nmr hyperpolarization 
 can temporarily increase nuclear spin polarization 
 by 48 orders of magnitude , translating into corresponding 
 increases in nmr and mri detection sensitivity .
the 
 sensitivity gains improve the detection limit , which enables new imaging 
 methodologies  including hyperpolarized mri for molecular imaging 
 of dilute metabolites and functional contrast 
 agents such as hyperpolarized xe .
parahydrogen - based hyperpolarization techniques are special in 
 that they transform the spin order of the parahydrogen gas singlet 
 state into observable nuclear spin polarization ( up to order unity ) 
 of other molecules .
conventional parahydrogen induced polarization 
 ( phip ) has relied on the molecular addition 
 of parahydrogen to an unsaturated molecular precursor across c = c 
 or cc bonds .
this technique was 
 further advanced by preserving the high nuclear spin order of nascent 
 parahydrogen pairs on c sites with long t1 relaxation times exceeding 1 min .
recent 
 advances in catalysis have also enabled phip hyperpolarization of c contrast agents in biologically compatible aqueous media 
 in seconds with successful application in 
 living organisms .
one of the main fundamental limitations of 
 conventional phip is 
 the requirement for an unsaturated precursor for molecular addition 
 of parahydrogen ( p - h2 ) .
this limitation 
 has been lifted by the introduction of the signal amplification by 
 reversible exchange ( sabre ) hyperpolarization technique in 2009 . as with conventional phip ,
sabre involves p - h2 and substrate exchange on metal complexes ; however , instead 
 of hydrogenation , hyperpolarization of the substrate molecule is achieved 
 by transfer of spin order originating from p - h2 during the lifetime of the transient complex . despite the rapid progress of this technique with an expanding list 
 of substrate molecules amenable to sabre and recent 
 demonstration of heterogeneous sabre ( het - sabre , where the solubilized 
 substrate exchanges on the metal complex tethered to solid - phase support ) , the chemistry and the mechanism of the sabre 
 process are still not fully understood . moreover , despite promising 
 efforts to mitigate the poor solubility of sabre 
 catalysts in water , this method has not been demonstrated in pure 
 aqueous media suitable for biomedical applications 
the work presented here takes advantage of in situ high - resolution nmr detection of sabre at 9.4 t , which unlike low - field in situ sabre detection offers an advantage 
 of exquisite chemical shift 
 dispersion and high - resolution for distinguishing chemical compounds 
 and their sites .
specifically , we demonstrate in situ detection of a hyperpolarized intermediate during activation steps 
 of the most potent sabre catalyst , [ ircl(cod)(imes ) ] ( imes = 1,3-bis(2,4,6-trimethyl - phenyl)imidazol-2-ylidene ; cod = 
 cyclooctadiene ) .
it is found that the sabre effect is activated only 
 when the cod moiety is removed via its hydrogenation to cyclooctene 
 and cyclooctane in agreement with previous studies .
furthermore , such activation requires the presence of excess 
 substrate in order to avoid side reactions that irreversibly deactivate 
 the catalyst ( likely involving catalyst oligomerization ) . in situ studies of high - field sabre with the activated catalyst 

are consistent with substrate enhancements resulting from cross - relaxation 
 with hyperpolarized hydride spins in a manner akin to the spin - polarization 
 induced nuclear overhauser effect ( spinoe ) . finally , because of the catalyst s chemical changes that 
 occur during activation , it is possible to solubilize the activated 
 catalyst in aqueous media , which previously has not been demonstrated 
 without the use of organic cosolvents .
sabre hyperpolarization 
 of nicotinamide ( the amide of vitamin b3 ) in water is shown 
 as a proof of principle for potential biological applications .
long , 5 mm medium - wall 
 ( 0.77 
 mm - thick ) nmr tubes . the end of the nmr tube ( 3/4 in .
wall thickness ) to provide high - pressure connection 
 to a wye ( for " y " shape connection ) 0.25 in .
push - to - connect 
 fitting 
 ( mcmaster carr , p / n 5779k44 ) ( figure 1 ) .
long 
 pieces of the same 0.25 in . od teflon tubing . on one end ,
a push - to - connect 
 reducing connector ( mcmaster carr , p / n 5779k352 ) was used to connect 
 a 0.125 in .
