Artemisinin from the plant Artemisia annua is the most potent pharmaceutical for the treatment of malaria. In the plant, the sesquiterpene cyclase amorphadiene synthase, a cytochrome‐dependent CYP450, and an aldehyde reductase convert farnesyl diphosphate (FDP) into dihydroartemisinic aldehyde (DHAAl), which is a key intermediate in the biosynthesis of artemisinin and a semisynthetic precursor for its chemical synthesis. Here, we report a chemoenzymatic process that is able to deliver DHAAl using only the sesquiterpene synthase from a carefully designed hydroxylated FDP derivative. This process, which reverses the natural order of cyclization of FDP and oxidation of the sesquiterpene hydrocarbon, provides a significant improvement in the synthesis of DHAAl and demonstrates the potential of substrate engineering in the terpene synthase mediated synthesis of high‐value natural products. In this complex reaction cascade, two 6‐membered rings, four stereocentres, and two double bonds are formed with exquisite regio‐ and stereochemical control in one step.4) can be made from amorpha‐4,11‐diene (3) either through a three‐step chemical synthesis or by combining a biooxidation with two chemical steps.4 can then be converted into 1 chemically or enzymatically in four well‐established steps.5), which is then converted into dihydroartemisinic acid (6) by transition metal‐catalyzed hydrogenation. The pharmaceutical company Sanofi scaled up this process in 2014 but the manufacture was discontinued owing to strong market forces,The sesquiterpenoid endoperoxide artemisinin  is widel7 into the synthetic intermediate DHAAl (4). In contrast to existing procedures, this process, which reverses the natural order of cyclization of FDP and oxidation of the sesquiterpene hydrocarbon, significantly shortens the synthesis of dihydroartemisinic aldehyde (4), in that it uses only one enzyme and requires a single oxidation step that occurs prior to the ADS‐catalyzed cyclization to 4. The process avoids several redox steps after the cyclization since it bypasses the formation of the intermediate amorphadiene (3) altogether  along a complex reaction path that involves isomerization of the 2,3‐double bond from the E to the Z configuration and 1,6‐cyclization to generate a bisabolyl cation (8), followed by a 1,3‐hydride shift from C1 to C7 and 1,10‐cyclization to the amorphyl cation (9). Finally, deprotonation at C12 or C13 generates amorpha‐4,11‐diene  catalyzes the Mgl cation , followe7;4) via a 12‐hydroxyamorphyl cation ‐farnesyl chloride ‐dihydroartemisinic acid (11R)‐6 .Diphosphate 1H‐NMR spectrum of the purified enzymatic products  and 9.57 ppm . The identity of the minor product was determined to be (11R)‐4 based on 1H‐NMR data and comparison with an authentic sample of 4 .S)‐4 epimer based on its diagnostic 1H‐NMR signals at δH 5.27 , 1.64 , 1.07 , and 0.87 ppm , which are complementary to those of (11R)‐4 at δH 5.13 , 1.64 , 1.06 , and 0.87 ppm ‐4 and (11S)‐4 is further supported by the epimerization of the corresponding esters that is described later in Scheme S)‐4 and (11R)‐4, in addition to approximately 10 % of a minor product  of aldehyde 4 based on its identical MS spectrum  with that of (11S)‐4. The MS spectra as well as the GC elution times of (11R)‐4 (faster) and (11S)‐4 are consistent with those reported previously.12 may initially be formed from carbocation OH‐9 through deprotonation of either Ha or Hb (path a‐b in Scheme 12 equilibrates to the observed mixture of epimers of 4 outside the active site of ADS. However incubations of diphosphate 7 with ADS in D2O led to the exclusive production of deuterated (11S)‐4 ; no deuterated (11R)‐4 was detected by GC–MS. This observation indicates that only (11S)‐4 can be formed via enol 12.The 13) was prepared in three steps  from known 1‐O‐THP‐protected 12‐hydroxyfarnesol7 ‐4 after its release from the active site. Control experiments excluded the presence of ester hydrolase activity in our enzyme preparations (see the Supporting Information). Remarkably, the incubation of 13‐acetoxyfarnesyl diphosphate  with ADS (Scheme S)‐4 (93 %) and enol 12 ‐epimer of 4 implies that enol 12 does not revert to (11R)‐4 under the reaction conditions  most likely proceeds through an oxygen‐assisted non‐stereospecific intramolecular ‐hydride shift from carbocation OH‐9 , deprotonation of 17 via enol 12 to (11S)‐4 appears unlikely. Hence a stereospecific ‐hydride shift of Ha from carbocation 17 most likely accounts for the exclusive formation of (11S)‐4  and (11S)‐4 was oxidized to the corresponding dihydroartemisinic acids (6) in 93 % yield (Scheme 18) in 94 % yield .R)‐ and (11S)‐methyl esters . (11R)‐dihydroartemisinic methyl ester is the desired intermediate for the synthesis of artemisinin.To further advance this novel chemoenzymatic approach towards the production of artemisinin, the 2:3 mixture of (11ic acids  in 93 % d Scheme  in 94 % 4), a major intermediate in the production of artemisinin (1), the most important drug for the treatment of malaria. We have for the first time shown that hydroxylated FDP analogues can be accepted as substrates by sesquiterpene synthases. Hence our work offers a novel “reversed biosynthetic” approach for the synthesis of functionally diversified hydroxylated terpenoids. The chemical synthesis of such products is often difficult owing to the need to make several small rings with high stereo‐ and regiocontrol. The relatively high substrate promiscuity and the templating effect of the active site allow terpene synthases to chaperone unnatural substrates along well‐defined reaction paths to specific products with high fidelity. This design offers a promising approach for the production of high‐value terpenoids and terpene alkaloidsIn conclusion, we have developed an efficient chemoenzymatic route to dihydroartemisinic aldehyde  should be addressed to the authors.SupplementaryClick here for additional data file.


Bladder afferent nerve responses to the purinergic agonist αβMethylene-ATP were depressed in TRPV1−/− mice (p ≤ 0.001) and also in TRPV1+/+ mice treated with the TRPV1-antagonist capsazepine . These effects were independent of changes in bladder compliance or contractility. Responses of DRG neuron to αβMethylene-ATP (30 µM) were unchanged in the TRPV1−/− mice, but the proportion of responsive neurones was reduced (p ≤ 0.01). Although the TRPV1 agonist capsaicin (1 µM) did not evoke intracellular responses in PMUCs from TRPV1+/+ mice, luminal ATP levels were reduced in the TRPV1−/− mice (p ≤ 0.001) compared to wildtype. TRPV1 modulates P2X mediated afferent responses and provides a mechanistic basis for the decrease in sensory symptoms observed following resiniferatoxin and capsaicin treatment for lower urinary tract symptoms.Both TRPV1 and P2X receptors present on bladder sensory nerve fibres have been implicated in mechanosensation during bladder filling. The aim of this study was to determine possible interactions between these receptors in modulating afferent nerve activity. In wildtype (TRPV1 As the bladder fills, the degree of distension is detected by mechanosensitive ion channels located either on sensory nerve terminals or on the specialised epithelial lining of the bladder, the urothelium. This evokes an afferent signal that is conveyed to the CNS via the dorsal root ganglia. Initially this signal lies below the level of consciousness, supplying the autonomic reflexes responsible for controlled bladder filling. As bladder volume increases, however, a sensation of fullness and a desire to void is perceived. In continent individuals this desire can be deferred until it is convenient to empty the bladder, however in patients with lower urinary tract conditions such as overactive bladder syndrome (OAB), bladder pain syndrome (BPS) or interstitial cystitis (IC), this sensation is difficult to defer, occurs when the bladder is not yet full and can even be accompanied by pain (BPS/IC) suggesting that a defect in the ability of the bladder to detect filling may underlie these conditions.1. However, the complex mechanisms which determine how the afferent nerves detect bladder filling remain poorly understood, limiting the development of effective treatments for these bladder sensory defects.Sensory innervation from the bladder is conveyed by myelinated low threshold Aδ fibres and unmyelinated high threshold C fibres carried in the pelvic, hypogastric and pudendal afferent nerves3. Thirdly, the P2X1 and P2X3 agonist, αβMethylene-ATP increases firing in both nociceptive and non-nociceptive populations of bladder afferents4. Finally, it has been shown that stretch or distension of the urothelium evokes the graded release of non-neuronal ATP5 which activates purinergic receptors located on the afferent terminal to control afferent firing. Previous studies using P2X3 or P2X2/3 null mice found profound attenuation of the afferent response to bladder filling, increased bladder capacity and decreased voiding frequency, supporting a role for ATP in bladder sensation6. Purinergic receptors are likely to play a role in human urinary bladder physiology and pathophysiology, as P2X receptors are expressed in bladder smooth muscle7 and have been shown to mediate a non-adrenergic non-cholinergic component of contraction from patients with idiopathic detrusor instability8. P2X receptors have also been identified in myofibroblasts, urothelium, and sub-urothelial nerve fibres of human bladders11.There is compelling evidence that purinergic receptors play an active role in mechanosensation of the bladder in rodents. Firstly bladder projecting afferents express a number of P2X puringeric receptors, of which P2X2 and P2X3 are the main subtype. Secondly, ATP is able to activate pelvic nerve afferents arising from the rat urinary bladder and initiate bladder overactivity13. In the urinary tract, TRPV1 is predominantly expressed on primary sensory afferent nerve fibres, whilst contradictory evidence both support and negate a functional role for TRPV1 in the urothelium20.The transient receptor potential (TRP)V1 receptor is a member of the TRP superfamily, activated by noxious heat ≥ 43 °C, pH ≤ 6.0, and the vanilloids capsaicin and resiniferatoxin. TRPV1 is mostly confined to unmyelinated, small diameter primary afferent fibres and has been consistently implicated in nociception, and