. The anionic chains are further linked by multiple ion–dipole interactions involving the 12-coordinate cations bonded to two pyrazole N-atoms and all of the eight nitro O-atoms. The resulting ionic networks follow the CsCl topological archetype, with either metal or organic ions residing in an environment of eight counter-ions. Weak lone pair–π-hole interactions are also relevant to the packing. 4-4--3,5-dinitropyrazol-1-ido]rubidium], [Rb(C6HN8O8)]n, (1), and its isostructural caesium analogue  governs the self-assembly of the translation-related anions in a predictable fashion. Such one-component modular construction of the organic subtopology supports the utility of the crystal-engineering approach towards designing the structures of polynitro energetic materials. The anionic chains are further linked by multiple ion–dipole interactions involving the 12-coordinate cations bonded to two pyrazole N-atoms  and all of the eight nitro O-atoms . The resulting ionic networks follow the CsCl topological archetype, with either metal or organic ions residing in an environment of eight counter-ions. Weak lone pair–π-hole interactions  are also relevant to the packing. The Hirshfeld surfaces and percentage two-dimensional fingerprint plots for (1) and (2) are described.In the structures of the title salts, poly[[μ These materials may give an insight into the development of flame colorants in pyrotechnics: rubidium and caesium compounds exhibit, respectively, purple and orange colors when burned.Following the above findings, we now describe the synthesis and structure of rubidium and caesium 4--3,5-dinitropyrazolates C2/c. The molecular structure of the rubidium salt (1) is shown in Fig. 1− (or C6HN8O8−) and two cations situated on a crystallographic twofold axis [Rb1] or on a center of inversion [Rb2]. The easy formation of such salts is conditioned by the appreciable acidity of polynitropyrazoles, c.f. pKa = 3.14 for 3,5-dinitropyrazole versus 14.63 for the parent pyrazole }− anions, which maintain supramolecular boxes with a small internal cavity for the cation –3.6985 (16) Å; Cs—O 3.071 (2)–3.811 (2) Å] and two N atoms of the pyrazole rings  Tables 1 and 2 ▸.2(TNBP)] , the protolytic inequivalency of two pyrazole halves is reflected by the ring C—N distances, which are almost the same for anionic ring A (atoms C4/C5/C6/N3/N4) [N3—C4 = 1.343 (2) and N4—C6 1.348 (2) Å] and are slightly differentiated for the neutral ring B (C1/C2/C3/N1/N2) [N1—C1 1.348 (2) and N2—C3 1.331 (2) Å]  Å for ring A. Even more sensitive parameters are the bond angles at the N atoms, which are perceptibly different for the former fragment , being much closer for the latter [106.38 (15) and 107.59 (15)°]. In the case of (2), the corresponding geometries are nearly identical for rings A and B . This situation agrees with the disorder of the H atoms between two positions [at the N1 or N4 carrier atoms] within the N—H⋯N hydrogen bond in (2) as discussed below.The main geometrical parameters of the organic anions are very similar to those of the parent [HÅ] Fig. 1. In addi− anions display twisted conformations, with the dihedral angles between the rings being 42.99 (8) and 44.86 (10)° for (1) and (2), respectively. These angles, however, are unusually small. For example, typical parameters for the structurally similar 3,3′,5,5′-tetramethyl-4,4′-bipyrazole unit are 65–90°  Å observed for (2). Indeed, the nitro/nitro stackings are energetically favorable, as a special kind of lone pair–π-hole bond }M⋯M edges are 5.2560 (3), 6.5962 (3), 8.8395 (8) and 5.4775 (4), 6.3932 (5), 9.1482 (12) Å for (1) and (2), respectively]. Every such cell is populated with the organic anion and, conversely, every cation resides inside the distorted prismatic box of eight anions  and 2.832 (3) Å; H⋯Nxiv = 1.93 and 1.99 Å; N1H⋯N4xiv = 166 and 163° for (1) and (2), respectively; symmetry code: (xiv) x, y − 1, z], arranging the latter into linear polar chains propagating along the b-axis direction }− salts  Å] originating in situation of the pyrazole N atoms almost exactly above the NO2 N atoms ] itself  and (2) mitigate against mutual interactions of nitro groups, which are totally eliminated from the suite of supramolecular bonds. The second type of interchain interaction is stacking between pairs of inversion-related pyrazole and pyrazolate rings  −x + y + z; (xiii) −x + y − z.] (Table 41) is antiparallel, while the above lone pair–π-hole interactions support coherent alignment of the contributing chains , the polarity of the chains is eliminated because of the disorder of the H atoms in the N—H⋯N/N⋯H—N bonds.The above hydrogen-bonded chains associate to yield layers lying parallel to the ns Fig. 5. There as Table 3. This pegs Fig. 6, with th] Table 4. As a rens Fig. 6. This rens Fig. 6. In the et al., 2004CrystalExplorer17  are shown in Fig. 7xiii = 2.741 (2) Å . We note that slight contraction of the O⋯M fraction in the case of M = Rb [13.6% for (1) and 13.0% for (2)] coincides with a larger contribution of less favorable O⋯O contacts [37.4% for (1) and 35.5% for (2)]. This may be an additional factor destabilizing the structure: the crystals of (1) eventually decompose under the mother solution, unlike the stable Cs analogue. The lone pair–π-hole pyrazole-NO2 interactions generate 5.3% (1) and 6.3% (2) of the contacts of the Hirshfeld surfaces, with the shortest N⋯N = 2.9 Å. The nature of the O⋯N/N⋯O and N⋯C/C⋯N contacts [in total 23.3% (1) and 22.8% (2)] is similar, since they correspond to the stacking of pyrazole and NO2 groups with shortest O⋯N and N⋯C distances of 3.2 and 3.3 Å, respectively. However, there are no pairs of the features that are characteristic for the mutual O⋯N/N⋯O interactions of NO2 groups themselves  and 5.2 and 6.6% for (2)], but only the latter correspond to hydrogen bonding, as is reflected in the plots. These bonds are responsible for a pair of very sharp features pointing to the lower left, with a shortest contact of 1.9 Å, whereas O⋯H/H⋯O contacts are identified only with a diffuse collection of points between the above features and with a shortest contact of 2.8 Å.The supramolecular interactions in the title structures were also assessed by Hirshfeld surface analysis ] was synthesized in 92% yield by nitration of 4,4′-bipyrazole in mixed acids and then crystallized from water as a monohydrate , 0.332 g (1.0 mmol) of H2(TNBP)·H2O was added to a solution of 0.116 g (0.5 mmol) of Rb2CO3 in 8 ml of water and the mixture was heated at 353–363 K until total dissolution was observed. The solution was cooled to room temperature and left for a few hours for crystallization. Pale-yellow crystals of Rb{H(TNBP)} were isolated in a yield of 0.325 g (82%) and dried in air. The compound is unstable when stored under the reaction solution as the initially formed crystals dissolve in a period of 10–15 d and colorless H2(TNBP)·H2O deposits. In a similar way, the reaction of 0.332 g (1.0 mmol) of H2(TNBP)·H2O and 0.163 g (0.5 mmol) of Cs2CO3 in 8 ml of water gives 0.415 g (93%) of pale-yellow Cs{H(TNBP)} (2). Unlike (1), this material is stable under the mother solution. Similar reactions with Na2CO3 and K2CO3 did not afford any hydrogen bipyrazolates and led to soluble M2{TNBP}  and precipitation of the excess amount of H2(TNBP)·H2O.To prepare the Rb salt (1), C6HN8O8Rb: C 18.08, H 0.25, N 28.12; found: C 17.93, H 0.44, N 28.49. IR : 590 w, 708 w, 838 m, 854 s, 996 m, 1024 m, 1308 s, 1352 vs, 1398 vs, 1432 m, 1490 vs, 1500 m, 1556 vs, 1636 w, 3448 br.Analysis (%) calculated for (2), C6HCsN8O8: C 16.15, H 0.23, N 25.13; found: C 16.01, H 0.38, N 28.11. IR : 516 w, 586 m, 708 m, 838 s, 852 s, 994 s, 1022 m, 1170 w, 1306 s, 1324 s, 1350 vs, 1396 vs, 1432 s, 1488 vs, 1512 vs, 1544 vs, 1634 m, 3024 br, 3442 br.Analysis (%) calculated for (Uiso(H) = 1.5Ueq(N). For (2), the H atom is equally disordered over two positions corresponding to the N1 and N4 carrier atoms.Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S2056989021010227/hb7988sup1.cifCrystal structure: contains datablock(s) global, 1, 2. DOI: 10.1107/S2056989021010227/hb79881sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989021010227/hb79882sup3.hklStructure factors: contains datablock(s) 2. DOI: 2113578, 2113577CCDC references:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5′-untranslated region (5′-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5′-UUUCGU-3′ hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5′- and 3′-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured The online version contains supplementary material available at 10.1007/s12104-021-10053-4. The structured parts of the SARS-CoV-2 genome have been divided into fragments in a ‘divide and conquer’ approach, allowing us to determine the secondary structures of these RNA elements.  is caused by the human Betacoronavirus SARS-CoV-2 (SCoV2), a close relative of the severe acute respiratory syndrome (SARS) causing agent SARS-CoV. Betacoronaviruses have large positive sense, single-stranded RNA genomes, with highly conserved 5′- and 3′-untranslated regions (UTRs) that do not code for viral proteins. These structured UTRs are highly conserved among Betacoronaviruses and are important for the replication, balanced transcription of subgenomic mRNAs and translation of viral proteins.  