od teflon tubing line to provide an exhaust path for  used  p - h2 gas exiting the solution . on the other top 
 end ,
connectors ( mcmaster carr , p / n 9087k121 and western analytical , 
 p / n u-51001 ) were used to allow 0.125 in .
i d ) 
 tubing , connected via a short flangeless nut ( western analytical , 
 p / n xp-208x ) , to extend all the way through the length of 5 mm nmr 
 tube to reach to the bottom of 5 mm nmr tube to deliver fresh p - h2 gas to the bottom of nmr tube for efficient 
 bubbling through the entire content of the nmr tube .
high - pressure 
 experiments were conducted by attaching a safety valve ( one - way , calibrated 
 to the exact pressure rating ) to the exhaust gas line to allow p - h2 pressure of up to 5.1 bar .
all nmr experiments 
 were conducted using a 9.4 t ( 400 mhz ) bruker avance iii spectrometer .
the iridium [ ircl(cod)(imes ) ] ( ir - imes ) 
 catalyst was synthesized as described previously .
all sabre studies of pyridine ( py ) at high magnetic field were conducted 
 with the ir - imes catalyst dissolved in either methanol - d4 , ethanol - d6 , or deuterated 
 water . for the methanol - d4-based solutions , 
 the complex consisted of 8 mm ir - imes and 47 mm py in 1 ml of methanol - d4 .
the ethanol - d6-based solutions contained 8 mm ir - imes and 32 mm py in 1 ml of ethanol - d6 . all nmr experiments with py
were performed 
 as high - field sabre experiments wherein p - h2 was bubbled through the solution and nmr 
 detection was performed at 9.4 t. for in situ high - field 
 sabre detection with py , the nmr tube was placed into the magnet similar 
 to a typical solution nmr experiment .
p - h2 gas ( > 90% para - state prepared using previously described instrumentation ) was bubbled through a given solution at a flow 
 rate of 0.7 ml / s under atmospheric pressure for durations ranging 
 from 30 to 60 s , depending upon the experiment .
h nmr 
 spectra were acquired immediately ( 3  2 s ) after the bubbling 
 was stopped .
experiments with nicotinamide utilized a solution 
 of 3.5 mm ir - imes 
 combined with 35 mm nicotinamide in 0.7 ml of ethanol - d6 , d2o , or a mixture of both .
solutions of 
 33% and 50% d2o in ethanol - d6 were obtained by diluting the activated ir - imes / nicotinamide solution 
 in ethanol - d6 ( 0.7 ml volume ) 
 with either one or two aliquots of d2o ( 0.35 ml 
 each ) , respectively . all sabre experiments with nicotinamide
were 
 conventional ( low - field ) sabre , where p - h2 was bubbled in the fringe field of the 9.4 t magnet 
 ( the solution in the nmr tube experienced 6  4 mt sabre polarization 
 field ; this field strength was measured using a portable gauss meter ) . 

once p - h2 bubbling was stopped , the sample 
 was quickly transferred inside a 9.4 t nmr magnet for h spectra acquisition with only a 5  2 s delay between the 
 end of p - h2 bubbling and the beginning 
 of the nmr spectra acquisition . during activation ,
p - h2 bubbling was performed under atmospheric pressure 
 with a flow rate of 0.7 ml / s .
once the nicotinamide solution was fully 
 activated , p - h2 bubbling occurred under 
 5.1 bar of pressure with a flow rate of 1.2 ml / s .
the experiments conducted in situ ( within an 
 9.4 t nmr magnet ) utilized no active temperature control , with the 
 room - temperature air supply providing temperature stabilization .
experimental 
 setup for sabre with controlled parahydrogen ( p - h2 ) bubbling through a catalytic solution in 
 a 5 mm nmr tube .
the solution sits in a medium - wall nmr tube where p - h2 is delivered via 1/16 in .
a wye push - to - connect adapter allows parahydrogen to flow into the 
 nmr tube , while allowing for the expended gas to leave the detection 
 volume via the exhaust line .
the exhaust line can be capped with a 
 pressure - calibrated safety valve to conduct sabre at a higher p - h2 pressure .
the sabre activation of ir - imes with pyridine 
 ( py ) in methanol - d4 was investigated using 
 high - resolution h nmr spectroscopy at 9.4 t ( figure 2 ) .