inflammatory pain−/−) mice have reduced afferent responses to bladder distension, reduced reflex bladder activity and increased bladder capacity22. Moreover, application of capsaicin or RTX causes activation of bladder sensory nerves and increased reflex activity23, while systemic administration of a TRPV1 antagonist increases micturition threshold volume and decreases bladder contraction amplitude24. Previous studies have also shown that in experimental models of cystitis, genetic deletion or pharmacological blockade of TRPV1 prevents bladder hyperactivity27. However, TRPV1 is not considered to be a mechanically gated ion channel and thus the mechanism by which it is able to alter bladder sensitivity to filling remains unclear.There is evidence to suggest that the TRPV1 receptor contributes to afferent nerve responses during bladder filling. TRPV1 knockout  produced concentration-related increases in afferent nerve activity and intravesical pressure, indicating detrusor contraction Fig. . The aff−/− mice compared to TRPV1+/+ control mice    Fig. . In ordeine Fig. . Capsaze 9) Fig. . Moreove−/− mice, peak afferent nerve responses to the nicotinic agonist DMPP was examined .To determine if the decreased response to αβMe-ATP was due to a general change in neuronal excitability in the TRPV1ned Fig. . In cont−/− mice was secondary to altered detrusor muscle activity. However, this would appear not to be the case since there was no significant difference in αβMe-ATP induced bladder contraction between preparations from TRPV1+/+ and TRPV1−/− mice despite markedly different afferent firing profiles. Furthermore capsazepine (10 µM) failed to alter contractile responses to αβMe-ATP  .The afferent nerve response to αβMe-ATP occurred concurrent with a moderate contraction of the detrusor smooth muscle. It is possible therefore that the changes in afferent nerve firing in the TRPV1 4) Fig. . Bladder 4) Fig. . To furt 4) Fig. . However 4) Fig. . Moreove−/− failed to exhibit responses to capsaicin. There was no significant difference in maximal αβMe-ATP (30 µM) induced calcium rise in DRG neurons (24.4 ± 2.4% Vs 28.1 ± 3.2%) from TRPV1+/+ and TRPV1−/− mice respectively   Fig. . However+/+ fig.  . There was no significant difference in the sensitivity to ATP between PMUC from TRPV1+/+ and TRPV1−/− animals. Interestingly direct application of capsaicin to PMUC failed to elicit a rise in intracellular calcium  was absent in the TRPV1−/− mice. In contrast the release of acetylcholine at rest or during bladder distension was unchanged in TRPV1−/− mice   Fig. .6. Moreover, P2X receptors have been shown to co-localise on afferents expressing TRPV131, and co-immunoprecipitation studies have demonstrated a physical association of TRPV1 and P2X3 receptors in the DRG32. P2X3 immunoreactivity on bladder projecting neurons is significantly increased in patients with neurogenic detrusor overactivity33 and is significantly reduced in those patients who responded positively to intravesical application of the TRPV1 agonist, resiniferatoxin, which causes receptor desensitisation, further suggesting there may also be a functional association between the TRPV1 and P2X3 receptors on bladder afferent neurons innervating the bladder33. The results presented in this study provide the first direct evidence that the TRPV1 receptor is able to influence bladder afferent responses to P2X stimulation, a process considered essential to mechanosensation during bladder filling.A number of previous studies have shown that P2X3 sensitive afferent nerves are essential for normal bladder function and that purinergic responses in sensory neurons are largely mediated by P2X2, P2X3 and P2X2/3 receptors34 and P2X3 receptors located on the afferent nerves play a role in mechanosensation6. Bladders from wildtype mice responded to the application of αβMe-ATP with a transient bladder contraction and a profound afferent response. In calcium free Krebs, bladder contractions were lost but nerve firing persisted, albeit diminished. This suggests that the receptors responsible for these two components are distinct, and, given the body of evidence in the literature is seems likely that P2X1 receptors on the muscle mediate the contractile response while P2X3 receptors34 at the nerve terminal drive the direct nerve response. Membrane bound ionotropic P2X1 and P2X3 receptors, when bound by ATP, mediate non-selective cation influx of Na+, and Ca2+, and the efflux of K+ across the cell membrane under physiological ionic distribution to initiate cell depolarisation35. The degree of afferent firing that is maintained in Ca2+ free buffer in our experiments is likely a consequence of direct Na+ induced membrane depolarisation. Whilst P2X1 receptor mediated influx of both Na+ and Ca2+ influx in smooth muscle cells creates an excitatory junction potential, it is the subsequent opening of voltage gated calcium channels and calcium induced calcium release from intracellular stores which create the concentrations of intracellular calcium necessary for smooth muscle contraction36. Afferent responses to αβMe-ATP were also tested in the presence of the L-type calcium channel blocker nifedipine. Nifedipine significantly attenuated bladder muscle contraction with no effect on afferent firing (unpublished data), further supporting differential dependence of voltage gated calcium channels for muscle contraction and afferent firing in response to P2X receptor activation.αβMe-ATP is a non-selective agonist which activates P2X1 and p2X3 receptor subtypes. Previous studies have provided compelling evidence that P2X1 receptors located on the smooth muscle mediate bladder contraction−/−, thereby affecting the sensitivity to αβMe-ATP. However, the effect of TRPV1 deletion was replicated by the acute treatment with capsazepine, indicating that the attenuated response to αβMe-ATP is unlikely due to a reduction in P2X receptor expression. The similar attenuation observed in the TRPV1−/− and capsazepine treated wildtype mice is also consistent with capsazepine’s pharmacological antagonism of the TRPV1 receptor rather than as a consequence of any non-specific or smooth muscle actions. Interestingly, knockout or pharmacological blockade of the TRPV1 receptor had no effect on detrusor muscle contraction stimulated by αβMe-ATP suggesting that the interaction was at the level of the afferent nerve or urothelium rather than secondary to changes in muscle tone. The effect of capsazepine on afferent firing persists after contractile responses are attenuated in calcium free conditions, again ruling out any effect on bladder contractility. These results are supported by a previous study from our laboratory showing that mechanosensitive responses to ramp distension are significantly attenuated by removal or block of TRPV1 with no concurrent effect on bladder compliance during ramp distension21.Loss of TRPV1 reduced the bladder afferent response to αβMe-ATP application. It is possible that P2XR expression on bladder afferent endings is altered in the TRPV137 through extensive phosphorylation of intracellular signalling molecules, which in the case of TRPV1 and P2X3/P2X2/3 have been shown to be regulated by the phosphoinositides PiP2 and PiP3 in peripheral neurons39. Thus it is possible that removal or blockade of the TRPV1 receptor reduces the sensitivity of P2X3 receptors by alterations to its intracellular signalling cascade.TRP channels have been recently shown to modulate the sensitivity of other ion channels40. In order to test whether this would explain the phenomenon observed in our study we examined the effect of TRPV1 knockout on the bladder afferent responses to a nicotinic agonist, DMPP, as nicotinic agonists have previously been shown to stimulate capsaicin sensitive bladder afferents41. The rationale behind this was to stimulate the bladder by a non-purinergic mechanism in order to assess whether the loss of TRPV1 caused a reduction in nerve sensitivity to both agonists or if the response was specific for αβMeATP. We found no significant difference in the response to DMPP between the wild-type and knock-out mice suggesting that loss of TRPV1 did not result in an overall loss of bladder sensitivity, but that inhibited P2X receptor responses in the TRPV1 knockout is due to a specific interaction between the TRPV1 and P2X3 receptors.A previous study into the role of TRPV1 in intestinal mechanosensitivity suggested that loss of TRPV1 function leads to an overall reduction in the pre-tuning of the afferents, thereby reducing excitability+/+ and TRPV1−/− mice. DRG neurons from TRPV1+/+ responded to αβMe-ATP with an increase in intracellular calcium with similar proportions to those reported by others42. We found that the magnitude of the calcium signal was not different between neurons from TRPV1+/+ and TRPV1−/− mice. The opening of P2X receptors following αβMe-ATP binding permits the influx of Na+, as well as Ca2+ ions. Membrane depolarization of sensory neurons is dependent on Na+ influx, and results in the opening of voltage-gated calcium channels to act as a second messenger of electric signalling36. Sensory neuronal signalling through calcium influx is further amplified by calcium-induced calcium release from intracellular stores which is much larger than the initial calcium influx through P2X receptor ion channels. Thus, it is likely that any difference in P2X-receptor excitability between TRPV1−/− and TRPV1+/+ mice is masked by the much larger calcium signals associated with the opening of calcium channels. The proportion of DRG neurons responding to αβMe-ATP was significantly reduced in the TRPV1−/− neurons compared to the wildtypes. In a previous study, Kiyatkin et al. found that 30% of DRG neurons exhibit functional responses to both αβMe-ATP and capsaicin confirming that a subpopulation of neurons in the DRG co-express functional TRPV1 and P2X3 receptors43. We found the number of responsive DRG neurons in the TRPV1−/− mice was reduced by about 25%, which likely represents the proportion of co-expressing cells in the wild type animals whose absence in the knockout animals would account for the attenuated ex-vivo afferent firing in response to αβMe-ATP. Since a similar attenuation was seen following the acute (30 min) treatment with capsazepine it would imply that P2X receptor expression is