and the basal stem.  encoding an HDV ribozyme  according to the manufacturer’s instructions and linearized with HindIII prior to in-vitro transcription by T7 RNA polymerase  were performed to obtain sufficient amount of RNA. Preparative transcription reactions (6 h at 37 °C and 70 rpm) were terminated by addition of 150 mM EDTA. SL5b + c and 5SL5b_GC RNAs were purified as follows: RNAs were precipitated with one volume of 2-propanol at − 20 °C overnight. RNA fragments were separated on 12–15% denaturing polyacrylamide (PAA) gels and visualized by UV shadowing at 254 nm. SL5b + c and SL5b_GC containing RNA bands were excised from the gel and then incubated for 30 min at − 80 °C, followed by 15 min at 65 °C. The elution was done overnight by passive diffusion into 0.3 M NaOAc, precipitated with EtOH and desalted via PD10 columns . Residual PAA was removed by reversed-phase HPLC using a Kromasil RP 18 column and a gradient of 0–40% 0.1 M triethylammonium acetate in acetonitrile. After freeze-drying of RNA-containing fractions and cation exchange by LiClO4 precipitation (2% in acetone), the RNA was folded in water by heating to 80 °C followed by rapid cooling on ice. Buffer exchange into NMR buffer  was performed using Vivaspin centrifugal concentrators (2 kDa molecular weight cut-off). The purity of SL5b + c and SL5b_GC was verified by denaturing PAA gel electrophoresis and homogeneous folding was monitored by native PAA gel electrophoresis, loading the same RNA concentration as used in NMR experiments. 100% D2O samples were prepared by lyophilisation and redissolving in identical volumes of pure D2O to keep the buffer salt concentration constant.RNA synthesis for NMR experiments: For DNA template production, the sequences of SL5b + c (genomic nucleotides 227 to 263) and SL5b_GC, (5′-G-(genomic 227 to 252)-CC-3′) Fig. C, togeth15N- and two uniformly 13C, 15N-labelled samples (750 µM in buffer with 95% H2O/5% D2O and 430 µM in buffer with 100% D2O). In addition, two uniformly 13C, 15N-labelled samples of SL5b_GC were prepared: an H2O sample at a concentration of 510 µM in buffer with 95% H2O/5% D2O and a D2O sample at a concentration of 300 µM in buffer with 100% D2O. For the divide-and-conquer approach, the unlabelled single stem-loop 5c .Using this protocol, three NMR samples of SL5b + c were prepared: a 350 µM uniformly 1H -TCI prodigy probe and a Bruker AVANCE Neo 1 GHz spectrometer equipped with a 5 mm, z-axis gradient 1H -TCI cryogenic probe and at the Center for Biomolecular Magnetic Resonance (BMRZ) at the Goethe University Frankfurt using Bruker NMR spectrometers from 600 to 800 MHz, which are equipped with AVANCE Neo, AVIIIHD, AVIII and AVI consoles and the following cryogenic probes: 5 mm, z-axis gradient 1H -TCI cryogenic probe (600 MHz), 5 mm, z-axis gradient 1H/19F -TCI prodigy probe (600 MHz), 5 mm, z-axis gradient 1H -TCI cryogenic probe (600 MHz), 5 mm, z-axis gradient 1H -QCI cryogenic probe (700 MHz) and 13C-optimized 5 mm, z-axis gradient 13C -TXO cryogenic probe (800 MHz).NMR experiments were carried out at the Weizmann Institute (WIS) using a Bruker AVIII 600 MHz NMR spectrometer equipped with a 5 mm, z-axis gradient www.logs.repository.com). 1H chemical shifts were referenced externally to DSS and 13C and 15N chemical shifts were indirectly referenced from the 1H chemical shift as previously described. , and chemical shift assignment was conducted using Sparky.  .The imino, aromatic and ribose resonances of the SL5b + c were assigned using a 1H, 15N-TROSY , 1H,1H-NOESY  and HNN-COSY  spectra, the U-C2 and -C4 as well as G-C2 and -C6 could be assigned in the 1H, 13C-HNCO, and as well for aromatic carbon resonances U-H3/C6 and G-H1/C8 in the 1H, 13C-HCCNH . Additionally, chemical shift assignments for the nitrogens N1 or N9 could be assigned in the 1H,15N-HCN experiment.The assignment of SL5b_GC is described in the following using the experiments summarized in SI Table SL5b_GC. From the imino proton chemical shift assignment by  SI Fig. A, 1H,1H- SI Fig. A, 1H,1H- SI Fig. A, 1H,1H- SI Fig. A, 1H,1H- SI Fig. A, 1H,1H-1H,15N-TROSY, 1H,1H-NOESY, 1H,13C-HNCO and 1H,13C-HSQC spectra of SL5b + c . The sequential walk was only interrupted by missing cross peaks between G256-H8 to A257-H8 in the loop region in both the 95% H2O and the D2O  by interaction with a phosphate oxygen in the backbone for a GAAA tetraloop. While we could not assign crosspeaks between the loop guanosine (G256) and the loop adenosine (A259) that would have confirmed the reported loop geometry in SL5c, we found an additional imino crosspeak assigned to G256. In addition to the imino and amino proton assignment, which is consistent with the published chemical shifts for SL5b + c  and pyrimidines (C2: 29% and C4: 24%). The assignment of the nitrogen atoms includes 70% N1 for purines and 65% N3 for pyrimidines, which represent mostly those involved in hydrogen bonding interactions. With the assignment transfer 90% of H1′–C1′ chemical shift assignment was obtained. Further ribose resonances for H2′ to H5″ and C2′ to C5′ are partially assigned in ranges of 10 to 30%. In summary, an assignment of 65% of the 1H, 53% of the 13C and 63% of the 15N atoms in the nucleobases of SL5b + c has been achieved.We herein present the Supplementary file1 (DOCX 500 KB)Below is the link to the electronic supplementary material.


In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity.   •Mtb is inhibited by indole-4-carboxamides-exogenous tryptophan intermediates rescue•amiC result in decreased hydrolysis of indole-4-carboxamidesMutations in •trpE mutants lose feedback inhibition by L-Trp increasing flux through the pathway•4-Aminoindole is incorporated into 4-amino-L-tryptophan poisoning cellular metabolism Mycobacterium tuberculosis liberating 4-aminoindole that is subsequently converted into toxic 4-amino-L-tryptophan by tryptophan synthase. Mutation in the amidase and two enzymes involved in tryptophan biogenesis confer resistance and illuminate the complexity of metabolic control of tryptophan levels.Libardo et al. demonstrate that indole-4-carboxamides are cleaved by an intracellular amidase of  Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), continues to take its toll on humanity with about 7 million new infections and 1.5 million people succumbing to the disease in 2018  in six enzymatic steps A. The fi in mice , establi in mice . This statent TB , further, a sulfolane, and an indoline-5-sulfonamide all tightly bind to the interfacial region of TrpAB, stabilizing the closed, active state of the complex and inhibiting both subunits of the enzyme . Second,Here, we report a series of antimycobacterial indole-4-carboxamide prodrugs that liberate 4-aminoindole, which inhibits neither TrpE nor TrpAB, but rather is metabolically incorporated to form 4-aminotryptophan. We uncovered resistance mechanisms that illustrate how modulation of metabolic flux effectively rescues Mtb from the toxic incorporation of 4-aminoindole.M. bovis BCG at low μM concentrations in BSA-free media . Spontaneous resistance was observed at frequencies that varied depending on the number of bacilli plated; that is, when 107 CFU (colony-forming unit) was plated, the frequency of resistance (FoR) was ~3 × 10−6, whereas at higher cell densities (109 CFU), the FoR was ~3 × 10−8. Determination of C1 MICs against these mutants revealed high-level (>12-fold) resistance from the first group of mutants (5A-5I) and intermediate-level (4- to 8-fold) resistance in the latter group (5J-5O)  . These mrv2888c). On the other hand, the low FoR mutants that appeared with higher cell density had missense mutations in either anthranilate synthase subunit I , or the α or β subunit of tryptophan synthase , all of which are essential enzymes in the tryptophan biosynthetic pathway  potentially conferred resistance against C1 . The mut pathway .amiC in M. smegmatis (Msm) (msmeg_2521) was shown to function as an inducer of acetamidase expression to allow growth using amides as a sole carbon source  (The homolog of n source . While M (Psmyc) , did not (Psmyc) A, suggesamiC mutants had lost the ability to metabolize C1 while mutants in L-Trp biosynthesis had no effect on metabolism . We observed complete disappearance of the parent compound peak from C1 after 2 h with a concomitant appearance of a peak with an m/z corresponding to the free carboxylic acid B. The m/tabolism C. This smegmatis .amiC mutations resulting in abrogated amide hydrolysis leads to high-level resistance also suggests that C1 is a pro-drug liberating an active metabolite from amide hydrolysis. We therefore determined whether the amine or the carboxylic acid that would result from hydrolysis of C1 were responsible for the observed activity. All four representative compounds share the 4-aminoindole (4-AI) moiety but differ in the carboxylic acid coupling partner. Measuring the MIC of these building blocks showed that only 4-AI was active against Mtb (MIC = 4.68 μM), while the free acids had little to no potency (amiC mutants were not cross-resistant to 4-AI (a molecule that bypasses the AmiC hydrolysis requirement), while the trpABE mutants were highly resistant to it , TrpB (L-Trp), or TrpE (anthranilate) rescued Mtb from the inhibitory activity of the indole-4-carboxamides and 4-AI, increasing MICs 4- to 16-fold B. Interein vitro enzyme assay showed that neither 4-AI  nor C1 (IC50 = 242 ± 53 μM) inhibited TrpE at concentrations near their MICs  was 250 times less sensitive to allosteric inhibition by L-Trp than WT enzyme (IC50 = 0.87 ± 0.11 μM)  C. This sR mutant was more susceptible to C5 relative to WT, suggesting a potential synergistic effect. We independently raised resistant mutants against C5 and, in agreement with previously published results , αAsp54 is >22 Ǻ away from the α subunit catalytic residue αAsp68  at concentrations as low as that of the bona fide TrpAB inhibitor C5 (IC50α = 0.10 ± 0.06 μM and IC50β = 0.37 ± 0.07 μM)  D and 5E,We studied the effect of representative mutations on the enzymatic activity of TrpAB to unravel the molecular basis of resistance B and 