the spectrum of the nonactivated catalytic ir 
 complex ( figure 2a ) clearly displays the h chemical shift 
 signatures of the ortho- , meta- , 
 and para - protons of py , and the methanol solvent 
 ( 4.8 ppm ) . upon hydrogenation by p - h2 bubbling 
 ( figure 2b ) ,
the formation of three resonances 
 ( at 12.3 , 17.4 , and 22.8 ppm ) from hyperpolarized 
 hydride species are observed , in addition to that from hyperpolarized 
 orthohydrogen ( o - h2 ) at 4.5 ppm . during catalyst activation the high - field sabre 
 signal of py
was monitored in situ . as the catalyst 
 is subjected to additional p - h2 bubbling , 
 the hydride intermediate
is quickly depleted , leaving a single hyperpolarized 
 hydride signal at 22.8 ppm ( figure 2c ) .
once the ir - imes - py complex is fully activated , 
 high - field sabre signal enhancement is observed on the ortho - protons of pyridine ( manifested by the large  negative  
 or emissive signals ) , o - h2 ( manifested 
 by  positive  or absorptive enhanced signals ) , and the 
 hydride peaks .
therefore , the introduction of p - h2 to the catalyst solution initiates the hydrogenation of the 
 cod moiety followed by its complete removal as detected here by high - field 
 sabre .
this conclusion is further supported by the disappearance of 
 cod proton nmr resonances in the spectra of the activated catalyst 
 producing sabre effect ( figures s1a , s1c - d , and 1d in ref ( 26 ) ) a single intermediate 
 hyperpolarized hydride species ( seen as nmr resonances at 12.3 
 and 17.4 ppm ) is likely to be short - lived ( both chemically 
 and hyperpolarization - wise with low h t1 ) and would be difficult to detect by performing the 
 conventional sabre hyperpolarization procedure in low magnetic field 
 followed by sample transfer to the high magnetic field in a high - resolution 
 nmr spectrometer ( as well as by conventional nmr without p - h2 ) . indeed , figure 3b shows that 
 after 12 min of p - h2 bubbling , 
 all signatures of the hyperpolarized hydride intermediate species 
 are absent from the h nmr spectrum .
the hyperpolarized 
 intermediate hydride species is likely the result of p - h2 pair being added in the axial and equatorial positions 
 ( vs both p - h2 exchanging in equatorial 
 position only as shown in structure 3 , figure 3a , of the fully activated ir  catalyst complex ) . 

this observation is in agreement with pioneering work by bowers and 
 weitekamp , who detected similar dispersive 
 signatures with wilkinson s catalyst after addition of p - h2 in equatorial and axial positions of hexacoordinate 
 rh(i ) complex with similar geometry .
moreover , the phase of these 
 dispersive resonances uniquely reports on the sign of homonuclear j - coupling between two hydride protons in this intermediate 
 species , and it is negative .
series of h nmr spectra illustrating the hyperpolarization 
 of the ir - imes - py complex in methanol - d4 by high - field sabre at 9.4 t. ( a ) thermally 
 polarized spectrum of the catalytic complex and py before p - h2 is introduced to initiate high - field sabre . 

( b ) spectrum recorded immediately after the introduction of p - h2 ( first point in figure 3c ) to the catalyst / py solution .
note the dispersive peaks 
 from the intermediate hydride species at 12.3 ppm and 17.4 
 ppm .
( c ) spectrum of the ir - imes - py reaction mixture after the catalyst 
 has been completely activated through p - h2 bubbling .
note that only one hydride species is seen , manifested 
 by an absorptive peak at 22.8 ppm .
additionally , the selective 
 high - field sabre hyperpolarization of ortho - h - py protons is apparent with the opposite phase of 
 these proton peaks .
it should be pointed 
 out that the high - field sabre of py ortho - protons 
 effect correlates well with formation of hyperpolarized 
 hydride at 22.8 ppm ( estimated qualitatively through correlation 
 of peak intensities of sabre enhancement of ortho - h - py peak with the rise of the ir 
dihydride peak ) , likely 
 indicating that this species is responsible for the high - field sabre 
 effect .
the proposed mechanism for catalyst activation / formation is shown in figure 3a , 
 where the catalyst proceeds through hydrogenation steps ( intermediate 
 ir  hydride species corresponding to transient nmr resonances 1 and 2 ) before the fully activated species ( 3 ) is formed to yield high - field sabre hyperpolarization of 
 py .
additional evidence is provided by detection of cyclooctane / cyclooctene 
 vs cyclooctadiene in the h nmr spectra before and after full catalyst activation , respectively 
 ( data not shown ) .
the in situ high - field sabre may 
 also be a potentially useful tool for studies related to identification 
 and discrimination of classical and nonclassical hydrides in the context 
 of sabre .
we also note that while the 
 enhancements of in situ high - field sabre described 
 here and previously are relatively small , 
 much larger enhancements can be observed with the application of appropriate 
 pulse sequences .