unchanged in the knockout but sensitivity to ligand is blunted. Together this data suggests that TRPV1 interacts with the P2X3 receptor to modify its sensitivity to agonist stimulation rather than the loss of TRPV1 alters P2X3 receptor expression and further experiments are planned to determine if this is indeed the case. The exact mechanisms involved in this interaction and down-stream signalling pathway still remain to be determined.To investigate this relationship in the absence of other potentially confounding factor in the bladder wall, responses to αβMeATP were determined in primary cultures of DRG neurons from lumbosacral (L6/S2) and thoracolumbar (T11/L2) regions from TRPV118 suggesting that TRPV1 may not be functionally coupled to calcium entry or release from intracellular stores in the mouse urothelium, at least under baseline conditions. Paradoxically, however, ATP release from the bladder was attenuated in the TRPV1−/− mouse, an effect which has previously been reported using isolated rat urothelial cells22.The bladder urothelium plays a key role in modulating the excitability of bladder afferent nerves. This is achieved by the non-neuronal release of inhibitory (such as NO) and excitatory (such as ATP) mediators. In this study, we examined whether the absence of TRPV1 also influenced the ability of the urothelium to respond to purinergic receptor stimulation or alter transmitter release. In concordance with a number of previous studies, we found no evidence that capsaicin caused a calcium response from urothelial cells20.As such, the role of TRPV1 in the urothelium remains controversial, and the detection of a functional TRPV1 receptor from a range of species, including human, appears to be dependent on the specific experimental procedures used, and perhaps an over reliance on immunocytochemistry in the absence of confirmatory assays44. Genetic deletion of TRPV1 can lead to compensatory changes that attenuate ATP release. Birder et al. (2002) showed that capacitance of the urothelium was blunted in the TRPV1−/− mouse suggesting that urothelial trafficking mechanisms might be disrupted following deletion of TRPV122. Importantly, however there was no difference in the sensitivity of PMUCs to ATP as indicated by similar pEC50s suggesting that the interaction between TRPV1 and purinergic receptors observed in the afferent recordings are unlikely to be due to a mechanism involving the urothelium.In the present study TRPV1 mRNA was identified in the urothelium, albeit in low levels, suggesting that TRPV1 might be expressed in urothelial cells despite the failure of capsaicin to evoke a response. This suggests that the levels of TRPV1 mRNA translation may be insufficient to elicit a calcium response, yet high enough to affect ATP release. Immunohistochemical localisation of TRPV1 in the urothelium has been questioned, citing a lack of knockout studies and relevant controls46. The fact that ATP release was changed by the loss of TRPV1 but that ACh release was not suggests that this feedback mechanism was uncoupled. This may be as a direct result of the differential release mechanism for the two mediators 47 or a developmental compensation aimed at maintaining control over micturition. However, until further studies are conducted to understand how these two transmitters are coupled interpreting this data can only be speculative.In contrast to ATP, acetylcholine release was unaffected by the loss of TRPV1. This was surprising since several previous studies have shown that ATP mediates a positive feedback mechanism on ACh outflow from the urothelium21. Importantly it has been shown that the relative density of suburothelial P2X3 and TRPV1 immunoreactive nerves increases in disease33 and is significantly reduced following successful treatment with intravesical instillation of resiniferatoxin or capsaicin to cause receptor desensitization. This pathway, therefore, may contribute a larger proportion of the mechanosensory response to distension during pathophysiological conditions. The present results provide a mechanistic basis for the decrease in sensory symptoms seen following resiniferatoxin and capsaicin treatment for lower urinary tract symptoms49, although the relative contribution of the urothelium and the sub-urothelial afferent nerves remains unknown.The TRPV1 receptor may have two synergistic roles in mediating bladder mechanosensitivity. A modulatory role in regulating the release of ATP from the urothelium, coupled with a decrease in afferent nerve sensitivity to ATP could combine to result in the deficit in mechanosensitivity observed during bladder distension+/+) and TRPV1 Knockout (TRPV1−/−) mice with a genetic background of C57/BL6 were obtained from GlaxoSmithKline . Trans-membrane domains 2–4 of the mouse VR1 gene (i.e. DNA encoding amino acids 460–555) were replaced by the neo gene50. Mating pairs of TRPV1−/− and TRPV1+/+ N1F1 littermates were used to generate separate colonies of TRPV1+/+ and TRPV1−/− mice at the University of Sheffield. In the course of the study, genotyping was periodically performed to confirm the absence of the TRPV1 gene in the TRPV1−/− mice. There were no overt differences in feeding behaviour, litter size, growth rate and body weight between TRPV1−/− and TRPV1+/+ groups.All experiments were performed in accordance with the University of Sheffield’s Animal Care Committee under a project license issued by the UK Animals (Scientific Procedures) Act 1986. TRPV1 wildytpe , continually perfused with gassed  Krebs-bicarbonate solution  at 35 °C. The urethra and dome were cannulated to enable recording of intravesical pressure and enable evacuation of fluid. Pelvic and hypogastric nerves were dissected into fine multiunit branches, and afferent activity was recorded by a neurolog headstage , amplified, filtered  and captured by a computer via a power 1401 interface and Spike 2 software .Nerve recording was conducted using an DRG’s from pelvic (L6-S1) and hypogastric (L1-L2) regions of the mouse spinal cord were isolated and incubated in HBSS (pH 7.4) containing collagenase (4 mg/ml), and dispase (4.5 mg/ml), at 37 °C for 20 minutes. The collagenase/dispase solution was aspirated and replaced with Hanks balanced salt solution (HBSS) containing papain (2 mg/ml) for 20 minutes at 37 °C. The papain solution was aspirated, replaced with 500 µl DMEM F12 (Gibco) and cells were dissociated via trituration with a Pasteur pipette. Cells were plated onto matrigel coated coverslips (corning) and left for 2 hrs before flooding with DMEM containing 10% FBS and Penstrep (100 IU/ml of penicillin and 100 ug/ml of streptomycin) (Sigma).−/− and TRPV1+/+ mice were dissected, pinned urothelial side up and incubated with MEM media containing 2.5 mg/ml dispase (2–3 hours at 21 °C). Cells were collected by gentle scraping and dissociated in 0.025% trypsin EDTA at 37 °C (10 minutes) and re-suspended in keratinocyte serum free media (KSFM) before plating overnight onto collagen (IV) coated coverslips.Bladders from TRPV122 mM and MgCl2 1 mM, pH 7.4) for 1 hr prior to imaging. Cells were stimulated with either ATP, αβMethylene ATP, or capsaicin for 3–5 minutes and changes in intracellular calcium [Ca2+]i, were monitored in real-time. Ionomycin (Sigma) (5 µM) was applied as a positive control in DRG experiments. Results are expressed either as relative fluorescence (RF) or normalised to percentage of maximum ionomycin-induced calcium influx to overcome intra-experimental variability. [Ca2+]i is represented as the ratio between the fluorescence signal at 340/380 nm. Numbers are quoted as: N = number of mice and n = number of cells.Cultured DRG neurones and urothelial cells (20–24 hours) were loaded with 2 μM fura-2-acetoxymethyl ester (fura-2AM) for 30 minutes at 37 °C and washed  with the outflow tap closed and left to equilibrate until bladder pressure and baseline nerve activity were stable (approx. 45 mins). αβMethylene-ATP (30 µM) or dimethyl-4-phenylpiperazinium (DMPP) (100 µM) were applied to the bath by bolus dose and washed out with Krebs buffer after 60 seconds with the perfusion system. Krebs buffer was continually perfused for one hour to minimize receptor desensitisation before a second administration of experimental compound. In some experiments, capsazepine (CPZ) (10 µM), or vehicle control (0.01% DMSO) was perfused in the Krebs solution for the period prior to the second stimulus. Bladder contraction was measured as a change in intravesical pressure (mmHg) and latency was calculated as the time (in seconds) from drug application to maximal nerve firing.In nerve recording experiments, the preparation was allowed an equilibration period of 30 min before any experimental protocol commenced. Control bladder distensions were always carried out using isotonic saline  at a rate of 100 µl minSequential filling (ramp distension) of a catheterised bladder (as described above) was performed . Intraluminal fluids were collected and the amount of ATP and Acetylcholine (ACh) in the samples was determined using the luciferin-luciferase ATP bioluminescent assay and the Amplex red ACh/ACh esterase assay kit (Molecular Probes).−ΔΔCt method using β-actin as a housekeeping gene.mRNA from cultured urothelial cells and DRG neurones (20–24 hrs) was isolated  and cDNA was synthesised by reverse transcription using superscript III (Invitrogen) from mRNA following the manufacturers protocol. cDNA was amplified by PCR for 35 cycles with the following primers and Sybr green fluorescence (Biorad). Primer sequence for the TRPV1 receptor was forward 5′ CAAGGCTCTATGATCGCAGG 3′ and reverse 5′GAGCAATGGTGTCGTTCTGC 3′. All samples were assayed in triplicate in the same plate. The relative amount of target mRNA was calculated by the 2Whole nerve multi-fibre afferent nerve activity was quantified using a Spike2 software which counted number of action potentials crossing a pre-set threshold .For afferent nerve recording experiments, baseline activity was subtracted from stimulus induced response. Data are presented as means ± S.E.M. Statistical analysis was carried out using either a 1 or 2-way ANOVA and appropriate post-hoc test or a Student’s T-test, where appropriate. Tukey post-hoc test was used for 2-way ANOVA when n values for groups was the same, and Bonferroni for all other ANOVA, with significance confirmed at P < 0.05.