5C.Motivated by the lack of enzymatic inhibition, and the fact that 4-AI is an analog of indole, we explored the possibility of 4-AI being incorporated into L-Trp biosynthesis by TrpB. We incubated purified TrpAB with 4-AI instead of indole under saturating L-Ser concentrations and observed the appearance of a product peak with an m/z of 220, which we hypothesized was 4-amino-L-Trp (4-aTrp) . To confcat values for the β reaction for the WT enzyme with 4-AI  for 5 min, quenched the reaction with 0.1% formic acid in methanol, and detected the formation of 4-aTrp via LC-MS. The kith 4-AI E appeareith 4-AI I. We fouith 4-AI D and 6E,ith 4-AI I. Therefith 4-AI B.600] = 3.0). We then extracted the metabolites in Mtb using 1:1 ACN:MeOH and injected aliquots in the LC-MS. We detected formation of 4-aTrp in WT cells  , but transport of this molecule into Mtb cells may not be very efficient. Incubating Mtb cells with 10 or 100 μM 4-AI over a 3-day period followed by detection of either L-Trp or 4-aTrp via LC-MS  . While dia LC-MS  showed tmegmatis .amiC mutations at a frequency of 10−6, 100-fold more common than the frequency of trpABE mutations. The frequency of resistance was dependent on the size of the inoculum; thus, when 109 cells were plated, there were probably enough bacilli on the plate to hydrolyze all C1 to 4-AI, and we were inadvertently raising mutants against 4-AI. Whereas, when 107 cells were plated, hydrolysis may not have been as extensive; hence, the mutants were selected against the parent compound C1. AmiC-mediated hydrolysis must have a certain degree of substrate promiscuity since derivatives that varied greatly in the size, polarity, and position of the carboxylic acid coupling partner relative to indoleamine displayed low μM MICs against Mtb   , coupledn <5 μM) , which an <5 μM) . Therefon <5 μM) .In this work, we describe a series of indole-4-carboxamides that, upon AmiC-mediated hydrolysis, liberate 4-AI, which gets incorporated into the L-Trp biosynthetic pathway to form 4-aTrp. The tolerance of the L-Trp biosynthetic enzymes in Mtb is demonstrated by a recent study that shows that fluoroanthranilates produce fluorotryptophans, but none of the fluorinated intermediates , indicatEven though TrpB can utilize an assortment of indole substitutions, the downstream effects of the substituted tryptophans in Mtb may vary greatly. For instance, positional isomers of C4 that would liberate 5- and 6-aminoindole were inactive, while those that liberate 7-aminoindole had only weak activity  overexpression of the target to overcome metabolic blockage by stoichiometrically limiting the number of enzymes deactivated by the drug . (2) UsiIn conclusion, we demonstrated the mechanism of action of indole-4-carboxamides and the molecular basis of Mtb resistance against these compounds. We validated the idea that modulation of metabolism—either by maintaining flux through resistance from feedback inhibition (mutation in TrpE) or reducing flux through enzymatic attenuation (mutations in TrpAB)—affords antimicrobial resistance in Mtb. Poisoning tryptophan metabolism by deliberately engineering novel prodrugs of 4-AI with suitable pharmaceutical properties represents a promising avenue for the development of a novel antituberculosis agent.Mycobacterium tuberculosis (Mtb) comes the urgent need to identify potential new drug targets. Tryptophan biosynthesis has previously been identified as a potent target for chemotherapy due to synergistic effects with current drug regimens. Here, we describe the mechanism of action of a series of antimycobacterial indole-4-carboxamides and the resulting mechanism of resistance Mtb deploys to subvert their cidal effect. We found the series of indole-4-carboxamides to be prodrugs of an antimetabolite, 4-aminoindole, which becomes metabolically incorporated by tryptophan synthase to form the cytotoxic 4-aminotryptophan. Mtb acquired resistance by deploying three discrete mechanisms (1) decrease in drug metabolism, (2) increased biosynthetic flux of tryptophan, and (3) in situ enzymatic attenuation of tryptophan synthase. This third method, we propose to be a bona fide resistance mechanism in mycobacteria. Ultimately, these results further demonstrate the complexity involved in Mtb modulation of tryptophan biosynthesis and enhance our understanding of this valuable drug target.With the rise of multidrug-resistant cbarry@niaid.nih.gov)Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Clifton E Barry, III , referred to as BSA-free media in the main text. Some MIC measurements were also done in 7H9/ADC  or GAST-Fe medium (0.3 g Bacto Casitone (Difco), 4.0 g K2HPO4, 2.0 g citric acid, 1.0 g L-alanine, 1.2 g MgCl2 6H2O, 0.6 g K2SO4, 2.0 g NH4Cl, 1.80 ml 10 NaOH, 10.0 ml glycerol, 0.05% Tween 80 and 0.05 g ferric ammonium citrate adjusted to pH 6.6). Solid medium used where appropriate was Middlebrook 7H11 agar supplemented with 5 g/L dextrose, 2 g/L NaCl, 0.5% glycerol, and 0.06% oleic acid .2O to 10 μM. The 10 μM solutions were used for all cloning protocols. All cloning protocols and plasmid isolations were done using commercially available kits from Zymo Research Company.All primers used in this study were obtained from Eurofins in their dry and desalted form and reconstituted (as received) in 1X TE buffer to make 100 μM stocks and further diluted in mQ HM. tuberculosis and M. bovis were performed in 7H9/ADC, while those for the resistant mutants (and the corresponding WT control) were done in 7H9/Gcas. Bacteria were grown in the corresponding media up to an OD600 of 0.2 to 0.4. A two-fold serial dilution series (50 μL) of the test compounds was placed in each well of a sterile 96-well round bottom plate, and then 50 μL of the bacterial suspension diluted to OD600 of 0.0002 was added. The plates were incubated at 37°C for 2 weeks prior to visual determination of the MIC. The MIC is defined here as the drug concentration that completely inhibited growth of cells. MICs measured in the presence of supplementation were done by supplementing the growth medium with 1 mM (or 70 μM) of the corresponding metabolite.The antimicrobial susceptibility testing against ex-vivo efficacy testing, J774.1 cells (2x105 cells/well) were seeded in flat-bottom 24-well plates (Corning Inc.) in DMEM GlutaMAX supplemented with 10% FBS, 20 mM HEPES, and 0.5 mM sodium pyruvate (hereafter abbreviated as DMEM/FBS). The cells were infected with M. tuberculosis H37Rv  for 24 hrs, followed by removal of the medium and washing twice with Dulbecco PBS. Infected cells were exposed to test compounds at the specified concentrations in fresh growth medium (1 mL/well). Cells were incubated at 37°C, 95% humidity, 5% CO2 incubator for 7 days. The medium was changed every 2 days and the infected cells were treated with a fresh batch of drugs each time. After 7 days of incubation, the medium was removed and the cells were washed with PBS twice. Then, 500 μL of 0.1% SDS was added to each well to lyse the J774 cells. After 5 mins, the lysate was thoroughly mixed and diluted in 7H9/ADC and appropriate dilutions were plated in duplicate on 7H11/OADC plates to calculate the most probable number of bacteria. Colonies were counted manually in each plate following 6-8 weeks of incubation at 37°C.For the in vitro cytotoxicity of the indole-4-carboxamides were measured in DMED supplemented with 10% FBS. 1x104 J774.1 cells were seeded onto each well of a sterile opaque 96-well plate and left to attach overnight. The next day, the media was aspirated and replaced with a two-fold serial dilution of compounds in the same media. The cells were treated for 24 hrs prior to viability determination using CellTiter-Glo (Promega) according to the manufacturer instructions.The 7, 108, and 109 cells of M. tuberculosis H37Rv were plated on 7H11/OADC plates containing 5x the in vitro MIC of C1. Drug-free plates were used to enumerate bacterial load. The plates were incubated at 37°C for 4-6 weeks until colonies grew to an appreciable size. The colonies were picked and inoculated into drug-free 7H9/Gcas medium and the MIC were tested (according to the above protocol) to confirm true resistance against C1. The genomic DNA of the mutants were isolated using the CTAB method  and 1004 (R) with terminal BamHI restriction sites. Furthermore, the hsp60 promoter (amiC gene was amplified from M. tuberculosis H37Rv genomic DNA using primers 1007 (F) and 1008 (R) and the Q5 Hot-start high-fidelity 2X Master Mix (New England Biolabs). The amplicon and pL5-Psmyc was double digested with NcoI and ClaI; the vector was also treated with alkaline phosphatase  for 30 mins. The DNA fragments were gel purified and the amiC fragment was ligated to the vector backbone overnight at room temperature using T4 DNA ligase (New England Biolabs). The ligation reaction was transformed into E. coli NEB5α competent cells and the transformants were plated on LB agar containing 50 μg/mL kanamycin. Positive transformants were grown and the plasmid was extracted and confirmed by Sanger sequencing (Eurofins).The mycobacterial shuttle vector pMV361  was deripromoter  was exchpromoter  exploitiM. tuberculosis H37Rv (400 μL) were transferred into a 2mm-gap electroporation cuvette and mixed with 1 μg of pL5-Psmyc-AmiC. The cells were electroporated using a Bio-Rad GenePulser II electroporator set at the following paramters: 2.5 kV, 1000 Ω, 25 μF. Immediately after electroporation, 1 mL of 7H9/ADC was added to the cells and the mixture was transferred into 10 mL of sterile 7H9/ADC and the cells were allowed to recover for 24 hrs at 37°C. The cells were diluted and plated into 7H11/OADC plates containing 25 μg/mL kanamycin and incubated at 37°C for 4-6 weeks. Electroporation