( a ) schematic showing the tentative mechanisms 
 underlying activation 
 and sabre hyperpolarization with imes - ir catalyst and py .
the hydrogenation 
 of cod containing catalyst forms intermediate species corresponding 
 to transient nmr resonances ( 1 ) and ( 2 ) , 
 before forming the hyperpolarized catalyst - pyridine complex corresponding 
 to nmr resonance ( 3 ) .
additionally , without the presence 
 of pyridine , the activation mechanism reverts to forming an inactive 
 species , possibly a catalyst dimer ( cat : cat)/oligomer , see supporting information ) that does not hyperpolarize 
 via sabre .
( b ) nmr spectra demonstrating transient dispersive nmr 
 resonances ( 1 and 2 ) of intermediate hydride 
 species during the activation process .
( c ) plot showing the decay 
 ( measured as the integrated nmr signal in magnitude mode ) of the transient 
 nmr resonances ( 1 and 2 ) ] and the rise of 
 hyperpolarized ir 
the proper activation 
 of ir - imes requires not only h2 , but also the presence 
 of sufficient substrate to form a fully coordinated 
 ir  hydride complex with sabre properties . in the absence of the ligating substrate ( py ) ,
addition 
 of h2 to the ir - imes catalyst likely results in ( i ) cod 
 removal via hydrogenation and ( ii ) irreversible formation of inactive 
 species ( potentially dimer or trimer species ) .
our attempts to observe 
 sabre hyperpolarization with such catalyst states were unsuccessful  even 
 with subsequent additions of py and p - h2 .
additional evidence of possible dimer / trimer formation is provided 
 in the supporting information .
the potential 
 oligomerization would be in agreement with previous observation of 
 dimer formation of crabtree s catalyst ( similar to the ir - imes catalyst used here ) as well as observation 
 of oligomerization in catalysts from the same family . from the perspective of practical consideration ,
deactivation 
 of this catalyst occurs when the substrate ( py or others ) is either 
 absent or present in insufficient concentrations , yielding a solution 
 that remains yellow at the concentrations studied , while the properly 
 activated system turns clear upon complete activation . in the 
 high - field sabre effect , hyperpolarization occurred within the 9.4 
 t field of the high - resolution nmr spectrometer , and the following 
 signal enhancements occur : ir  hydride ( at 22.8 ppm ) 
 and o - h2 ( both with absorptive signals ) , 
 and the selective ( emissive ) enhancement of the ortho - protons of pyridine ( ortho - h - py ) in free and catalyst - bound 
 forms , figure 2c . here ,
the activated catalytic complex ( 3 ) was further 
 probed to study chemical and polarization exchange in three hyperpolarized 
 species by applying frequency selective rf irradiation during p - h2 bubbling ( i.e. , rf irradiation was applied 
 throughout the entire 60 s - long bubbling step ) during the build - up 
 phase of the high - field sabre effect .
application of a frequency - selective 
 saturation rf pulse at the hyperpolarized hydride resonance frequency 
 during sabre polarization ( figure 4a ) revealed 
 that the hyperpolarized o - h2 signal ( at 
 4.5 ppm ) was suppressed along with the hydride signal ( at 22.8 
 ppm ) even with very soft ( b1  
 7.5 hz ) rf saturation .
importantly , the ortho - h - py 
 hyperpolarized signal intensities were suppressed such that the high - field 
 sabre enhancements were significantly reduced under conditions of 
 a relatively strong ( b1  2.4  
 10 hz ) rf saturation on the hydride spectral band ( figure 4a , black trace ) .
when rf saturation is applied at 
 the o - h2 frequency ( figure s1 , supporting information ) , a similar trend is observed , 
 with nearly full depletion of the hyperpolarized ir 
hydride 
 peak , and partial depletion of ortho - h - py peaks as 
 in figure 4a , black trace .