An unprecedented 3D hetero‐triple‐walled metal‐organic framework is obtained by straightforward elaboration of the mixed molecular building block (MBB) strategy. In this approach, multiple individual flexible and rigid MBBs are integrated into one composite building block as separate layers, which are of the same shape but different sizes. This MOF shows exceptional water stability and the application of Li‐ion battery electrodes. SchemeMetal‐organic frameworks (MOFs) are highly impressive for their remarkable diversity in terms of compositions and structures,Conceptually, the multiwalled MOF frameworks can be built up from the assembly of multilayered building blocks with metal centers or clusters as vertices. In general, such multilayered configuration usually requires both the shape‐ and size‐matching between individual layers. Thus, the simplest way to simultaneously meet these two requirements is to build up a parallel stacking of multi‐identical MBBs. In line with this principle, several double‐walled MOFs have been reported recently, the constituent double‐layered building blocks of which are primarily constructed with two identical linearAlternatively, the rational design of multilayered building block can be implemented in a different manner, by assigning the shape‐matching MBBs of different sizes and rigidities as individual layers. In this way, the longer, flexible organic ligands are exploited because of their considerable tunability in geometry and tend to form the exterior layers of target composite building block, while the shorter, rigid linkers can be encapsulated between them to serve as the interior layers for stabilizing the framework. It is worth mentioning that this strategy of integrating individual flexible and rigid MBBs into one composite building block, called the mixed MBB strategy,Intrinsically, the mixed MBB strategy can easily address the two aforementioned limitations in developing multiwalled MOFs with the strategy based on multi‐identical rigid MBBs. First, the mixed MBB strategy promises a high degree of structural flexibility due to the participation of flexible organic ligands. The tunable geometries of these involved flexible MBBs could not only facilitate the size‐matching requirement by adapting their lengths and configurations upon conformational alternation, but could also effectively reduce the steric hindrance between adjacent MBBs, and thus the size penalty for metal‐cluster vertices. This then makes it possible to employ a conventional metal‐carboxylate cluster of relatively small size as vertex to gather and dispose multiple MBBs, thus enabling the commonly used carboxylate‐linker approach in the design of multiwalled MOFs. Second, the rigidity of individual framework wall is determined by its constituent MBB, which thus leads to a significant diversity in the character of the resulting framework walls, i.e., those featuring different compositions and rigidities. This unique structural characteristic may engender new functions and/or significantly improve the inherent properties of MOF materials.6L4(TPT)2(μ3‐OH)2]·Co(H2O)6·xG}n (1) ‐1,3,5‐triazine, H3L = 2,4,6‐tris[1‐(3‐carboxylphenoxy)ylmethyl]mesitylene, G = guest molecules). Here, two trigonal planar ligands of different sizes and rigidities were employed, namely TPT and H3L, bearing electron‐deficient triazine ring and electron‐rich benzene ring, respectively. Evidently, such a shape complementarity between ligands involving mutually attractive species will largely facilitate the integration of multiple MBBs. Then, the well‐known trigonal prismatic secondary building unit (SBU), [M3(μ3‐O)(O2CR)6], is selected so as to coincide with the C3‐symmetry of ligands, which consists of octahedrally coordinated metal ions and is more beneficial to form the highly symmetrical structures.3L ligands and one rigid TPT ligand are “pinched together” at three trinuclear cobalt‐carboxylate SBUs, Co3O(COO)6, with their central parts packed in parallel to form a “sandwich‐like” hetero‐triple‐layered building block 2μ3‐OH2]·Co1 was obtained by the solvothermal reaction of H3L, TPT, and Co(NO3)2·6H2O in DMF/C2H5OH/H2O at 95 °C for 72 h. Single‐crystal structural analysis reveals that 1 crystallizes in the cubic space group Pa‐3 and possesses a 3D coordination network consisting of the hetero‐triple‐layered building blocks interconnected via the trinuclear Co3 SBUs. In 1, there exist two crystallographically independent Co2+ ions , denoted as Co12+ and Co22+, respectively. Each Co12+ ion is surrounded by four carboxylate oxygen atoms in equatorial positions, as well as one μ3‐O13 atom and one pyridine nitrogen atom in axial positions, thus exhibiting a slightly distorted octahedral geometry. Based on this coordination configuration, three equivalent Co12+ ions (Co···Co: 3.488 Å) are linked together by six carboxylate groups from six individual L3− ligands and one μ3‐O13 atom to form a trinuclear Co3O(COO)6 SBU, with all the Co—O distances falling in the range of 1.98(3)–2.23(2) Å. On the other hand, each Co22+ ion is coordinated by six water molecules and lies right above the trinuclear Co3 SBU with the distance of about 5.717 Å between Co2 and O13 .Complex 3−‐TPT‐L3−; Figure 3O(COO)6 SBUs as vertices that are arranged in an equilateral triangle in accordance with the trigonal symmetry of both L3− and TPT ligands .Each hetero‐triple‐layered building block  and are mirror located at the two sides of one TPT ligand with their central parts arranged in parallel, thus resembling a compressed chamber with high symmetry . The spontaneously adopted parallel stacking of the “sandwich‐like” triple layers indicates the presence of a weak π···π interaction (3.61–4.01 Å) between the central electron‐deficient triazine and electron‐rich benzene ring, from adjacent TPT and L3− ligands, respectively .Close examination of the formed L3 SBU connects with three L3−‐TPT‐L3− pairs to form a left‐ or right‐handed chiral propeller‐type unit  when considering both L3−‐TPT‐L3− and Co3O(COO)6 as 3‐connected nodes . Moreover, each of the resulting chiral 3D networks, either left‐ or right‐handed, involves two different types of helix chains (left‐ versus right‐handed) with the same pitch length of ≈27 Å . Through self‐assembly of these 3D chiral nets, the first hetero‐triple‐walled MOF (1) is then formed but found to be racemic due to the interpenetration of the chiral nets of opposite handedness .From another point of view, each Cot Figure c. The 3D3L ligands were used in our previous study to construct the cage‐in‐cage double‐walled MOFs, [M3L2(TPT)2·xG]n (M = Ni or Co),2(CO2)4, serves as the common vertex for two hetero‐double‐layered building blocks (L3−‐TPT), bearing in mind that in 1, each trinuclear Co3O(COO)6 SBU gathers three L3−‐TPT‐L3− pairs. In comparison, it is observed that every two TPT ligands at the same SBU are non‐coplanar, for either Co2(CO2)4 or Co3O(COO)6, but associated with different dihedral angles between the two central triazine rings . The significant difference in dihedral angles consequently leads to two very distinct multiwalled architectures: the L3− and TPT MBBs linked through Co2(CO2)4 SBUs adapt to form a closed polyhedral structure, while those at Co3O(COO)6 SBUs tend to produce an open network structure  and variable‐temperature PXRD measurements. The TGA profiles reveal that the framework of 1 is thermally stable below 350 °C , and that a weight loss corresponding to the removal of encapsulated solvents occurs in accompany with a interpenetrated net sliding phenomenon, which also results in the slight peak shifts in the low‐angle region of the variable temperature PXRD patterns .1, as confirmed by the TGA profiles shown in Figure S11 (Supporting Information), thus indicating that the hetero‐triple‐walled framework of 1 is structurally robust upon removal of encapsulated solvent molecules. In order to justify the influence of multiwalled organization on the framework stability, we also compared the chemical stability of the triple‐walled MOF with its double‐walled ([Co3L2(TPT)2·xG]n)2)3(TPT)4·xG]nThe aesthetically pleasing multiwalled architecture is a unique structural attribute that may result in a high degree of structural robustness. h Figure3, thus1 as anode material. Figure S14 (Supporting Information) displays the representative charge–discharge curves for various cycles at a current density of 50 mA g−1 in potential range between 0.01 and 3.00 V. During the initial cycle, moderate specific discharge and charge capacities of ≈1108 and 345 mAh g−1 were achieved. The large irreversible capacity observed here is probably due to inevitable formation of solid electrolyte interface (SEI) film and extended electrolyte degradation,Recently, there has been growing interest in developing MOFs as a new class of electrode materials for Li‐ion batteries (LIBs),1. As seen from the inset of Figure1.1 indicates that, despite a decline of reversible capacity in the initial several cycles, a nearly invariable capacity of about 300 mAh g−1 can be obtained in the following cycles .Detailed analysis of the cyclic voltammograms (CVs) was also conducted to examine the electrochemical behavior of s Figure , with th1 exhibits a superior or comparable Li storage performance, especially in cyclability, as compared with many developed MOF electrodes,2 material.1 not only helps to avoid the capacity decay and thus improve the cyclability owing to its high chemical stability, but also provides more redox‐active sites from multiple ligands within a confined pore space,The results presented above reveal that In summary, we elaborate an alternative method, i.e., the mixed MBB strategy, for constructing the multiwalled MOFs of high structural complexity and aesthetic appeal, under which the shape‐matching flexible and rigid MBBs are integrated into one composite building block as separate layers. Compared to the strategy based on multi‐identical rigid MBBs, this new strategy not only enables more compositional diversity, especially in the character of framework walls, but also promises higher structural flexibility that could effectively reduce the dependence of multilayered building block on the size of metal‐cluster vertices. With the methodological development in engineering of multiwalled MOFs, we obtained an unprecedented 3D hetero‐triple‐walled MOF with remarkable structural flexibility and chemical stability, which could significantly improve the cyclability of MOF materials in the application of LIB electrodes. Therefore, we anticipate that the proposed mixed MBB strategy could not only offer a new platform for developing novel MOF structures, but also provide new insights into the functionalization of this class of crystalline materials.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


A long lifetime of 3750 μs demonstrates complete loss of hydration, consequently Eu must have been incorporated into the bulk crystal. The results show a strong dependence of the incorporation kinetics on the recrystallization rate of the different calcites. Furthermore the investigation of the effect of different background electrolytes (NaCl and KCl) demonstrate that the incorporation process under recrystallization conditions strongly depends on the availability of Na+. These findings emphasize the different retention potential of calcite as a primary and secondary mineral e.g. in a nuclear waste disposal site.The interaction of calcite with trivalent europium under recrystallization conditions was studied on the molecular level using site-selective time-resolved laser fluorescence spectroscopy (TRLFS). We conducted batch studies with a reaction time from seven days up to three years with three calcite powders, which differed in their specific surface area, recrystallization rates and impurities content. With increase of the recrystallization rate incorporation of Eu The long-term safety of geological nuclear waste disposals has to rely on a detailed and thorough understanding of the processes governing the migration of radionuclides through the geo/biosphere. These reactions can include adsorption and surface precipitation as well as incorporation within the bulk of a material. Calcite, one of earth’s most abundant minerals, has long been known for its potential to incorporate other cations than calcium1262+  under growth, phase transformation and even dissolution conditions was already evidenced in the past131314151617181920212223242+ or Cd2+, but also trivalent actinides  relevant to long term nuclear waste storage. In these previous studies it was shown that trivalent rare earth elements and actinides can be taken up by the calcite host lattice structurally. Natural mineral specimen