using an empty vector was used as control. The resulting colonies were grown in 7H9/GCas containing 25 μg/mL kanamycin and the MICs were measured using the protocol described above.Electrocompetent M. tubeculosis and the C1R mutants, an LC-MS-based protocol was employed. The cells were grown in 7H9/Gcas to an OD600 of 1.0, washed twice with PBS, and resuspended in PBS at 1/3 the original culture volume to afford a suspension at OD600 of 3.0. Then, 1 mL of the cell suspension was added to enough compound in DMSO to create a final compound concentration at 10X the in vitro MIC. A 200 μL aliquot was removed for the 0 hr time point (immediately after addition of the cells to the drug) and mixed with an equal volume of acetonitrile (ACN) and kept at 4°C. After 24 hrs, another 200 μL aliquot was removed and mixed with ACN. The cells in these aliquots were pelleted and the supernatant was filtered through a 0.2 μm centrifuge filter prior to taking the samples out of the BSL3. A 20 μL aliquot was injected directly into the LC-MS. LC gradient used to separate the metabolites was 5-95% ACN over 20 mins. Chromatograms obtained at 270 nm were used to determine % compound remaining after 24 hrs, and were normalized relative to the 0 hr time point.To determine the degree of drug metabolism in live M. tuberculosis (100 mL) was grown up in 7H9/ADC to an OD600 of 0.8. Cells were harvested, washed twice with 20 mL PBS and resuspended in 5.6 mL modified Sauton’s medium. The Sauton’s medium consisted of magnesium sulfate heptahydrate (0.5 g/L), potassium dihydrogen phosphate (0.5 g/L), potassium sulfate (725 mg/L), citric acid (2 g/L), ferric ammonium citrate (50 mg/L), L-asparagine (4 g/L), zinc sulfate (0.1 mg/L), glycerol (20 g/L), with pH adjusted to 6.5 with sodium hydroxide (10 M). Half of the cell suspension was treated with 100 mM of 4-aminoindole and the other half with an equivalent amount of DMSO. The samples were divided into 600 mL aliquots and incubated at 37°C in a shaking incubator with 600 mL acetonitrile added at the indicated time points. Samples were stored at -80°C prior to further processing. In a separate experiment, M. tuberculosis (700 mL) was grown up in 7H9/Gcas to an OD600 of 0.5. Cells were harvested, washed and treated with 10 mM and 100 mM of 4-aminoindole as above and quenched with 600 mL acetonitrile prior to storage at -80°C.+ adduct were detected with the product ions m/z 157.1 (CEV 20), 188 (CEV 7), and 181.1 (CEV 10) for 4-aminotryptophan, tryptophan, and IS respectively (CEV = collision energy voltage in volts).For tryptophan and 4-aminotryptophan concentration determination , a stocktrpB and trpE with the mutant alleles, a previously described single-stranded DNA-mediated recombineering protocol was employed  to a final concentration of 500 ng/mL and allowing the cells incubate overnight at 37°C. Then, 3 mL of a 2M sterile glycine solution was added and the cells were incubated for an additional 16 hrs at 37°C. The cells were collected and washed twice with 10% glycerol and finally resuspended in 3 mL of sterile 10% glycerol. 400 μL of the electrocompetent and recombinogenic M. tuberculosis were transferred to a 2mm-gap electroporation cuvette, into which 500 ng of oligos were added. The cells were electroporated using a Bio-Rad Gene Pulser II set at the following parameters: 2.5 kV, 1000 Ω, 25 μF. The cells were removed from the electroporation cuvette and added to 10 mL of 7H9/ADC and allowed to recover at 37°C for 3 days. Then the cells were subcultured 1:100 into 7H9/ADC containing 20 μg/mL kanamycin and allowed to grow to an OD600 of ~1.0. The cells were diluted and finally plated on 7H11/OADC plates containing 20 μg/mL streptomycin and incubated at 37°C for 4-6 weeks. Several clones for each recombineering target were selected and genomic DNA was extracted using the CTAB method. The clones were screened for the desired mutations via PCR using primers 1013 – 1018  was read using a ClarioStar multiwell plate reader. The fluorescence of the treated wells were normalized to that of the corresponding untreated well and presented as % cell viability.The resistance of the recombineering and WT strain against NMMV03 compounds were measured using the Alamar Blue Assay. The bacterial strains were grown in 7H9/GCas and the susceptibility testing were done according to the MIC protocol described above. After 7 days of drug treatment, 10 μL of Alamar Blue (ThermoFisher) was added to each well and the plates were returned to the 37°C incubator for 24 hrs. The fluorescence (λM. tuberculosis H37Rv trpE gene was amplified from a genomic DNA template using the Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primers 1019 (F) and 1020 (R). The trpE amplicon was cloned between the NdeI and HindIII sites of pET28a, resulting in an N-terminal His6 tag. The PCR product and pET28a were separately digested with NdeI and HindIII and ligated overnight at room temperature using T4 DNA ligase. The ligation mixture was directly transformed into E. coli NEB5α competent cells and the cells were plated onto LB agar + 50 μg/mL kanamycin. The plasmid was extracted and analyzed by Sanger sequencing. To introduce the H170R, the Q5 site-directed mutagenesis kit (New England Biolabs) was used using the pET28a-TrpE construct as template. Primers 1021 (F) and 1022 (R) were used and the resulting Q5 product was transformed into competent NEB5α cells. The H170R mutation was confirmed by Sanger sequencing. The pET28a-TrpE WT and H170R plasmids carrying the correct sequence were then transformed into competent E. coli BL21(DE3) and the transformants were plated onto LB agar plates containing 50 μg/mL kanamycin. Positive transformants were inoculated into 20 mL LB + 50 μg/mL kanamycin and grown overnight. The entire overnight culture was transferred into 2 L of LB containing 50 μg/mL kanamycin and allowed to grow until OD600 of ~0.6. Protein expression was induced by addition of IPTG to a final concentration of 1 mM. The cells were incubated for an additional 3 hrs at 37°C prior to harvesting by centrifugation at 4000xg for 10 mins. The cells were resuspended in 30 mL of lysis buffer ) and disrupted via sonication (cooling on ice for 1 min every 30 sec sonic bursts). The lysate was centrifuged at 45000xg for 1 hour and the supernatant was loaded onto a Ni-NTA column equilibrated with 25 mL of column buffer . The column was washed with 30 mL of column buffer with increasing imidazole concentrations . The protein was eluted using 10 mL of column buffer containing 300 mM imidazole and collected in 1 mL fractions. The fractions containing purified TrpE (as seen from a Coomassie-stained polyacrylamide gel) were pooled together and concentrated to ~2.5 mL. The protein was buffer exchanged using a PD-10 (Sigma-Aldrich) column equilibrated using 30 mL of storage buffer  and eluted from the PD-10 column using 3 mL of storage buffer. The protein was split into 300 μL aliquots and flash frozen using a dry ice/isopropanol bath and stored at -80°C until use. The concentration of the protein preparations was established using the Pierce BCA Assay kit (ThermoFisher) using BSA as a standard, and the concentrations were converted to μM using the calculated molecular weight of TrpE WT and H170R.The ex = 320 nm and λem = 460 nm using a ClarioStar multiwell plate reader. The total volume was 200 μL in all cases and the assay buffer used contained 20 mM Tris-HCl pH 8.0, 100 mM NH4Cl, 10 mM MgCl2, 100 mM EDTA. The Michaelis-Menten curves were obtained by incubating the enzyme with increasing concentrations of chorismate (dissolved in the assay buffer) where the reaction was started by addition of 0.67 μM of TrpE WT or the H170R mutant. For the inhibition studies, a fixed 50 mM chorismate concentration was used, and increasing concentration of the drugs or L-Trp was added. The fluorescence of anthranilate was converted into concentration values using a standard calibration curve prepared in the same buffer used for kinetic studies. The kcat, and IC50 values were all calculated using nonlinear curve fitting in GraphPad Prism 8.The TrpE enzyme kinetics were studied by direct detection of the fluorescent product, anthranilate at λ6 epitope in MCS1 and cloning TrpA in MCS2. trpB was amplified from H37Rv genomic DNA using primers 1023 (F) and 1024 (R) and Q5 Hot-start high-fidelity 2X master mix (New England Biolabs). The amplicon and pRSFDuet-1 were digested with HindIII for 1 hr; the vector was also treated with alkaline phosphatase  for 30 mins. The DNA fragments were gel purified and ligated using T4 DNA ligase (New England Biolabs) overnight at room temperature. The ligation mixture was transformed into competent E. coli NEB5α cells, the plasmid was extracted, and the sequence was verified using Sanger sequencing (Eurofins). Then, trpA was cloned into pRSFDuet-TrpBMCS1 in a similar manner between the NdeI and XhoI sites. The resulting pRSFDuet-TrpBMCS1-TrpAMCS2 construct was fully sequence to verify the sequence of both coding regions and the overall integrity of the plasmid. Initial expression studies in BL21(DE3) cells using this construct showed significant difference in the expression level between TrpB and TrpA (~5:1 TrpB:TrpA).The TrpAB enzyme complex were co-expressed using the pRSFDuet-1 vector tagging TrpB with an N-terminal HiR marker of pRSFDuet-1 was replaced by amplifying the AmpR cassette from pUC19 using primers 1027 (F) and 1028 (R) and cloning the amplicon between the AgeI and SphI restriction sites of pRSFDuet-1. The resulting pRSFDuetAmpR was used as a backbone for TrpA. TrpA was amplified from H37Rv genomic DNA using primers 1029 (F) and 1030 (R) and Q5 Hot-start high-fidelity 2X master mix (New England Biolabs). The amplicon and pRSFDuetAmpR was double digested with NcoI and HindIII; the vector was also treated with alkaline phsphatase . The DNA digests were gel purified and ligated using T4 DNA ligase (New England Biolabs) overnight at room temperature. The ligation was transformed into competent E. coli NEB5α cells and plated on LB agar containing 100 μg/mL Ampicillin. The positive transformants were grown, the resulting plasmid (pRSFDuetAmpR-TrpAMCS1) extracted and analyzed by Sanger sequencing (Eurofins).To increase expression of TrpA, we prepared another construct and co-introduced the two plasmids into the expression vector. The TrpA supplemental expression plasmid was also made using the pRSFDuet-1 vector. Briefly, the native KanMCS1-TrpAMCS2 construct as template. Primers pairs 1031 (F) – 1032 (R) and 1033 (F) – 1034 (R) were used and the resulting Q5 product was transformed into competent NEB5α cells. Introduction of the mutations were confirmed by Sanger sequencing (Eurofins).To introduce the βA168V and the βD261G mutations, the Q5 site-directed mutagenesis kit (New England Biolabs) was used using the pRSFDuet-TrpBE. coli BL21(DE3) were co-transformed with pRSFDuet-TrpBMCS1-TrpAMCS2 and pRSFDuetAmpR-TrpAMCS1 and grown in LB + 50 μg/mL kanamycin and + 100 μg/mL ampicillin. The transformed BL21(DE3) cells were grown in LB media at 37°C until an OD600 of 0.6-0.7. Expression was induced with IPTG  and incubated for 3 hours at 37°C. Cells were pelleted by centrifugation at 4000rpm for 20 minutes and then resuspended in lysis buffer ). After sonication, the lysed cells were pelleted by centrifugation at 15,000 rpm for 60 minutes. The supernatant was loaded onto a Ni-NTA column, and then washed with a wash buffer . After washing, the loaded protein was eluted with an elution buffer (Same as wash buffer but with 300 mM Imidazole). The eluted protein was dialyzed overnight against a storage buffer . TrpAB were identified using SDS-PAGE gel, aliquoted, and stored at -80°C until use. The concentration of the complex was measured spectrophotometrically using the Pierce BCA Assay kit (ThermoFisher) and the accompanying BSA standards. The concentrations calculated from the calibration curve were converted to μM units using the molecular weight of the complex. Due to the higher expression of TrpB in comparison to TrpA (~2:1 TrpB:TrpA), concentration of the TrpAB complex was adjusted to the percent TrpA present, assuming any extra TrpB was inactive enzyme.Competent E. coli indole-3-glycerol phosphate synthase (TrpCF) to convert 1-(o-carboxyphenylamino)-deoxyribulose 5-phosphate (CdRP) to IGP via an irreversible ring closure reaction spurred by decarboxylation and dehydration steps. The trpCF gene was amplified from E. coli MG1655 genomic DNA using primers 1035 (F) and 1036 (R) and Q5 Hot-start high-fidelity 2X master mix (New England Biolabs). The amplicon and pET28a was double digested with NdeI and HindIII; the vector was also treated with alkaline phsphatase . The DNA digests were gel purified and ligated using T4 DNA ligase (New England Biolabs) overnight at room temperature. The ligation mixture was transformed into competent E. coli NEB5α, the pET28a-(Ec)TrpCF plasmid extracted and analyzed via Sanger sequencing (Eurofins). The plasmid carrying the correct sequence was transformed into E. coli BL21(DE3) cells grown in the presence of 50 μg/mL kanamycin. An overnight culture of BL21 expressing TrpCF was transferred to 2 L of LB + 50 μg/mL kanamycin and allowed to grow to an OD600 of 0.6. Protein expression was induced by addition of IPTG to a final concentration of 500 μM and incubation for an additional 3 hrs at 37°C. The cells were harvested by centrifugation at 4000xg for 10 mins. The cells were resuspended in 30 mL of lysis buffer ) and disrupted via sonication (cooling on ice for 1 min every 30 sec sonic bursts). The lysate was centrifuged at 45000xg for 1 hour and the supernatant was loaded onto a Ni-NTA column equilibrated with 25 mL of column buffer . The column was washed with 30 mL of column buffer with increasing imidazole concentrations . The protein was eluted using 10 mL of column buffer containing 300 mM imidazole and collected in 1 mL fractions. The fractions containing purified TrpCF (as seen from a Coomassie-stained polyacrylamide gel) were pooled together and concentrated to ~2.5 mL. The protein was buffer exchanged using a PD-10 (Sigma-Aldrich) column equilibrated using 30 mL of storage buffer  and eluted from the PD-10 column using 3 mL of storage buffer. The protein were split into 300 μL aliquots and flash frozen using a dry ice/isopropanol bath and stored at -80°C until use. The protein concentration was measured spectrophotometrically using the Pierce BCA Assay kit (ThermoFisher) and the accompanying BSA standards.To test the α-reaction of TrpAB, isolation and purification of the substrate indole-3-glycerol phosphate (IGP) was necessary. IGP is not available commercially and no chemical synthesis method has been published, so we developed a method to enzymatically synthesize IGP. We used E. coli TrpCF overnight at 37°C. After incubation, to separate out the enzyme from IGP, the solution was loaded onto a 10,000MW filter and centrifuged. The resulting elute was aliquoted and stored at -20C. Conversion of CdRP to IGP was confirmed via LC-MS. Because no CdRP could be detected via LC-MS after overnight incubation, the concentration of resulting IGP was calculated by incubating IGP overnight with excess TrpAB and quantifying the amount of L-Trp present with LC-MS-based L-Trp standard curve.To generate CdRP, we followed chemical synthesis protocols previously reported . After d+, 30 mM sodium arsenate, 1 μg GAPDH, and increasing amounts of either IGP or L-Serine. The KM and kcat values for the α-reaction were calculated using the rates obtained from varying IGP concentrations. Because the activity of the TrpAB subunits is tightly coupled, such that no TrpA product will be produced without the binding of L-Ser to TrpB, we utilized as an analog, the formation of G3P from TrpA (under varying concentrations of L-Ser) to indirectly monitor the progress of the β-reaction. Therefore, KM and kcat values for the β-reaction were calculated using the rates obtained from increasing concentrations of L-Ser. The enzyme mix was aliquoted into the wells of clear, flat bottom 96-well plate. The reaction was initiated with the addition of substrate and monitored by following the NADH absorbance at 340nm (εNADH = 6300 M-1 cm-1).The TrpAB kinetics was monitored with an absorbance-based assay using a ClarioStar multiwell plate reader. The activity of the α-subunit was coupled to the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 100 nM TrpAB was prepared in the TrpAB storage buffer with 2.5 mM NAD-1 cm-1) (To monitor the β-reaction exclusively (Indole + L-Ser → L-Trp) a spectrophotometric monitoring was employed  using a -1 cm-1) . All kin+, 30 mM sodium arsenate, 1 μg GAPDH, and increasing concentrations of inhibitors. The reaction was initiated by addition of the enzyme and progress was monitored by following the NADH absorbance at 340nm (εNADH = 6300 M-1 cm-1). Inhibition of the β-reaction was monitored at 290 nm and performed as described above. The mixtures  were prepared in TrpAB storage buffer and included 100 nM TrpAB, 0.1 mM Indole, and 3 mM L-Ser  in the presence of increasing inhibitor concentrations. The reaction was initiated by addition of the enzyme and the progress was monitored by recording the absorbance at 290 nm. All inhibition studies were done at 25°C.Inhibition of the α-reaction was monitored by coupling the activity of the α-subunit to that of GAPDH. The mixtures  were prepared in TrpAB storage buffer and included 100 nM TrpAB, 50 μM IGP, 20 mM L-Ser, 2.5 mM NAD+) for the M+H+ adduct at 220m/z. Response units obtained from the LC-MS were converted to μM units using a separately determined calibration curve using synthetic 4-aTrp.A LC-MS-based assay was developed to detect and quantify the conversion of 4-AI to 4-aTrp. 50 nM TrpAB WT and mutant TrpAB were prepared in TrpAB buffer with 30 mM L-Serine. The reaction was initiated by the addition of increasing amounts of 4-aminoindole. After 5 minutes of incubation, the reaction was quenched with an equal volume of 0.1% formic acid in methanol followed by centrifugation. 2 μL of the collected supernatant was injected into the LC-MS. 4-aminotryptophan was separated and detected on an Agilent 1100 HPLC system with a single quadrupole mass selective detector (G1946) using a Waters Acquity BEH Amide 2.1 mm by 50mm 1.7mm particle column. The mobile phase was aqueous with 0.1% formic acid (channel A) and Acetonitrile with 0.1% formic acid (channel B). Gradient of 95% B was initiated and linearly changed to 50% B over 10 minutes at flow rate 0.3 mL/min. Compounds were quantified by diode array detector with absorbance at 220nm. Tandem confirmation was implemented by scanning in positive mode with electrospray ionization (ESIFurther confirmation of 4-aTrp by TrpAB WT was determined on an Agilent 1290 infinity with triple quadrupole mass selective detector (6460C). Confirmation was determined by retention time (same column / mobile phase) and energy resolved mass spectrometry (erms) profile with CEV 0-70V (E = 0 to 7.9eV) for both enzymatic product and synthetic standard.The 4-aTrp concentration following 4-AI treatment of Mtb was done exactly the same as the compound metabolism studies described above, except that to quench the metabolism, the aliquots were transferred to an equal volume of 1:1 ACN:MeOH mixture prior to filtering out the cells in the mixture. The 4-aTrp was detected and quantified using the LC-MS equipped with BEH amide column. Concentrations were normalized against the WT run of the first trial.AcOH-acetic acidDCM-dichloromethaneDIPEA-N,N-diisopropylethylamineDMAP- 4-DimethylaminopyridineDMF-N,N-dimethylformamideDMSO-dimethyl sulfoxideEDCI- 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideEtOAc-ethyl acetateEtOH-ethanolFA-formic acidHATU-1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolopyridinium-3-oxide hexafluorophosphateHOBt-1-hydroxybenzotriazoleMeCN-acetonitrileMeOH-methanolTHF-tetrahydrofuan•HP Agilent LC/MS series 1100 system equipped with a reverse phase column  and photodiode array detector coupled to an Agilent 1946 DSL quadrupole mass selective detector using electrospray ionization (ESI).