shifting the rf 
 saturation to the ortho - h - py ( corresponding to free 
 py in solution , h resonance at 8.55 ppm ) resonances 
 leads to a different trend ( figure s1c , 
 where only the ortho - h - py resonances of free py and 
 exchangeable py ( 8.33 ppm ) are clearly suppressed .
rf saturation 
 of free ortho - h - py has little observable effect on 
 the hyperpolarization enhancements for ortho - h2 , ir 
hydride , and catalyst - bound ( nonexchangeable in 
 cis- position ) ortho - h - py .
the one - h - phip mechanism is a potential candidate to explain 
 the current and previous observation 
 of high - field sabre effect , when at least one of the hydrogen atoms 
 from p - h2 molecule is incorporated via 
 hydrogen exchange .
however , while such exchange is indeed present , 
 and is concurrent with the sabre processes , it occurs on a much longer 
 time scale of days and therefore can not explain high- or low - field 
 sabre effects .
on the basis of 
 the rf saturation experimental results described 
 above the following high - field sabre model is proposed ( figure 4b ) .
hyperpolarization of exchangeable and free ortho - h - py ( the residence time of exchangeable py sites 
 is on the order of 0.10.2 s ) 
 is induced by the hyperpolarization pool of o - h2 and ir  hydride , but not the other way around .
this 
 model is in agreement with previously proposed spin polarization - induced 
 nuclear overhauser effect ( spinoe ) : the catalyst s 
 interaction with p - h2 produces high z - magnetization in the form of hyperpolarized hydride and o - h2 .
the close proximity of hyperpolarized hydride 
 spins then drives the bound ortho - h - py spins out 
 of thermal equilibrium via nuclear dipolar cross - relaxation ( which 
 then results in enhanced spectra of free ortho - h - py 
 as well ; note that emissive enhancements would be consistent with 
 expectation that the double - quantum term of the noe should dominate 
 in the extreme - narrowing regime ) . on the other hand , reduction or 
 destruction of the hydride / o - h2
consistent with 
 the conclusion that p - h2 itself is not 
 the direct source of spin order for high - field sabre
. high - resolution 
 proton nmr rf - saturation study via in situ detection 
 of high - field sabre .
( a ) nmr spectra were recorded 3  
 2 s after p - h2 bubbling was stopped inside 
 the 9.4 t magnet of the nmr spectrometer .
the color - coded nmr spectra 
 were recorded under conditions of variable rf - power saturation (  soft  
 ( green trace ) with b1  7.5 hz ; 
  hard 
( black trace ) with b1  2.4  10 hz ; and  no  ( red 
 trace ) rf saturation pulses ) applied at the ir  hydride resonance 
 frequency during in situ p - h2 bubbling
. 
 continuous rf irradiation on the hydride peak diminishes the polarization 
 enhancements at both the o - h2 peak ( 4.5 
 ppm ) , and the ortho - h - py peaks ( 8.058.55 
 ppm ) in addition to destroying the hydride hyperpolarized signal . 

the same trend is observed for rf - saturation at o - h2 ( see supporting information ) as depicted in the scheme shown in part b. however , rf saturation 
 on the ortho - h - py has little observable effect on 
 the polarization of ir  hydride and o - h2 ( see supporting information ) . 

frequency - selective rf saturation was applied during high - field sabre 
 hyperpolarization ( p - h2 bubbling for 60 
 s at 9.4 t ) .
prior to activation , 
 the ir - imes catalyst ( with its cod ligand intact ) is insoluble in 
 water , even with the presence of sabre substrates such as py or nicotinamide 
 added to the solution .
therefore , organic solvents such as methanol - d4 , ethanol - d6 or 
 dmso are typically used to solubilize the ir - imes catalyst for sabre 
 hyperpolarization .
however , it was found 
 that when the activated , hexacoordinate ir - imes complex generated 
 in methanol or ethanol is dried , the resulting activated complex could 
 be dissolved in pure water .