contain considerable amounts of rare earths depending strongly on their origin. These findings imply that under natural conditions, mechanisms must be at work by which dissolved metal ions in contact with calcite at equilibrium incorporate into the material over time.Plutonium and the minor actinides neptunium, americium, and curium are expected to contribute most significantly to the long-term radiotoxicity according to performance safety assessments8102 in geological formations calcite and other carbonates with their slow kinetics of mineral-fluid reactions play a significant roleMoreover, the dissolution and precipitation behavior of abundant minerals like calcite under different fluid compositions is important for the evaluation of geothermal energy production sites263+ as well as Cm3+ with calcite synthesized in mixed flow reactors3137F0 → 5D0 transition at 578.1 nm. Species B (λexc(Eu) = 578.4 nm) was found to be incorporated within the strongly distorted calcite structure. Finally, species C (λexc(Eu) = 579.6 nm) was found to be located on the Ca2+ site on the calcite lattice, with an almost undisturbed octahedral symmetry, identifiable by its characteristic splitting pattern. For both species B and C, Na+ was required for charge compensation during the incorporation, suggesting a mechanism where two Ca2+ ions were replaced by one Eu3+ atom and one Na+ atom. Atomistic simulations by Vinograd et al.3)2 end member, and they suggest the formation of such domains in the solid solution. The Ca2+ lattice site in dolomite has a D3d point symmetry, as opposed to the C3i symmetry of the same lattice site in calcite. Therefore, Eu in such a crystallographic environment should exhibit a splitting pattern similar to that of species C in calcite.The interaction of Eu3+ or Cm3+ with calcite under recrystallization conditions are scarce. Our previous work3+ on a single calcite powder in 0.01 mol L−1 NaClO4 at different contact times, i.e. one day, two weeks and one month at recrystallization conditions. In the excitation spectra, a broad peak centered at 579.3 nm with at least two species exhibiting partially preserved hydration shells were observed. Interaction was found to be dominated by a continuum of chemically similar adsorbed species as well as beginning incorporation into the bulk structure. An impact of the reaction time on the number of species and their lifetimes was noticed, but the transition was too slow to be probed meaningfully in the allotted time.Where calcite occurs as a primary phase, interaction will occur close to equilibrium. Studies dedicated to the interaction of Eu3+ interaction with calcite under recrystallization conditions. Via TRLFS of curium’s 6D7/2 → 8S7/2 transition they observed a sorption species with two and an incorporation species with no water molecules bound to the Cm3+, respectively. Over a contact time of 6 months the incorporation of Cm3+ in to the crystal bulk progressed at the cost of the sorption species. The incorporation species identified by Stumpf and FanghänelEarlier Stumpf and Fanghänelet al.3+ with calcite. Batch sorption experiments were conducted at 50 °C and samples subsequently analyzed by site-selective TRLFS. Eu3+ was found to be incorporated into a hydrated and/or hydroxylated surface layer, with two distinct site families. Substitution of Eu3+ for Ca2+ in the lattice framework was also in evidence. Nevertheless, sites A and B revealed by Schmidt et al.et al.et al.3+ and Cm3+ with calcite evidently is strongly affected by experimental conditions.Piriou 3+ three calcite powders differing in their specific surface area (SSA) and impurities content were chosen and their reactions at near-equilibrium conditions studied. The evolution of the speciation was followed as a function of reaction time, starting from one week up to two months, and for some samples up to three years. In order to probe the relevance of the coupled substitution mechanism identified by Schmidt et al.To further investigate the impact of surface reactivity and recrystallization kinetics of calcite on the incorporation of EuThree calcite powders  were chosen for this study. C1 and C3 were obtained as powders from Merck, Germany and Solvay, Germany, respectively. C2 was obtained from Ward’s Science, USA as a single crystal and ground to a grain size < 63 μm. All solids were characterized by powder XRD, BET, and SEM. In addition the impurity inventory was characterized by ICP-MS after dissolution of the minerals . Concerning C2, impurities in Na, Mg, Si, K, Sr, Cs, Fe and Zn (10.4 to 142 μg g−1) were present. In addition to the aforementioned elements for C2, material C3 had also minor impurities in Ba and Mn. The highest concentrations were found for Na (109 μg g−1), Mg (1390 μg g−1) and Si (389 μg g−1).Sample C1 exhibited the overall lowest amount of impurities, the highest being Mg, Sr and Fe  is used for data fitting, both calcites C2 and C3 exhibit significantly higher rates compared to C1. Material C1 recrystallizes much slower and equilibrium is not reached within the reaction time. Clearly, C1’s recrystallization rate is significantly slower compared to the other materials, and we would expect this material to show the slowest surface reaction kinetics. Rate determination here is only slightly above detection limit. The rates of calcites C2 and C3 are rather high and equilibrium is reached rapidly. The specific rates, however, differ from each other with C3 being the most reactive.As also seen in the study by Berner 3+ of the solution independent of the reaction time (see 5D0 → 7F0) transition for all samples recorded at various reaction times are presented in The 3 different calcites  sorbed nearly all Eutime see  in SI. TAlready after ca. one week the spectra show significant differences between C1, C2, and C3, while all spectra show a broad distribution. For the calcite C1 with the lowest recrystallization rate, only one broad peak  ~1 nm) is found at ~579.4 nm, which was assigned to species δ. The spectrum of sample C2 is dominated by the same species δ, but is slightly shifted to lower excitation wavelengths. The sample with the highest recrystallization rate C3, however, shows a significantly broader peak (FWHM ~ 1.6 nm), as well as a more strongly blue-shifted spectrum. A weak peak at 578.8 nm is assigned to species γ. The shoulder with higher excitation wavelength shows the presence of species δ, and the intensity at lower excitation wavelength points to one or more species present, which are not clearly resolved here. A general trend of more strongly blue-shifted spectra for the calcites with higher recrystallization rate is visible.After one month the different progress in the reactions becomes more apparent. For C1 the excitation spectrum shows only minor changes. A slight shoulder is now visible at lower excitation wavelength which may indicate the presence of small quantities of species γ, but the speciation remains dominated by species δ. The same applies to C2, but weak peaks indicate contributions by species γ and species β, visible at 578.4 nm. The spectrum of C3 shows more clearly resolved peaks now, dominated by species γ. Both, the shoulder at the spectral range of species δ, and the shoulder at lower excitation wavelength decrease. At 578.2 nm a small peak becomes visible, which is attributed to a fourth species α.Due to the slow progress in the reaction of C1 from one week to one month, only C2 and C3 were probed after two months. The excitation spectra exhibit significant differences between C2 and C3. The excitation spectrum of C2 again shows a broad distribution with the maximum at species δ. Species γ is present, but because of the stronger blueshift of the whole spectrum species γ is not predominant. In addition, species β is recognizable as a small, but well-defined peak.The different reaction kinetics comes out clearly at this point. The excitation spectrum of C3 now shows well defined peaks for species α and γ, as well as a shoulder corresponding to species δ. The shoulder is decreased in intensity compared to one month earlier and appears to be divided into two peaks.Additional long-term experiments were conducted with C1 and C2, after 450 d and 1150 d, and 380 d, respectively to probe their slower reaction kinetics. The excitation spectrum of C1 is still broad and essentially featureless after 1.5 years (450 d), but starts to exhibit more clearly resolved features after ~3 years (1150 d). The speciation is still dominated by species δ, but the shoulder corresponding to species γ is now clearly visible. Also a small shoulder may be present, which would indicate the formation of species α. As expected the reaction proceeds quicker for C2. After one year (380 d) the excitation spectrum of C2 shows four distinguishable peaks. The dominating species is now γ, similar to C3 at the previous time steps. Species β is characterized by a sharp, narrow peak, while species δ shows a differentiation into two features similar to the same species observed for C3 after 2 months.Four unique species were identified throughout the reaction, which can be characterized by their emission spectra and fluorescence lifetimes. The emission spectra after direct excitation of the four different species are shown in 7F1 band shows a 3-fold splitting indicating a low symmetry7F2 band exhibits only a 3-fold splitting. Ligand field considerations dictate that a full splitting of the 7F1 band must be accompanied by a full splitting (5-fold) of the 7F2 band, which is likely not sufficiently resolved in our spectra, as the peaks are noticeably broadened. The long lifetime of 3700 ± 350 μs corresponds to the complete loss of the hydration of Eu3+ 3+ into the calcite crystal bulk.Species α was identified in C3 after 30 and 60 days, and may also be present in C1 after ~3 years. The 7F1 band and a 5-fold split 7F2 band, this maximum splitting pattern implies a low symmetry of the ligand field surrounding Eu3+. The lifetime of 2450 ± 150 μs here also points at complete loss of hydration in the first coordination sphere of Eu3+ and therefore an incorporation into the crystal bulk. Comparison of emission spectrum and lifetime clearly identifies β as species B, which had been previously identified in coprecipitation experiments3Species β has its excitation maximum at 578.4 nm and appears only in material C2 as a shoulder after one and two month and as a sharp and narrow peak after more than one year of reaction time. The corresponding emission spectrum reveals a 3-fold splitting in the 7F1 and a 5-fold 7F2 band can be determined, so site symmetry of species γ is low as well. Due to the inherent overlap of the emission spectra, additional interpretation of the observed splitting pattern is however not expedient. The same problem occurs in determination of the lifetimes of both species. We determine the respective lifetimes, where either species is most dominant, and we can be most confident in the determined value, but overlap remains an issue . Despite these concerns, a lifetime of 3750 ± 450 μs clearly shows a water free coordination shell of Eu3+, and hence bulk incorporation.Species γ is unique to the incorporation process close to equilibrium, and was identified in all 3 calcite materials. Its excitation maximum was found at 578.8 nm. To interpret its emission spectrum we have to consider an apparent overlap with species δ. This results from the broadness of the excitation peak of species δ and the small distance between the two excitation maxima. While no single species emission spectrum could be obtained, it appears a 3-fold 7F1 and 7F2 indicates a low symmetry.Species δ also occurs in all three calcites and dominates speciation of all samples early on, before being successively replaced by species γ, as well as α or β. The excitation maximum is always broad within the range of 579.2–579.5 nm. In the last spectrum of C2 (380 d) and C3 (60 d), respectively, the excitation peak is divided into two distinct peaks, but neither the emission spectra nor the lifetimes exhibit significant differences, upon excitation at either of the two maxima. The full splitting of 2O in Eu’s first coordination sphere. Species δ therefore can be identified as an Eu inner-sphere sorption complex on the calcite surface, the expected starting point of the metal/mineral interaction. However, with increased reaction time we determine continuously longer lifetimes up to a value of ~ 2300 ± 150 μs, which would imply no water molecules surrounding Eu3+, with no apparent changes in the emission spectra. It seems unlikely that an incorporation species would form so rapidly, and with such predominance even in the otherwise slowly reacting calcites C1 and C2. Therefore, we