•Bruker MicrOTOF II focus ESI Mass Spectrometer connected in parallel to Dionex Ultimate 3000 RSLC system with diode array detector equipped with a reverse phase column  using ESI.•HP Agilent LC/MS series 1100 system equipped with a reverse phase column  and photodiode array detector coupled to an Agilent G1956A quadrupole mass selective detector using electrospray ionization (ESI)•HP Agilent LC/MS series 1260 system equipped with a reverse phase column  and photodiode array detector coupled to an Agilent G6110A quadrupole mass selective detector using electrospray ionization (ESI)•Shimadzu LC20A system equipped with a reverse phase column  and photodiode array detector coupled to a Shimadzu LCMS-2020 mass selective detector using electrospray ionization (ESI)•The compounds were eluted with a gradient of 5 to 95% MeCN/water +0.025 or 0.1% Ammonia, or +0.1% FA or 0.0375% TFA.All reagents including solvents used in the synthetic procedure were purchased from Sigma-Aldrich and used without further purification unless otherwise mentioned. NMR was performed by either a Bruker AVANCE III HD NanoBay 400 MHz, Spectrometer or Bruker Avance DPX 400, Bruker Avance DPX 500 spectrometer, and chemical shifts were measured in ppm relative to specific solvent signal. Routine mass and purity analyses (LRMS) were performed on:•Method A. Varian preparative HPLC system equipped with a reverse phase column  and photodiode array detector.•Method B: Gilson preparative HPLC system equipped with a reverse phase column (Column: Phenomenex Synergi C18 150∗25∗10 μmum) and a photodiode array detector; Mmobile phase: [water (0.225% FA)-MeCN]; B%: 35%-65%, 10 min)•Method C: Gilson preparative HPLC system equipped with a reverse phase column Column: PPhenomenex Synergi C18 150∗30 mm∗4 μmum) and a photodiode array detector; Mmobile phase: [water (0.225% FA)-MeCN]; B%: 45%-72%, 12 min)•Method D: Gilson preparative HPLC system equipped with a reverse phase column Column: Phenomenex Synergi C18 150∗25∗10 μmum) and a photodiode array detector; Mobile phase: [water (0.225% FA)-MeCN]; B%: 20%-50%, 10 min)•Method E: Gilson preparative HPLC system equipped with a reverse phase column Column: Phenomenex Gemini 150∗25 mm∗10 μmum) and a photodiode array detector; Mobile phase: [water (0.04% NH4OH+10mM NH4HCO3)-MeCN]; B%: 35%-65%, 10 min•Method F: Gilson preparative HPLC system equipped with a reverse phase column ( Phenomenex Synergi C18 150∗25∗10 μm) and a photodiode array detector; Mobile phase: [water (0.225% FA)-MeCN]; B%: 50-80%, 10 min)•Method G: Gilson preparative HPLC system equipped with a reverse phase column (Phenomenex column: Gemini 150∗25 5μmu) and a photodiode array detector; mMobile phase: [water (0.05% ammonia hydroxide v/v)-MeCN]; B%: 46%-76%, 12 min•Method H: Gilson preparative HPLC system equipped with a reverse phase column: Phenomenex Gemini 150∗25mm∗10μmum) and a photodiode array detector; mMobile phase: [water (0.04%NH4OH+10mM NH4HCO3)-MeCN]; B%: 37%-67%, 9 min•Method I: Gilson preparative HPLC system equipped with a reverse phase column Phenomenex Gemini 150∗25mm∗10uμmm) and a photodiode array detector; mobile phase: [water (0.04%NH4OH+10mM NH4HCO3)-MeCN]; B%: 40%-70%, 9.5 min•Method J: Gilson preparative HPLC system equipped with a reverse phase column column: Phenomenex Synergi C18 150∗25∗10μmum) and a photodiode array detector; mobile phase: [water (0.225%FA)-MeCN]; B%: 40%-70%, 9min•Method K: Enamine Instrument: Agilent 1260 Infinity HPLC systems equipped with a reverse phase column DAD and mass-detector Column:  Detection: DAD – DAD1A 215 nm, DAD1B 254 nm. MSD – single quadrupole, AP-ESI Mobile phases: A - Deionized water (100%). B - HPLC-grade MeOH (100%) Gradient: from A-50%: B-50% to A-0%: B-100%.•Method L: Gilson preparative HPLC system equipped with a reverse phase column column: Phenomenex Synergi C18 150∗25∗10μmum) and a photodiode array detector; Mmobile phase: [water (0.225%FA)-MeCN]; B%: 45%-75%, 9minThe purification of compounds was performed by flash column purification using SiliaFlash® P60 (40–60 μm) or by preparative HPLC using one of the following methods:1H NMR) unless otherwise stated.All reagents including solvents used in the synthetic procedure were purchased from Sigma-Aldrich and used without further purification unless otherwise mentioned. Synthesized compounds were ≥95% pure . The reaction mixture was stirred at room T for about 4 h. After evaporation, it was purified by preparative HPLC system in the condition of 20 mL/min of flow rate with linear gradient over 30 min . A yellow solid mixture of 4- and 6-nitro-L-tryptophan was obtained : 1H NMR  δ 8.34 , 7.97−7.93 , 7.79−7.76 , 7.56 , 7.50 , 7.26 , 4.11−4.03 , 3.74 , 3.49 , 3.37−3.30 ; LRMS (ESI) m/z 250 [M + H]+.To a suspension of L-tryptophan  in 4 mL AcOH and 1 mL H3 . The reaction mixture was stirred overnight at room. The reaction mixture was diluted with EtOH and washed with 0.1N HCl and brine. The organic layer was dried over Na2SO4 and evaporated in vacuo. The crude mixture was purified by flash column chromatography (EtOAc / hexane = 4 / 1 with 0.4% AcOH) to obtain the desired product 2 : 1H NMR  δ 8.56 , 7.88 , 7.72 , 7.36 , 5.14 , 4.59 , 3.47 , 3.15 , 1.67 , 1.33 ; LRMS (ESI) m/z 448 [M - H]-.To a solution of 1  in 5 mL of MC were added Boc anhydride , DMAP , and NEt2 balloon. After filtration of the reaction mixture through a pad of celite, it was evaporated and used in the next reaction without further purification (3). The evaporated residue (3) was dissolved in 1 mL of MC and 1 mL of TFA. The reaction mixture was stirred for about 2 h at room T. After evaporation, it was purified by preparative HPLC system in the condition of 20 mL/min of flow rate with A / B = 95 / 5 over 10 min  to obtain desired product 4 as a TFA salt : 1H NMR  δ 7.58 , 7.43 , 7.25 , 7.11 , 4.23 , 3.57 , 3.46 ; 13C NMR  δ 162.9, 162.5, 137.9, 127.3, 121.9, 121.8, 121.6, 119.4, 114.7, 113.2, 104.4, 25.8; LRMS (ESI) m/z 220 [M + H]+.To a solution of 1 (20 mg) in 3 mL MeOH was added 5 mg of 10 wt.% Pd/C. The reaction mixture was stirred for about 2 h at room temperature under a HC2  as a dark solid. 1H NMR  δ 1.18 , 9.76 , 7.54 , 7.39 - 7.28 , 7.20 , 7.08 - 6.95 , 6.60 , 4.82 ; LRMS (ESI) m/z 267.0 [M + H]+.To a solution of 1H-indol-4-amine  and 2-phenoxyacetic acid  was added EDCI , HOBt  and DIPEA  and the solution was stirred at 25°C for 20 hours. The mixture was poured into an ice-water (5 mL), the resulting precipitate collected by filtration, washed with water (2 mL×3) and dried under vacuum. The crude product was purified by Prep-HPLC (Method B) to give the desired compound C3  as a gray solid. 1H NMR : δ 11.07 , 9.40 , 7.57 , 7.27 , 7.12 , 7.04 – 6.95 , 6.70 , 2.53 , 1.86 – 1.75 , 1.67 , 1.50 – 1.39 , 1.34 – 1.18 ; LRMS (ESI) m/z 243.1 [M + H]+To a solution of cyclohexanecarboxylic acid  and 1H-indol-4-amine  in DMF (2 mL) was added HATU  and DIPEA  and the solution was stirred at 25°C for 4 hours. The mixture was poured into an ice-water (5 mL), the resulting precipitate collected by filtration, washed with water (2 mL×3) and dried under vacuum. The crude product was purified by Prep-HPLC (Method B) to give the desired compound in vacuo to remove most of the solvent to give a crude residue which was poured into saturated ammonium chloride solution (200 mL) and stirred for 5 min. The pH of the mixture was adjusted to pH=3 with HCl  and the aqueous phase was extracted with EtOAc (30 mL×3). The combined organic phases were washed with brine (20 mL), dried with anhydrous anhydrous sodium sulfate, filtered and concentrated in vacuo to give methyl 5-picolinate  as a white solid which was used without further purification. 1H NMR  δ= 8.56 , 8.14 , 7.73 , 5.05 , 3.91 ; LRMS (ESI) m/z 236.1 [M + H]+To a mixture of methyl 5-hydroxypicolinate  and 2,2,2-trifluoroethyl trifluoromethanesulfonate  in MeCN (5 mL) was added cesium carbonate  at 0°C and the mixture was stirred at 25°C for 10 hours. The reaction was concentrated in vacuo to give 5-picolinic acid  as a white solid. LRMS (ESI) m/z 222.0 [M + H]+To a mixture of give methyl 5-picolinate  in MeOH (6 mL) was added a solution of lithium hydroxide monohydrate  in water (2 mL) at 25°C and the mixture was stirred at 25°C for 12 hours. The pH of the mixture was adjusted to pH=5 with AcOH (0.15 mL), stirred for 20 min and extracted with EtOAc (30 mL×3). The combined organic phase was washed with brine (30 mL), dried with anhydrous anhydrous sodium sulfate, filtered and concentrated in vacuo to give a crude residue which was purified by prep-HPLC (Method C) to give the desired compound C4  as a white solid. 1H NMR  δ = 11.28 , 10.26 , 8.58 , 8.21 , 7.83 - 7.76 , 7.39 , 7.24 , 7.11 , 6.52 , 5.04 ; LRMS (ESI) m/z 336.1 [M + H]+To a mixture of 5-picolinic acid  and 1H-indol-4-amine  in DMF (5 mL) was added HATU  and DIPEA  in one portion at 25°C and the mixture was stirred for 10 hours. The mixture was filtered and the filtrate was concentrated C6  was obtained as a white solid. 