this highly desirable characteristic is 
 likely endowed by the chemical changes that accompany catalyst activation , 
 including the loss of the hydrophobic cod moiety . motivated 
 by the biomedical utility of hyperpolarization techniques ,
sabre hyperpolarization 
 of nicotinamide in aqueous media was tested owing to the molecule s 
 biological relevance ( it belongs to the vitamin b group ) . here ,
ir - imes 
 was activated with nicotinamide in ethanol - d6 , achieving hyperpolarization on the four aromatic protons 
 located on the pyridine ring via sabre at 6  4 mt detected at 
 9.4 t ( figure 5a ) .
the sabre polarization enhancements 
 for free nicotinamide protons approach   100 and are 
 shown in table 1 .
two sequential aliquots of 
 d2o were added that resulted in reductions of sabre hyperpolarization 
 enhancements ( table 1 ) for all protons except 
 for hd , which exhibited low polarization enhancements .
a separately 
 prepared ( after full activation with h2 in ethanol ) catalyst / nicotinamide 
 sample was dried and then reconstituted in pure d2o .
the 
 structure of this complex is expected to be similar to that described 
 by cowley and co - workers for this catalyst and pyridine .
sabre polarization enhancements approaching 
   30 were observed for most nicotinamide aromatic proton 
 peaks in 100% d2o ( figure 5c ) .
the 
 preparation procedure for the solution corresponding to the spectrum 
 shown in figure 5c was different from those 
 reported earlier by zeng and co - workers , mewis and coworkers , and hvener 
 and co - workers in that once the sabre 
 catalyst activation is achieved in ethanol , the activated complex 
 solution is dried on a rotovap first , and the catalyst / nicotinamide 
 mixture is then reconstituted in 100% d2o .
first , the water - insoluble cod and its 
 hydrogenation products do not migrate into the aqueous phase .
moreover , 
 the use of 10% alcohol in previous works prevents potential in vivo injections , while 100% 
 d2o solutions prepared using the method described here 
 potentially allows for in vivo use of sabre hyperpolarized 
 contrast agents via intravenous administration .
the smaller 
 polarization enhancements achieved in d2o versus the organic 
 solvents can be largely attributed to the reduced 
 solubility of hydrogen gas in water .
previous studies have shown that 
 h2 is up to 14 times more soluble by molar fraction in 
 organic solvents than in water .
the reduced solubility of h2 in aqueous media limited the effectiveness of p - h2 bubbling in our setup and hindered the ability of p - h2 to interact with the catalytic ir complex 
 needed for sabre to occur .
however , potential solutions such as increasing 
 the partial p - h2 pressure may recover 
 the enhancement reduction in water versus that observed in ethanol . 
 moreover , the decrease in the sabre signal enhancements in water vs 
 ethanol may be also caused by the difference in t1 relaxation times , which can be lower in aqueous media 
 compared to that in organic solvent media . furthermore , the 
 low - field sabre proton signal enhancements for 
 nicotinamide in ethanol were significantly lower than sabre enhancements 
 reported by other groups , when using more advanced specialized sabre 
 equipment . the experimental limitations 
 ( including the lack of precise field control , introduction of p - h2 via bubbling and potentially longer nmr 
 sample shuttling time ) resulted in significantly reduced sabre signal 
 enhancements in the presented study .
therefore , the reported enhancements 
 for nicotinamide in water can be potentially significantly improved 
 using more advanced hyperpolarization hardware as well the optimization 
 of temperature and p - h2 pressure , because these experimental variables are 
 related to nicotinamide residency / sabre contact time on this ir - dihydride 
 complex . conventional ( low - field ) sabre hyperpolarization of nicotinamide 
 in ethanol - d6 ( a ) and in d2o ( c ) yields signal enhancement of four aromatic proton peaks of 
 interest ( ha , hb , hc , and hd ) , with the position of each proton labeled 
 in the structure inset .
the enhancement (  ) values for sabre 
 polarization of nicotinamide using the ir - imes catalyst can be found 
 in table 1 .
the respective thermally polarized 
 reference h nmr spectra of nicotinamide in ethanol - d6 and d2o are shown in parts b and 
 d ; these thermal spectra are magnified by 20-fold relative to the 
 corresponding hyperpolarized spectra .
peak heights in thermal spectrum of aqueous sample vs thermal spectrum 
 of the sample in ethanol - d6 is likely 
 the result of partial sample reconstitution from the dried solid into 
 aqueous medium .