suggest the long lifetime occurs for different reasons. First of all, it is likely that strong overlap with the long-lived species γ contributes significantly to an apparently longer lifetime of δ, especially as γ becomes more dominant with longer reaction times. It is also possible that with the advancing recrystallization reaction more CO32− becomes available in the near-surface solution, which could potentially substitute the coordinating water molecules to form a ternary surface complex as an intermediate step to full structural incorporation. Such a substitution could also potentially cause the observed splitting of species δ’s broad excitation peak into two narrower (yet still strongly overlapping) peaks. Despite the uncertainties regarding species δ, it is evident that this species is not identical to the incorporation species C on the symmetrical Ca2+ lattice site3, which had been previously identified in our co-precipitation experiments with a similar excitation wavelength (λexc (C) = 579.6 nm).As mentioned above determination of the lifetime of δ is difficult, due to its broad excitation range, and thus overlap with γ, and therefore is most reliable for early samples. Here we find lifetimes of ~700 ± 50 μs, corresponding to ~0.9 ± 0.5 H3+ into calcite requires co-substitution with Na+ for charge compensation. With the fastest reacting calcite C3 we conducted a recrystallization experiment in which we replace NaCl by KCl as background electrolyte to test whether the same charge compensation mechanism plays a role under recrystallization conditions. The excitation spectra of C3 reacted with both electrolytes are displayed in Incorporation species γ had not been previously identified in our studies under growth conditions. However, those studies had shown that structural incorporation of Eu3+ and Na+ for two Ca2+ is required for incorporation. Nevertheless, incorporation remains the most significant interaction mechanism in the KCl system, likely due to a considerable amount of naturally occurring Na in calcite C3  are incorporated into the bulk, this indicates enhanced incorporation in the NaCl system relative to the KCl system. This may indicate that also under recrystallization conditions coupled substitution of Euvia recrystallization. Moreover, in the latter case speciation will also be impacted by the recrystallization rate, with a clear trend towards increased incorporation at faster recrystallization.Our study demonstrates that interaction of Eu and calcite will result in significantly different speciation depending on whether the interaction occurs under growth conditions, or 3+ inner sphere sorption species (species δ) on the calcite surface. Eu3+ is initially still in contact with water, which presumably is substituted by CO32− in a ternary surface complex, before Eu is buried under more crystal layers to become structurally incorporated.The initial step of the reaction is a Eu3)2-dolomite domain, based on symmetry considerations.The main product of the incorporation reaction is none of the species A, B, or C that were previously identified after co-precipitation of calcite in the presence of EuOne could speculate that the structural control of a local disturbance in the lattice is stronger in the slow process close to equilibrium, than in the faster reaction upon mineral growth. For example, Eu’s preference for specific sorption sites on the calcite surface could direct its final location in the lattice. During growth of calcite preferred sorption of cations would be expected to occur in crystallographic locations , while in the case of recrystallization sorption may be more likely at e.g. kink sites, step edges, or other similar surface defects. Thus, Eu would be found in a distorted or non-crystallographic site from the initial sorption step onwards.We observed two other low symmetry incorporation species, species α (C3) and species β (in C2). Species β has been observed in our previous co-precipitation experiments (as species B), but is less defined here than in the co-precipitation experiments. Species α was hitherto unknown and is always a minor species where it occurs at all. Species β only occurs for sample C2, and only after more than one year of reaction. Here, its corresponding peak is relatively strong and well defined, however. It remains unclear what the mechanism of formation of these minor species is, and whether they are transitory species, or will remain stable. At least species β only occurs in significant amounts after major contributions of γ have been found, and γ is not significantly reduced where it occurs, so evidently the two species form independently.3+ + Na+ ↔ 2 Ca2+) has been described beforeThe effect of coupled substitution , natural calcite single crystals purchased from Ward’s Science USA  and industrial calcite from Solvay  were used in this study. Materials C1 and C3 were used as delivered, while C2 was ground and sieved to grain size < 63 μm with a ceramic mortar. The three calcites were characterized by powder X-ray diffraction (XRD) see  in SI. T3+ stock solution was prepared by dissolving EuCl3⋅6H2O (Sigma Aldrich p.a.) in Milli-Q water (18.2 MΩ cm−1). The concentration of this stock solution, 1 × 10−4 mol L−1, was confirmed by ICP-MS. All experiments were then carried out using diluted fractions of this solution. 0.01 mol L−1 NaCl and KCl solutions were prepared from a Merck powder (p.a.).The Euet al.45Ca was used to measure the uptake of calcium ions and calculate the amount of calcite recrystallized. The formula developed by Berner et al.For the determination of recrystallization rates of calcite we applied a method previously shown by Berner 45Ca concentrations at t = 0 and t, the decay constant λ = ln(2)/t1/2 of 45Ca radioactive decay and reaction time t. Two series of experiments with different solid-to-liquid ratios were conducted to determine recrystallization rates of the three calcite powders considered in this study. Firstly, a high ratio of 20 g L−1 was chosen for better comparability with Berner’s data. Due to the high recrystallization rates of calcites C2 and C3, equilibrium of the exchange reaction was reached after several hours and no rate determination could be conducted. Therefore, we performed a second experimental series with a lower solid-to-liquid ratio of 2.5 g L−1. The suspensions for both series were pre-equilibrated for one day and spiked with 15.5 MBq 45Ca as CaCl2 in 600 μL deionized water and shaken constantly in an overhead shaker. Sampling was performed within the first five days by removing 50 μL of solution and measuring the activity of 45Ca by liquid scintillation counting . The rates were determined in the linear regime by fitting the slope of the amount of recrystallized calcite as a function of time. Since the exchange reaction exhibits two time-dependent phases with distinct rates, only the first rate was determined in this study, being more sensitive to experimental conditions.with n(t) as the time-dependent molar amount of recrystallized calcite, the total solution volume V, −1 NaCl under atmospheric CO2 until a pH of 8.3 ± 0.1 was reached. Afterwards, supernatants were collected by filtration or centrifugation at 6,800 g for 1.5 h (Avanti J-20 XP Beckman Coulter). 125 mg of each fresh calcite were suspended in 50 mL of these pre-equilibrated supernatants (in polypropylene tubes) to obtain a final solid-to-solution ratio of 2.5 g L−1. Required amounts of the Eu3+ stock solution were added to reach a final concentration of 10−6 or 5 × 10−7 mol L−1. The pH of the calcite suspensions was regularly checked and was found to be constant at 8.4 ± 0.1. All pH measurements (pH-meter Inolab WTW series pH720) were performed using a combination glass electrode (BlueLine 16 pH from Schott Instruments) in which an Ag/AgCl reference electrode was incorporated. The pH values were measured to an accuracy of ±0.05. Electrodes were calibrated using two NIST-traceable buffer solutions (pH 6.87 and pH 9.18 from WTW).Prior to the sorption experiments, all three calcites were equilibrated in 0.01 mol L3+. The calcite samples were dried at room temperature and subsequently analyzed by site-selective TRLFS. Their content in Ca and Eu at the end of the sorption experiments was determined by ICP-MS after dissolution. For the calcite C3, additional similar experiments were performed using calcite saturated solutions in 0.01 mol L−1 KCl.All experiments were performed at room temperature with a contact time of up to 3 years. After varying intervals, samples were centrifuged during 2 hours at 6,800 g and the remaining europium concentration in the supernatant was determined by ICP-MS. The difference to the initial europium content provided the amount of sorbed Eu−6–10−7 mbar). Three laser systems were used for these studies. The description of two systems can be found elsewhereThe solid samples collected at the end of the sorption experiments were cooled down in a cryostat chamber at ultra-high vacuum (105D0 → 7F0) transition (575–582 nm). For each step the integrated fluorescence intensity is recorded. As the transition is non degenerate (J = 0 for both states), the number of peaks in such an excitation spectrum corresponds directly to the number of Eu3+ species in the system. Subsequently, these species can be excited directly to obtain single species, emission spectra and fluorescence lifetimes so far as spectral overlap permits. All site-selective TRLFS measurements were carried out at low temperatures (<10 K), in order to increase the spectral resolution. A spectrograph  with polychromator grids  and intensified CCD detector  were used to record the luminescence emission. For lifetime measurements, the camera delay was increased gradually up to several milliseconds, until fluorescence intensity dropped to a minimum of 1/e.Eu speciation was determined by adjusting the laser’s emission wavelength in steps of down to 0.01 nm through the spectral range of the non-degenerate  into Calcite under Recrystallization conditions. Sci. Rep.6, 33137; doi: 10.1038/srep33137 (2016).


This leads to a widely varying melt undercooling and different crystal orientations in each joint, which results in a different resistance to electromigration, thermomechanical fatigue, and other failure modes in each joint. Here we identify a family of nucleants for tin, prove their effectiveness using a novel droplet solidification technique, and demonstrate an approach to incorporate the nucleants into solder joints to control the orientation of the tin nucleation event. With this approach, it is possible to change tin nucleation from a stochastic to a deterministic process, and to generate single-crystal joints with their  Control over the crystallographic orientation of solder joints based on βSn will improve the reliability of electronic interconnects. Using a technique based on droplet solidification and lattice matching, Ma et al. are able to control the βSn nucleation events, hence control the grain orientation. Nucleation is also central to the microstructures of electronic solder joints and plays an important role in determining the reliability of electronic systems7.The nucleation of crystals is a key step in numerous processes in biology, earth science, materials engineering, and beyond. Substantial research has focused on understanding and controlling crystal nucleation, for example: to trigger the nucleation of ice crystals in clouds and cause rain9. Each of these failure modes is a strong function of the crystallographic orientation of βSn in a solder joint because tetragonal βSn has highly anisotropic properties. For example, the diffusivities of common solutes  are up to four orders of magnitude faster along the c-axis than along the a-axis; the coefficient of thermal expansion (CTE)16 is approximately two times higher along c than a; βSn has its highest stiffness along its direction of maximum CTE (the c-axis)13; and βSn also has strong plastic anisotropy17. There is a consistent agreement that solder joints with the βSn c-axis parallel with the electron flow direction or temperature gradient suffer the most severe electromigration25 or thermomigration damage26. Thermomechanical fatigue is influenced by βSn grain orientations27 by inducing stresses between adjacent grains of different orientations31, and by inducing stresses between the substrate and solder, which are highest when the βSn c-axis is nearly parallel with the substrate plane as this induces the maximum CTE mismatch33. In shear fatigue, joints with the βSn c-axis nearly parallel with the substrate plane and at ~20–60° with the shear direction are more resistant than other βSn orientations34. From this, it can be seen that the