1H NMR : δ 11.06 , 10.17 , 8.82 , 8.35 , 7.97 , 7.40 - 7.30 , 7.14 , 6.45 - 6.39 , 5.11 ; LRMS (ESI) m/z 336.0 [M + H]+.To a solution of 1H-indol-5-amine  and 6-nicotinic acid  in DMF (0.5 mL) was added EDCI , HOBt  and DIPEA , the resulting precipitate collected by filtration, washed with water (2 mL×3) and dried under vacuum. The crude product was purified by Prep-HPLC (Method D) to give the desired compound C6, except using 1H-indol-6-amine  the desired compound C7  was obtained as a white solid. 1H NMR : δ 11.08 , 10.25 , 8.81 , 8.35 , 8.05 , 7.49 , 7.34 - 7.22 , 7.14 , 6.39 , 5.11 ; LRMS (ESI) m/z 336.0 [M + H]+.Using a similar method to C6, except using 1H-indol-7-amine , the desired compound C8  was obtained as a white solid. 1H NMR : δ 10.92 , 10.22 , 8.90 , 8.41 , 7.50 - 7.27 , 7.17 , 7.01 , 6.48 , 5.13 ; LRMS (ESI) m/z 336.0 [M + H]+.Using a similar method to C6, except using 1-methylindol-4-amine , the desired compound C9  was obtained as a white solid. 1H NMR : δ 10.20 , 8.85 , 8.37 , 7.49 - 7.09 , 6.61 , 5.12 , 3.81 ; LRMS (ESI) m/z 350.0 [M + H]+.Using a similar method to To a solution of 6-pyridine-3-carboxylic acid -6-nicotinamide  in DMF (1 mL) was added EDCI , HOBt  and DIPEA  and the solution was stirred at 25°C for 16 hours. The mixture was poured into an ice-water (5 mL), the resulting precipitate collected by filtration, washed with water (2 mL×3) and dried under vacuum to give N-(1H-indol-4-yl)-6-nicotinamide  as a black solid. LRMS (ESI) m/z 336.0 [M + H]+3•THF  at 25°C and the solution was stirred at 50°C for 3 hours. The reaction mixture was concentrated in vacuo to give a crude residue which was azetroped with EtOAc (2 mL×3). The crude product was purified by Prep-HPLC (Method F). to give the desired compound C10  as a gray solid. 1H NMR  δ 10.82 , 8.21 , 7.80 , 7.12 - 7.08 , 6.95 - 6.86 , 6.83 - 6.74 , 6.69 - 6.58 , 6.23 , 6.00 , 4.95 , 4.38 , 1.11 - 0.99 ; LRMS (ESI) m/z 322.1 [M + H]+To a solution of N-(1H-indol-4-yl)-6-nicotinamide  in THF (1 mL) was added BHin vacuo to give 5-nitro-2-pyridine  as a white solid. 1H NMR : δ 9.12 , 8.59 , 7.25 , 5.16 ; LRMS (ESI) m/z 223.0 [M + H]+To a mixture of sodium hydride  in DMF (10 mL) was added 2,2,2-trifluoroethanol  at 0°C and the mixture was stirred at 0°C for 1 hour. A solution of 2-chloro-5-nitropyridine  in DMF (10 mL) was added to the cooled reaction mixture at 0°C and the mixture was stirred at 25°C for 2 hours. The reaction was concentrated in vacuum to remove most of the solvent. The residue was poured into saturated of ammonium chloride solution (200 mL) and stirred for 5 min. The aqueous phase was extracted with EtOAc (30 mL×3). The combined organic phases were washed with brine (20 mL), dried with anhydrous anhydrous sodium sulfate, filtered and concentrated 2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20°C for 4 hours. The reaction was filtered and the filtrate concentrated in vacuo to give 6-pyridin-3-amine  as a yellow oil. 1H NMR : δ 7.51 , 7.07 , 6.69 , 4.93 , 4.80 To a solution of 5-nitro-2-pyridine  in MeOH (10 mL) was added 10% Pd/C (40 mg) under Hin vacuo to remove most of the solvent, the residue was poured into water (20 mL) and stirred for 5 minutes. The aqueous phase was extracted with EtOAc (30 mL×3), the combined organic phases were washed with brine (20 mL), dried with anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (Method B) to give the desired compound C11  as a white solid. 1H NMR : δ 11.41 , 10.31 , 8.62 , 8.21 , 7.64 , 7.59 , 7.50 , 7.23 , 7.03 , 6.91 - 6.81 , 4.99 ; LRMS (ESI) m/z 336.0 [M + H]+To a mixture of 6-pyridin-3-amine  and 1H-indole-4-carboxylic acid  in pyridine (10 mL) was added EDCI  in one portion at 25°C, the reaction warmed to 45°C and stirred for 10 hours. The reaction was concentrated EWTo a solution of 6-nicotinic acid  and 1H-indol-4-amine  in DMF (1 mL) was added EDCI , HOBt  and DIPEA , the resulting precipitate collected by filtration, washed with water (2 mL×3) and dried under vacuum to give N-(1H-indol-4-yl)-6-nicotinamide  was obtained as a black solid. LRMS (ESI) m/z 336.0 [M + H]+in vacuo. The crude product was purified by Prep-HPLC (Method D) to give N-(1-formylindolin-4-yl)-6-nicotinamide . (ESI) m/z 366.0 [M + H]+N-(1H-indol-4-yl)-6-nicotinamide  in AcOH (1 mL) was added sodium cyanoborohydride  and the reaction was stirred at 25°C for 4 hours. The mixture was poured into water (1 mL) and extracted with EtOAc (15 mL×4). The combined organic layers were dried over anhydrous sodium sulfate and concentrated C12  as an orange solid. 1H NMR : δ 9.93 , 8.78 , 8.30 , 7.13 , 6.91 , 6.64 , 6.38 , 5.56 , 5.10 , 3.46 - 3.39 , 2.88 ; LRMS (ESI) m/z 338.0 [M + H]+.To a solution of N-(1-formylindolin-4-yl)-6-nicotinamide  and EtOH (0.5 mL) was added aq.HCl  and the mixture was stirred for 6 hrs at 80°C. The mixture was cooled to room temperature, poured into an ice-water (5 mL), the resulting precipitate collected by filtration, washed with water (2 mL×3) and dried under vacuum. The crude product was purified by Prep-HPLC (Method E) to give the desired compound in vacuo to remove most of the solvent to give a residue which was poured into water (20 mL) and stirred for 5 min. The aqueous phase was extracted with EtOAc (30 mL∗3). The combined organic phases were washed with brine (20 mL), dried with anhydrous anhydrous sodium sulfate, filtered and concentrated in vacuo to give N-methyl-1H-indol-4-amine  as a white solid. LRMS (ESI) m/z 147.1 [M + H]+.To a mixture of 1H-indol-4-amine  in DMF (10 mL) was added sodium hydride  in one portion at 0°C and the mixture was stirred at 0°C for 1 hour. Methyl iodide  was added into the mixture which was stirred at 0°C for 1 hour. The reaction was concentrated in vacuo to remove most of the solvent to give a crude residue which was poured into water (20 mL) and stirred for 5 min. The aqueous phase was extracted with EtOAc (30 mL∗3). The combined organic phases were washed with brine (20 mL), dried with anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (Method F) to give the desired compound C13  as white solid. 1H NMR : δ 10.22 , 8.85 , 8.37 , 7.43 , 7.33 - 7.28 , 7.19 - 7.11 , 6.61 , 5.12 , 3.81 ; LRMS (ESI) m/z 350.1 [M + H]+.To a mixture of compound 6-nicotinic acid  and N-methyl-1H-indol-4-amine  in pyridine (5 mL) was added EDCI  in one portion at 25°C, the mixture was warmed to 45°C and stirred for 10 hours. The reaction was concentrated C14  as a grey solid. 1H NMR  δ 10.85 , 8.61 , 8.28 - 8.18 , 7.82 , 7.32 - 7.19 , 5.05 ; LRMS (ESI) m/z 337.1 [M + H]+.To a solution of 5-picolinic acid  and 1H-benzimidazol-4-amine  in DMF (2 mL) was added HATU  and DIPEA  and the solution was stirred at 40°C for 16 hours. The mixture was poured into an ice-water (5 mL), the resulting precipitate collected by filtration, washed with water (2 mL×3) and dried under vacuum. The crude product was purified by Prep-HPLC (Method H) to give the desired product in vacuo to give 5-picolinamide  as pink solid. 1H NMR  δ 8.42 , 8.04 , 7.68 , 7.54 , 4.98 ; LRMS (ESI) m/z 221.0 [M + H]+.To the solution of 5-picolinic acid  and ammonium chloride  in DMF (4 mL) was added EDCI , HOBt  and DIPEA  and the reaction mixture was stirred at 25°C for 12 hours. The reaction was quenched with water (4 mL) and extracted with EtOAc (8 mL∗3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo to give a crude residue which was purified by Prep-HPLC (Method I) to give the desired product C15  δ 11.73 , 10.47 , 8.59 , 8.26 - 8.19 , 7.88 , 7.82 , 7.49 - 7.44 , 6.63 , 5.09 - 5.03 ; LRMS (ESI) m/z 337.1 [M + H]+.To the solution of 1H-pyrrolopyridin-4-ylboronic acid  and 5-picolinamide  in DCM (2 mL) was added copper (II) acetate  and pyridine  and the reaction mixture was stirred at 25°C for 12 hours. The reaction mixture was concentrated in vacuo to give 6-pyridazine-3-carboxylic acid  as a yellow solid. LRMS (ESI) m/z 223.0 [M + H]+.To a suspension of sodium hydride  in DMF (1 mL) was added 2,2,2-trifluoroethanol  dropwise at 0°C and the mixture was stirred at 0°C for 30 minutes. Methyl 6-chloropyridazine-3-carboxylate  in DMF (1 mL) was added dropwise at 0°C and the mixture was stirred at 50°C for 2.5 hours. The mixture was poured into an ice-water (1 mL) and extracted with EtOAc (1 mL∗3). The combined organic layer was washed with brine (1 mL∗3), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated C16  as a grey solid. 1H NMR  δ 11.26 , 10.49 , 8.34 , 7.70 - 7.62 , 7.38 , 7.28 , 7.16 - 7.09 , 6.54 , 5.34 ; LRMS (ESI) m/z 337.0 [M + H]+.To a solution of 6-pyridazine-3-carboxylic acid  and 1H-indol-4-amine  in DMF (2 mL) was added HATU  and DIPEA  and the solution was stirred at 25°C for 4 hours. The mixture was poured into an ice-water (5 mL), the resulting precipitate collected by filtration, washed with water (2 mL×3) and dried under vacuum. The crude product was purified by Prep-HPLC (Method J) to give the desired product C17)  as an off-white solid. 1H NMR  δ 11.13 , 9.65 , 7.53 , 7.31 , 7.16 , 7.03 , 6.94 , 6.59 , 4.76 , 2.25 ; HRMS (ESI) m/z 281.1308 [M + H]+.To a stirred solution of 2-(p-tolyloxy)acetic acid  and HOBt  in DMSO (2 mL) was added 4-aminoindole  and the reaction stirred for 30 minutes. EDCI  was added and stirring continued for 24 hours. The crude reaction mixture was purified by preparative HPLC (Method K), to give the desired product acetic acid , the desired compound (C18)  was obtained as an off-white solid. 1H NMR  d 10.90 , 9.00 , 7.34 , 7.08 , 6.94 , 6.79 , 6.28 , 3.90 , 3.19 , 0.93 - 0.84 , 0.31 - 0.25 , 0.03 - 0.00 ; HRMS (ESI) m/z 245.1300 [M + H]+.Using a similar method to in vacuo. The crude residue was purified by Reverse phase flash column (water/MeCN+ 0.1% FA) to give -3-hydroxy-N-(1H-indol-4-yl)cyclobutane-1-carboxamide  as a pink solid. 