note that sabre was conducted conventionally at low 
 field ( 6  4 mt ) , and it was detected by high - resolution proton 
 nmr spectroscopy ( at 9.4 t ) using the activated ir - imes catalyst in 
 ethanol - d6 and d2o .
the activation and mechanism 
 for hyperpolarization of the ir - imes 
 catalyst in alcoholic and aqueous media was probed using in 
 situ high - field sabre .
introduction 
 of p - h2 to the catalyst in the presence 
 of pyridine substrate initiates a hydrogenation of the cod moiety , 
 leading to intermediate species .
sabre hyperpolarization of the activated 
 catalytic complex results in the enhancement of ir  hydride , o - h2 , and ortho - h - py protons 
 in free and catalyst - bound forms .
rf saturation studies probing polarization 
 and chemical exchange of the hyperpolarized species support the conclusion 
 that substrate hyperpolarization at high field ( 9.4 t ) arises from cross - relaxation with protons belonging 
 to the hyperpolarized hydride / o - h2 spin 
 bath . once activated and dried , the ir - imes catalyst was found 
 to be 
 soluble in aqueous media with either py and nicotinamide substrates 
 incorporated into its hexacoordinate structure .
sabre of nicotinamide 
 substrate in d2o was conducted using conventional low - field 
 sabre and detected 
 at 9.4 t. the sabre enhancements achieved in aqueous media , while 
 significant (   30 ) , were somewhat smaller than those 
 in ethanol - d6 ( table 1)which likely reflects the lower solubility of hydrogen 
 gas in water versus ethanol .
nevertheless , the sabre 
 enhancements in water can be potentially increased through the use 
 of higher p - h2 partial pressures ( e.g. , 
 10 bar ) frequently used in conventional phip .
furthermore , additional gains of aqueous sabre enhancements 
 may be realized through better mixing of catalyst / substrate solution 
 with p - h2 , which has already been shown 
 through the use of solution spray injection in p - h2 atmosphere , or hollow fiber membranes , and the optimization of other conditions ( e.g. , temperature and 
 magnetic field ) . in any case
, the ability 
 to conduct sabre hyperpolarization with 
 biologically relevant molecules in aqueous media significantly increases 
 the value of sabre to generate hyperpolarized contrast agents for in vivo molecular imaging  eliminating one of the 
 major shortcomings of the sabre hyperpolarization method .
furthermore , 
 other substrates beyond those studied here can be potentially hyperpolarized 
 in aqueous media , including recently reported tuberculosis drugs pyrazinamide 
 and isoniazid .
finally , it may be possible 
 to combine aqueous sabre hyperpolarization with het - sabre , potentially allowing the preparation of pure 
 aqueous hyperpolarized contrast agents with a recyclable catalyst . 

the latter can potentially enable not only clinical translation of 
 this hyperpolarization technique , but also high - speed and high - throughput 
 production of hyperpolarized contrast agents at relatively low cost .
 activation of a catalyst [ ircl(cod)(imes ) ] 
 ( imes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene ; 
 cod = cyclooctadiene ) ] for signal amplification by reversible exchange 
 ( sabre ) was monitored by in situ hyperpolarized proton 
 nmr at 9.4 t. during the catalyst - activation process , the cod moiety 
 undergoes hydrogenation that leads to its complete removal from the 
 ir complex . 
 a transient hydride intermediate of the catalyst is observed 
 via its hyperpolarized signatures , which could not be detected using 
 conventional nonhyperpolarized solution nmr . 
 sabre enhancement of 
 the pyridine substrate can be fully rendered only after removal of 
 the cod moiety ; failure to properly activate the catalyst in the presence 
 of sufficient substrate can lead to irreversible deactivation consistent 
 with oligomerization of the catalyst molecules . 
 following catalyst 
 activation , results from selective rf - saturation studies support the 
 hypothesis that substrate polarization at high field arises from nuclear 
 cross - relaxation with hyperpolarized 1h spins of the hydride / orthohydrogen 
 spin bath . 
 importantly , the chemical changes that accompanied the 
 catalyst s full activation were also found to endow the catalyst 
 with water solubility , here used to demonstrate sabre hyperpolarization 
 of nicotinamide in water without the need for any organic cosolvent  paving 
 the way to various biomedical applications of sabre hyperpolarization 
 methods . 
Introduction
Experimental
Section
Results and Discussion
Conclusions