optimum c-axis orientation is different for different failure modes.When electronics fail, the culprit is often the solder joints that interconnect the components. Most electronic solder joints contain at least 95% βSn phase to enable soldering at a temperature tolerable to the electronic components, but the βSn must then operate at up to 80% of its melting point while enduring a high current density, mechanical loading, and thermal cycling. Thus, solder joints are often the weakest link in an electronic system and, with the continuous miniaturization of electronics, the challenges of electromigration, thermomigration, thermomechanical fatigue, and mechanical fatigue are becoming ever more crucial32. Figure Z). Unit cell orientations are plotted on each grain using the Euler angles of the mean orientation of the grain. It can be seen that some of the joints contain a single βSn grain and some contain two or three βSn grains. The orientations of βSn grains in 33 such joints are plotted relative to the direction of current flow in Fig. c-axes, i.e., the <001>, are oriented differently in every joint and are at a wide range of angles from the plane of the substrate  and flip chip joints. The challenge is overviewed in Fig. ate Fig. . Thus, e37. The cyclic twinning can be seen from the superimposed unit cells on the two/three grain joints in Fig. 35 as indicated by the cross-hatched planes on the unit cells, and from Fig. 35. Since the joints are all either single grain or twinned, it can be inferred that βSn solidified from a single nucleation event. The location of this nucleation event is one of the Cu6Sn5 intermetallic compound (IMC) layers near where the solder ball meets the Cu pad as discussed in ref. 38. The orientation(s) of βSn in Sn-Ag-Cu solder joints are determined by a stochastic nucleation event and the key to controlling the βSn orientation(s) is to identify methods to tightly control this nucleation event.The joints in Fig. Here we develop a novel method to study heterogeneous nucleation mechanisms, where droplets of Sn are solidified on the facets of IMCs and the nucleation undercooling and resulting orientation relationships (ORs) are measured. Combining this method with a lattice matching approach to nucleant design, we identify a family of transition metal stannides that catalyze βSn nucleation and give useful orientation control. We next demonstrate a simple bonding technique to incorporate the nucleant particles into BGA solder joints to give precisely controlled βSn microstructures and grain orientations tailored to best resist a predefined failure mode.In order to control the orientation of βSn, we seek seed crystals that can be attached to the pads on the printed circuit board (PCB) side or the component side. The requirements of such a heterogeneous nucleant are: it must be the most potent nucleant in contact with the liquid solder, and generate a useful and reproducible OR with βSn; it must be bondable onto the pad or metallization; it should be a solderable surface when using existing electronic fluxes; and it must be thermodynamically stable in the liquid solder.44, and approaches to identify potent nucleants include the Turnbull–Vonnegut linear disregistry39, the Bramfitt planar disregistry40, and the Zhang–Kelly Edge-to-Edge model45. The former two approaches are ‘plane-on-plane’ matching, whereas, in the Edge-to-Edge model, the closest or nearly closest packed planes in the two phases are not necessarily the interfacial plane but their edges meet at the interface and the meeting rows have low linear disregistry41. All three approaches indicate that the lower the disregistry, the more potent the nucleation catalyst, and effective nucleation catalysts typically have disregistries of <10%45. In solders, the tetragonal βSn crystal is more complex than most metals and has a closest packed plane, {010}, that contains zig-zag rows along the closest packed direction, <100>, and straight rows along the next-closest packed direction, <001>, as shown in Fig. 8 distorted-square antiprisms  or [001]Sn||[010]βIrSn4 (yellow droplets) as indicated in the orientation map and pole figures. However, these are just two variants of the same OR since βIrSn4 is tetragonal  . However, importantly, the ORs in Table The nucleation potencies of these three IMCs were evaluated from DSC of ~20 µm tin droplets solidifying on the IMC facets. Typical DSC curves and the definition of the nucleation undercooling are given in Fig. 3, PtSn4, and βIrSn4 crystals are thin plates , where the IMC seed crystal remained solid during the TLPB. An immersion tin coating was applied to Cu pads and the nucleant IMC was laid flat with the main facet in the plane of the pad as shown in Fig. 3 as an example. The αCoSn3 was then TLPB to the pad by a reflow of 5 min at 240 °C, which resulted in the Cu/Cu3Sn/Cu6Sn5/αCoSn3 layers shown in Fig. 4, αCoSn3, and βIrSn4 are all solderable surfaces using a standard ROL-1 flux, these nucleant-modified pads were then used in the same manner as a Cu-OSP substrate and solder joints were made following the procedure in the Methods section. Figure 3 seed crystal and a 550 µm Sn-3.5Ag ball was used. The microstructure consists of primary Cu6Sn5, βSn dendrites with <110> growth directions indicated by the blue arrows, βSn + Ag3Sn + Cu6Sn5 interdendritic eutectic, and a bonded αCoSn3 particle on the bottom substrate. Note that a large αCoSn3 seed crystal has been used in this example to make it feasible to polish to a cross-section containing the seed crystal and measure the OR. The EBSD IPF-Z map of the βSn phase is shown in Fig. 3 phase. In both maps, the unit cell wireframes of these two phases are superimposed to show the OR, which is also indicated by pole figures in Fig. 3 particle, the nucleated single βSn grain is oriented with [100] or [010] across the joint and with [001] parallel with the substrate plane, i.e., the c-axis of βSn is in the substrate plane. Note that, even though primary and interfacial Cu6Sn5 are present prior to βSn nucleation, the nucleation of βSn always occurred on the seed crystal because they  are more potent nucleants than Cu6Sn5. Primary Ag3Sn plates were not observed here because the seed crystals require only a relatively small undercooling for βSn nucleation. Further examples of the phases are shown in Supplementary Fig. X, Y, and Z directions of the joint in Fig. Since the natural growth shape of primary αCoSntes Fig.  whose ma4 and βIrSn4 as seed crystals, although a longer time and higher temperature (180 min at 300 °C) were needed to TLPB these IMCs to Cu pads. Figure 3, PtSn4, or βIrSn4 seed crystals and either Sn-3.0Ag-0.5Cu or Sn-3.5Ag solder. The IMC seed crystals have a wide distribution of sizes from ~20–150 µm (length of the longest edge of the plate) and some are TLPB to the upper Cu substrates and others to the lower Cu substrate. In all cases, the IPF-Z maps show a single green z-orientation, indicating a single βSn grain with a <100>Sn across the joint and <001>Sn in the plane of the substrate in every joint. For those joints for which the seed crystals are in the sectioning plane, the seed crystal-βSn OR is the same as in Table X, Y, and Z directions of these joints in Fig. Similar results to Fig. c-axis of βSn grains and the substrate plane for all 67 joints. All joints show consistent control of the βSn orientation and the c-axis of βSn and the substrate plane are parallel to within ~10°. This misorientation between the [001]Sn and the substrate is mostly because the largest facets of the seed IMCs were not perfectly parallel with the substrate after TLPB, especially when the seed crystals are small . Despite this, Fig. The βSn orientation in 67 joints are summarized in <100> and <001> pole figures in Fig. The nucleation undercooling of 550 µm Cu/SAC305 + IMC/Cu joints for each type of IMC seed crystal were measured and are plotted vs. the lattice disregistry in Fig. c-axis orientation perpendicular to the direction of current flow which has been shown to give optimum resistance to electromigration and thermomigration in solder joints in past work25,26. For some applications, alternative orientations may be preferred and the βSn c-axis may need to be tailored to suit the application. For example, it has been shown by Arfaei et al. 34 that shear fatigue performance is best when the βSn c-axis is near-parallel with the substrate plane and at ~20–60° angle with the shear direction. This can be achieved with the current approach since the crystallographic orientation of the IMC plates can be readily determined from the macroscopic shape of the plate, and the IMC plates can be pick-and-placed to have a tightly controlled rotation angle. To demonstrate this, in Fig. 3 particles have been bonded along the edge of an array of Cu pads with edges parallel with the X direction (the assumed shear direction). This makes the c/b-axis of each αCoSn3 particle at ~45° with the X direction, as shown by the configuration in Fig. c-axis at ~45° with the X direction due to heterogeneous nucleation on the seed crystal. This can be seen in the EBSD IPF-X and IPF-Z maps in Fig. 34, assuming that X is the shear direction.The approach in Figs. c-axis at ~45° with the substrate plane, which would eliminate the worst situation for the thermomechanical fatigue . As the understanding of the role of crystal orientation on solder joint performance improves in the future, the approaches overviewed here open the possibility to tailor the c-axis orientation to best resist the dominant failure mode.For applications where thermomechanical fatigue is the dominant issue, the current technique could be altered to fix the nucleant IMC to have the largest facet perpendicular with the substrate plane and one edge parallel with the substrate plane. By doing this, the final joint would have the βSn 6Sn5 phase, which is a less potent nucleant than the seed crystals, and these Bi and In additions introduce extra (Bi) and ζ phases that form later during solidification and do not strongly affect the nucleation of βSn. The successful orientation control in these Ni-, Bi-, and In-containing solders indicates that this orientation control method is likely to be applicable to solder compositions developed in the future. Thus, it is suggested that the development of solders for optimum reliability could be tackled with a double approach of alloy design and orientation control.Here, orientation control has been demonstrated for the first-generation Pb-free solders, Sn-3Ag-0.5Cu and Sn-3.5Ag. The latest generation solders under development commonly additionally contain Ni, Bi, In, etc. It is shown in the Supplementary Fig. In summary, seed crystals for solder joints create new design opportunities for electronics manufacturing, opening the possibility of designing joints to combat different failure mechanisms and increasing the reproducibility of microstructures across solder arrays. The simple bonding step that incorporates the nucleants onto Cu pads is well suited to pick-and-place technology and the seed crystals are expected to be compatible with future improvements in solder alloy design. More broadly, the droplet solidification technique developed here has the potential to accelerate nucleant discovery in other systems by generating statistically significant datasets on nucleation ORs in a relatively short time.4, αCoSn3, and βIrSn4 single crystals were grown in Sn-rich liquid by cooling hypereutectic Sn-0.2Pt, Sn-0.1Co, and Sn-1Ir (mass%) alloys at 0.33 K s−1 from 400 °C. The IMC crystals were extracted by selective dissolution of the βSn phase in a solution of either 5% NaOH and 3.5% ortho-nitrophenol in distilled water or 50% HCl solution, and were then ultrasonically bathed in ethanol.PtSn4, αCoSn3, and βIrSn4 single crystals with a NH4Cl-ZnCl2 based flux . The Sn was melted on a hot plate with a peak temperature of 240 °C to ensure wetting and spreading on the facet, and then IMC crystals with Sn droplets spread on were ultrasonically bathed in ethanol to remove flux residues. Thermal cycles were then performed in a Mettler Toledo DSC1 under a nitrogen atmosphere with a heating rate of 0.17 K s−1, peak temperature 240 °C, time above the eutectic temperature of 180 s, and a cooling rate 0.33 K s−1. The nucleation undercooling was determined from the DSC data and the OR between the βSn droplet and IMC was measured by EBSD.A purity of 99.9% of Sn particles  between 1 and 50 μm were placed on the largest facets of the as-extracted (without further processing) PtSn−1, peak temperature 240 °C, time above the eutectic temperature ~180 s, and cooling rate 0.33 K s−1. For reflow under near-industrial conditions, a forced air convection Tornado LFR400 reflow oven  was used with the following thermal profile: heating rate 1 K s−1, peak temperature 250 °C, time above the eutectic temperature 80 s, and cooling rate ~3 K s−1.Sn-3.0Ag-0.5Cu and Sn-3.5Ag (mass%) alloys were prepared from commercial purity Sn (99.9%), Ag(99.9%), and Cu (99.9%) in a graphite crucible at 500 °C. After 1 h holding, the melts were stirred with a preheated graphite rod and 40 g samples were cast into a chemical analysis mold for X-ray fluorescence spectroscopy analysis. The measured compositions of these alloys are given in Supplementary Table 4, αCoSn3, and βIrSn4 single crystals were incorporated into BGA joints by two additional steps: (1) Cu pads were coated with a ~1 μm tin layer using a commercial immersion tin solution ; (2) PtSn4, αCoSn3, or βIrSn4 crystals were placed on the tin layer with NH4Cl-ZnCl2-based flux  coated on and their main facet near-parallel with the substrate at a desired rotation angle, and the nucleants were transient liquid phase bonded to the substrate by holding at 240 °C for 5 min (for the αCoSn3 case) or at 300 °C for 180 min (for the PtSn4 and IrSn4 cases). Since PtSn4, αCoSn3, and βIrSn4 are all solderable surfaces using a standard ROL-1 flux, these nucleant-modified pads were then used in the same manner as a Cu-OSP substrate. All solder joints were firstly made by reflowing in the Tornado LFR400 reflow oven. Some joints were reflowed subsequently in the DSC to measure the nucleation undercooling. Substrates were cut to ~3 × 3 mm2 for DSC measurements. N2 atmosphere was used in all reflowing cases.PtSnThe EBSD and DSC datasets generated during the current study are available in the Zenodo repository (doi: 10.5281/zenodo.884113).Supplementary InformationPeer Review File