1H NMR  δ 11.18 - 11.00 , 9.43 , 7.61 , 7.35 - 7.24 , 7.17 - 7.10 , 7.04 - 6.97 , 6.68 , 5.16 , 4.11 - 3.92 , 2.89 - 2.75 , 2.44 - 2.30 , 2.44 - 2.30 , 2.14 - 1.98 ; LRMS (ESI) m/z 231.1 [M + H]+.To a solution of -3-hydroxycyclobutane-1-carboxylic acid  and 1H-indol-4-amine  in DMF (3 mL) was added HATU  and DIPEA  and the mixture was stirred at 40°C for 16 hours. The reaction was quenched with water (6 mL) and extracted with EtOAc (12 mL∗3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The crude residue was purified by Reverse phase flash column (water/MeCN+ 0.1% FA) to give -3-((1H-indol-4-yl)carbamoyl)cyclobutyl methanesulfonate  as gray solid. 1H NMR  δ 11.10 , 9.63 , 7.58 , 7.28 , 7.16 , 7.07 - 6.95 , 6.66 , 4.99 , 3.22 - 3.05 , 2.67 , 2.34 ; LRMS (ESI) m/z 309.0 [M + H]+.To a solution of -3-hydroxy-N-(1H-indol-4-yl)cyclobutane-1-carboxamide  in DCM (4 mL) was added triethylamine  and methanesulfonyl chloride  and the mixture was stirred at 25°C for 12 hrs. The reaction was quenched with water (1 mL) and extracted with EtOAc (2 mL∗3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by Prep-HPLC (Method L) to give the desired product C19  as a white solid. 1H NMR  δ 7.65 , 7.33 - 7.26 , 7.14 , 7.05 - 6.98 , 6.93 , 6.84 , 6.70 , 4.90 , 3.51 , 2.74 - 2.67 , 2.39 - 2.32 ; LRMS (ESI) m/z 307.1 [M+H]+.To a solution of phenol  in DMF (1.25 mL) was added cesium carbonate  and the mixture was stirred at 80°C for 1 hour. -3-((1H-indol-4-yl)carbamoyl)cyclobutyl methanesulfonate  in DMF (1.25 mL) was then added and the reaction mixture stirred at 80°C for 1 hour. The reaction was quenched with water (2 mL) and extracted with EtOAc (3 mL∗3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated 3  under N2 and the reaction was stirred at 90°C for 12 hrs. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography  to give methyl 6-amino)nicotinate  as light yellow solid. 1H NMR  δ 8.79 , 8.05 , 6.50 , 5.03 , 4.20 , 3.89 ; LRMS (ESI) m/z 235.1 [M+H]+.To the reaction mixture of methyl 6-bromonicotinate , 2,2,2-trifluoroethan-1-amine  and t-BuONa  in dioxane (6 mL) was added tBuXPhos-Pd-Gin vacuo to give methyl 6-amino)nicotinate  as a grey solid. 1H NMR  δ 8.82 , 8.11 , 6.60 , 4.42 , 3.89 , 3.20 ); LRMS (ESI) m/z 249.1 [M+H]+.Methyl 6-amino)nicotinate , methyl iodide  and cesium carbonate  in DMF (2.5 mL) was stirred at 50°C for 3 hrs. The reaction mixture was poured into saturated ammonium chloride (5 mL) and extracted with EtOAc (2 mL∗3). The combined organic layer was washed with brine (5mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated 6-amino)nicotinate  and lithium hydroxide monohydrate  in THF (0.6 mL) and water (0.6 mL) was stirred at 25°C for 12 hrs. The reaction mixture was adjusted to pH=2 with aq.HCl (4M) and extracted with EtOAc (2 mL∗3). The combined organic layer was washed with brine (5 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give 6-amino)nicotinic acid  as a grey solid. LRMS (ESI) m/z 235.1 [M+H]+.in vacuo to give a crude residue which was purified by Prep-HPLC (Method L) to obtain the desired compound C20  as a grey solid. 1H NMR  δ 11.10 , 9.90 , 8.79 , 8.19 , 7.36 , 7.29 , 7.22 , 7.09 - 7.03 , 6.91 , 6.59 , 4.59 , 3.17 . LRMS (ESI) m/z 349.1 [M+H]+6-amino)nicotinic acid , 1H-indol-4-amine , HATU  and DIPEA  in DMF (1.5 mL) was stirred at 25°C for 12 hrs. The reaction mixture was concentrated in vacuo to give a crude residue which was purified by Prep-HPLC (Method L) to give the desired compound C21  as a grey solid. 1H NMR  δ 11.14 , 9.74 , 7.33 - 7.23 , 7.11 - 7.03 , 6.53 , 6.32 , 2.39 ; LRMS (ESI) m/z 241.1 [M+H]+.1H-indol-4-amine , 5-methylfuran-2-carboxylic acid , HOBt , EDCI  and DIPEA  in DMF (1 mL) was stirred at 25°C for 12 hours. The reaction mixture was concentrated www.graphpad.com. The number of biological replicates and statistical tests applied for each experiment is given in the corresponding figure legend.Statistical analysis was performed in GraphPad Prism version 8.0.1 for Widnows, GraphPad Software, San Diego, Califonia USA,


As far as compound I–azo–NH–MMA is concerned, a non-centrosymmetric pattern is formed due to a very rare I···π interaction involving the alkene group supplemented by hydrogen bonds. The Cambridge Structural Database contains only four structures showing the same I···CH2=C contact. For all compounds, an 19F-NMR spectroscopic analysis confirms the formation of halogen bonds in solution through a recognition process with chloride anion, and the reversible photo-responsiveness is demonstrated upon exposing a solution to UV light irradiation. Finally, the intermediate I–azo–NH2 also shows a pronounced color change due to pH variation. These azobenzenes are thereby attractive building blocks to design future multi-stimuli responsive materials for highly functional devices.Smart materials represent an elegant class of (macro)-molecules endowed with the ability to react to chemical/physical changes in the environment. Herein, we prepared new photo responsive azobenzenes possessing halogen bond donor groups. The X-ray structures of two molecules highlight supramolecular organizations governed by unusual noncovalent bonds. In azo dye I-azo-NO Dioxane is concentrated and the crude material is extracted three times with diethyl ether, washed with an aqueous solution of sodium carbonate, dried over sodium sulphate and the organic layer is concentrated affording a red solid. The crude material was purified by column chromatography using dichloromethane as eluent to afford HO–azo–NH2. Yield: 92%. Mp: 185 °C (decomposed). 1H-NMR  δ 7.68 , 7.65 , 6.86 , 6.74 . 13C-NMR  δ 160.66, 152.53, 147.75, 145.86, 125.48, 124.94, 116.56, 115.37. rocedure . HO–azo–2)4-phenyl)diazenyl)aniline  was dissolved in DMF (25 mL) at 50 °C. Potassium carbonate  was added to the reaction mixture and vigorously stirred for 20 min. Pentafluoroiodobenzene  was added dropwise to the reaction mixture and the reaction was stirred for 6 h. Upon complete conversion of HO–azo–NH2, the reaction was precipitated in water and the solid was washed with n-hexane yielding I–azo–NH2 as an orange solid. Furthermore, the aqueous layer was extracted with diethyl ether three times, and the organic layer was dried over anhydrous Na2SO4 and concentrated to afford I–azo–NH2. Yield: 91%. Mp: 165–168 °C. 1H-NMR  δ 7.85 , 7.78 , 7.06 , 6.73 , 4.05 . 13C-NMR  δ 157.98, 149.72, 149.52, 145.58, 125.20, 124.22, 116.07, 114.77. 19F-NMR  δ −119.35, −151.26. HR-MS (ESI) [M + H]+ Calculated: 487.9877; Found: 487.9900 (4.54 ppm)N-phenyl)diazenyl)phenyl)methacrylamide (I–azo–NH–MMA). To a stirred solution of I–azo–NH2  in dichloromethane (3 mL) were added triethylamine  and methacryloyl chloride  at 0 °C. After stirring for 5 min, the mixture was allowed to warm up to room temperature and stirred for 1 h. Then the reaction was quenched with H2O and extracted with dichloromethane (3 × 15 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated under reduced pressure and purified by column chromatography using dichloromethane as eluent to afford I–azo–NH–MMA. Yield: 87%. Mp: 200–202 °C. 1H-NMR  δ 7.93 , 7.929  7.74 , 7.67 , 7.09 , 5.84 , 5.52 , 2.09 . 13C-NMR  δ 166.66, 158.72, 149.07, 149.02, 140.96, 140.58, 124.83, 124.24, 120.45, 120.10, 116.14, 18.88. 19F-NMR  δ −119.17, −151.21. HR-MS (ESI) [M + H]+ Calculated: 556.0150; Found: 556.0149 (1.65 ppm)2 and I–azo–NH–MMA, X-ray intensity data were collected at 100 K, on a Rigaku Oxford Diffraction  Supernova Dual Source (Cu at zero) diffractometer equipped with an Atlas CCD detector using ω scans and CuKα (λ = 1.54184 Å) radiation. The images were interpreted and integrated with the program CrysAlisPro  [2 using the ShelXL program package [For the structures of I–azo–NOord, UK) . Using Oord, UK)  the stru package ,51. Non-2, I–azo–NH2 and I–azo–NH–MMA with the tetrabutylammonium chloride salt (TBACl) were monitored by 19F-NMR. Stock solutions of 25 mM (2 mL) and 100 mM (1 mL) of the azo dyes and TBACl were prepared in CDCl3. Bis ether was used as internal standard. Titration samples were prepared as follows  were added, leading to an orange solution.3 mg of I–azo–NH2 and I–azo–NH–MMA highlight supramolecular arrangements developing undulating chains due to non-covalent bonds. In I–azo–NO2, the nitro group is simultaneously engaged in orthogonal hydrogen and halogen bonds, which has been scarcely reported to date. In light of the literature, the self-complementary Ar–I···O2N–Ar synthon can self-assemble in a head-to-tail mode, arising thus as a very appealing motif to design supramolecular architectures in a high reliable fashion (2=C halogen bonding. A survey of the Cambridge Structural Database reveals that only four structures involve this contact. In addition to the crystallographic studies, 19F-NMR spectroscopic analyses have emphasized that such compounds can be involved in intermolecular recognition processes, as proposed through I···Cl− halogen bonding in solution. Subsequently, the photo-responsiveness of all XB-based compounds was studied by irradiation at 366 nm. As expected, a trans to cis isomerization was confirmed by UV-Vis analysis, this phenomenon being reversible. Finally, the synthetic intermediate I–azo–NH2 shows a light and pH responsive profile in combination with a halogen bond donor ability, with a potential application in anion sensing. Further works will aim at preparing polymeric materials using these stimuli responsive units and their application in the preparation of functional compounds.In summary, we have reported the synthesis of new azobenzene derivatives bearing halogen bond donor groups. The X-ray analyses of I–azo–NO fashion . Regardi