When the DCPD block was immersed in NaHCO3 or Na2CO3 solution at 80 °C, DCPD converted to CO3Ap within 3 days. β-Tricalcium phosphate was formed as an intermediate phase, and it was completely converted to CO3Ap within 2 weeks when the DCPD block was immersed in Na2CO3 solution. Although the crystal structures of the DCPD and CO3Ap blocks were different, the macroscopic structure was maintained during the compositional transformation through the dissolution–precipitation reaction. CO3Ap block fabricated in NaHCO3 or Na2CO3 solution contained 12.9 and 15.8 wt % carbonate, respectively. The diametral tensile strength of the CO3Ap block was 2 MPa, and the porosity was approximately 57% regardless of the carbonate solution. DCPD is a useful precursor for the fabrication of CO3Ap block.Carbonate apatite (CO Wobserved . Becausethan HAp . Moreove3Ap block is a promising artificial bone replacement, the studies conducted so far to identify the ideal precursor have been limited. There are requirements for the precursor involved in the fabrication of CO3Ap block. First, a precursor should be a block. Compositional transformation from the precursor to CO3Ap block occurs through a dissolution–precipitation reaction, maintaining the macroscopic structure of the precursor. Second, the precursor should have at least one component of CO3Ap such as calcium, phosphate or carbonate. Third, the precursor should be in a metastable phase and have suitable solubility in the solution used for the dissolution–precipitation reaction. If solubility is too low, it takes too long to fabricate the CO3Ap block from the precursor because of the rate of the dissolution–precipitation reaction. If the solubility is too high, the precursor cannot maintain its structure, and CO3Ap powder is formed instead of CO3Ap block. For example, calcium chloride cannot be a precursor because its solubility is too high even though it contains calcium.Although CO4·2H2O] is a candidate for the fabrication of CO3Ap block. DCPD block can be fabricated from a setting reaction, i.e., DCPD-forming cement .In this study, the feasibility of the fabrication of CO3 at 80 °C for 14 days. 2CO3 at 80 °C for 14 days. Samples maintained their macroscopic structure after the immersion in carbonate solutions regardless of the carbonate solution.3 at 80 °C for 14 days, and 2 M Na2CO3 at 80 °C for 14 days. Although the samples maintained their macroscopic structure as shown in 3 at 80 °C for 14 days, crystals with two different morphologies were present in the sample  2 M Na2CO3 at 80 °C for up to 14 days. The FTIR spectrum of (d) CO3Ap is shown for comparison. The frequencies at 567, 606, 1042, and 1092 cm−1 are assigned to PO43− groups [−1 are assigned to CO32− groups [−1 are assigned to OH− groups [3Ap  and 56.6%± 1.8% (Na2CO3 solution). No statically significant difference was observed between the carbonate solutions.3Ap block can be fabricated though a dissolution–precipitation reaction using DCPD block as a precursor. The DCPD block satisfied the requirements for fabricating the CO3Ap block.The results obtained in this study demonstrate that CO3Ap fabrication through a dissolution–precipitation reaction because the dissolution of the microporous block is faster than that of a dense block, and there is space for the formation of CO3Ap crystals.An ideal precursor is a block because the macroscopic structure is maintained during the dissolution–precipitation reaction. As shown in The setting time of the DCPD-forming cement was very short in the absence of a retarder. Although the setting time can be regulated by adding retarders such as citric acid, pyrophosphate, or sulfuric acid, no retarder was introduced to the mixture of β-TCP and MCPM in the present study to simplify the precursor. 3Ap block. Therefore, the DCPD block was immersed in NaHCO3 or Na2CO3 solution in the present study. The solution condition has a close relationship with the solubility. Although, the solubilities of DCPD in the NaHCO3 and Na2CO3 solutions are higher than other precursors such as calcite, calcium sulfate dihydrate, or α-TCP, the DCPD block was suitable for CO3Ap block fabrication because it was not dissolved during the reaction, and the macroscopic structure was retained as shown in 3Ap is reasonable because this reaction proceeds through a dissolution–precipitation mechanism. β-TCP was present 1 day after immersion in the carbonate solutions as an intermediate phase. The amount of β-TCP was higher when the DCPD block was immersed in NaHCO3 solution than in Na2CO3 solution. The amount of β-TCP decreased with immersion time and disappeared completely when the DCPD block was immersed in Na2CO3 solution for 2 weeks. However, β-TCP remained, even after 2 weeks when immersed in NaHCO3 solution. This difference may be because of the pH of the solution. In other words, β-TCP is more stable at neutral pH. The appearance of β-TCP as an intermediate phase demonstrates the possibility to use β-TCP block as a precursor for CO3Ap block fabrication even though its solubility is much lower than that of α-TCP. Moreover, the β-TCP block may be fabricated in the solution. The β-TCP block fabricated in the solution may have a higher osteoconductivity than β-TCP block fabricated using a sintering process.DCPD contains both calcium and phosphate; thus, only carbonate is required for the fabrication of the CO3 solution than in Na2CO3 solution may be the reason for this difference. In other words, DCPD crystals, shown in 3Ap, leading to the formation of more CO3Ap nuclei, increasing CO3Ap formation. When the DCPD block is immersed in NaHCO3 solution, DCPD dissolves slowly and results in a less supersaturated solution with respect to CO3Ap. Therefore, the precipitation of CO3Ap occurs only on the surface of DCPD crystals, and CO3Ap crystals maintain the crystal structure of DCPD.Different pH values of the carbonate solution cause different crystal morphologies as shown in 3Ap block was prepared through a dissolution-precipitation reaction at low temperature (80°C), the FTIR spectra of the obtained CO3Ap block had a broad band, indicating low crystalline CO3Ap that was also confirmed by XRD results. The large band made it difficult to quantitatively determine the types of CO3Ap. The band at 1413 cm−1 observed in the obtained CO3Ap is reported to be assigned to B-type CO3Ap [3Ap. However, no peaks corresponding to OH− groups were present in the spectra of the obtained CO3Ap. As a result, not only B-type but also A-type might co-exist in the obtained CO3Ap. Moreover, a broad band near 875 cm−1 assigned to the CO32− group consists of three components, such as A-type (878 cm−1), B-type (871 cm−1) and CO32− formed from apatitic lattice (866 cm−1) [32− from apatitic lattice such as that adsorbed on the surface of CO3Ap crystals.Since the CO66 cm−1) . Based o3Ap block in terms of DTS was higher (p < 0.05) when the DCPD block was immersed in NaHCO3 solution due to the crystal structure of CO3Ap. Plate-like CO3Ap shows high interlocking among the crystals. Although there was a slight difference in mechanical strength, there were no differences in porosity. The porosity of the CO3Ap block was approximately 57% regardless of the carbonate solution, which was lower than that of the DCPD block. The Ca/P ratio of DCPD is lower than that of CO3Ap, which has a Ca/P ratio around 2. Thus, DCPD needs to release PO4 and gain CO3 to transform its composition to CO3Ap. A high porosity indicates that the amount of released PO4 is higher than the amount of incorporated CO3 from the solution. Therefore, CO3Ap fabricated by compositional transformation through a dissolution–precipitation reaction using a precursor maintains its macroscopic structure but cannot maintain its microscopic structure.The mechanical strength of the COThe DCPD block was formed by the setting reaction of β-TCP and MCPM. β-TCP powder  and MCPM powder  were mixed with methanol  at a Ca/P molar ratio of 1.0. The methanol was allowed to evaporate at room temperature, and the mixture was placed into a split plastic steel mold 6 mm in diameter and 3 mm in height. Water was added dropwise until a water to powder weight ratio of 0.001 was reached. The samples were kept at 100% humidity for 24 h prior to testing.3 or 2 M Na2CO3 solution at 80 °C for up to 14 days. In this treatment, ten DCPD blocks were immersed in each sodium carbonate solution of 500 mL. After the treatment, the DCPD blocks were removed from the sodium carbonate solution and rinsed with distilled water.The obtained DCPD block was immersed in 2 M NaHCOThe crystal phase of the obtained samples was detected by powder X-ray diffraction (XRD) analysis. The specimen was ground into a fine powder and used for the analysis. XRD patterns were recorded with an X-ray diffractometer  using monochromatized X-ray (CuKα: λ = 0.1542 nm) operating at the condition of 40 kV and 40 mA. The diffraction angle was continuously scanned from 10° to 60° in 2θ at a scanning rate of 2°/min. A range of 10°–40° is shown in the figures because no relevant peaks were observed in the excluded region.−1. A spectral resolution of 4 cm−1 was employed to examine structural changes.Fourier transform infrared (FTIR) spectroscopy was performed with an FTIR spectrometer  using the KBr method over a wavenumber range of 400–2000 cmThe surface morphology of the obtained samples was observed by a scanning electron microscope  at 15 kV of accelerating voltage after gold–palladium coating by a magnetron sputtering machine . The fine structure of the obtained samples was observed by a transmission electron microscope  at 100 kV of accelerating voltage.dsample) and the theoretical density of HAp (dHAp 3.16 g/cm3) [The porosity the obtained specimen was calculated using the bulk density of the sample (6 g/cm3) , as show3Ap block. A CHN coder  was used to analyze the wt % of carbon.Carbonate contents were estimated from the wt % of carbon in the COThe mechanical strength of disk-shaped samples was evaluated in terms of their diametral tensile strength (DTS). After drying the samples at 60 °C for 24 h, their diameter and thickness were measured using a micrometer , and the samples were weighed using a microbalance. The samples were crushed with a universal testing machine  at a constant crosshead speed of 10 mm/min. The mean DTS value for eight samples was calculated and expressed as mean ± standard deviation.p < 0.05 is statistically significant.For statistical analysis, one-way analysis of variance and Fisher’s LSD method, as a post-hoc test, were performed using Kaleida Graph 4. We consider that 3Ap block was fabricated using DCPD block as a precursor by immersing the block in carbonate solutions. β-TCP forms as an intermediate phase during the transformation of the DCPD block to the CO3Ap block by compositional transformation through a dissolution–precipitation reaction. Further studies are awaited based on the results obtained in this initial study.CO