reaction 
 of nitric oxide ( no ) with a variety of aliphatic amines 
 generates diazeniumdiolate ions [ rrn  n(o)=no ] .
secondary amine derivatives ( r  h  r ) have become important 
 tools in studying the chemistry and biology of no by virtue of their 
 tunable rates and extents of hydrolysis to regenerate the starting 
 materials , as shown in eq 1 .
more recently , primary amine diazeniumdiolates have been 
 shown to hydrolyze according to two mechanisms running in parallel , 
 one mimicking the no - forming pathway of eq 1 and the other initiated by n - protonation at a different site to 
 generate similar amounts of a second nitrogenous bioeffector of current 
 intense interest , nitroxyl ( hno ) .
the 
 divergent primary amine pathways are shown in eq 2.1 if replacing one n - alkyl group 
 of the  pure  
 no donor shown in eq 1 with a proton converts 
 it to a mixed no / hno prodrug , one might wonder whether a  pure  

hno donor might result if both alkyl groups were removed , producing 
 the parent diazeniumdiolate structure ,  diazeniumdiolated ammonia  

indeed , recent calculations have indicated 
 that the species would produce no only very slowly , suggesting that 
 hno release would take over as the default hydrolysis pathway , potentially 
 producing hno with nontoxic dinitrogen and water as the only byproducts 
 ( eq 3 ) . here 
 we describe our initial attempts to prepare a derivative of 
 diazeniumdiolated ammonia for studies of its fundamental physicochemical 
 properties .
unlike secondary 
 and primary amines , 
 ammonia has shown no tendency to react directly with no . placing a 
 solution of ammonia in methanol or tetrahydrofuran in contact with 
 3 atm of no led to no observable gas uptake , precipitation , or development 
 of an ultraviolet peak at the characteristic diazeniumdiolate wavelength 
 of 250 nm .
even if such a direct no / nh3 reaction 
 were to occur , the product might be too reactive to isolate . while 
 secondary amine diazeniumdiolate salts are generally stable in the 
 absence of protonation sources , their primary amine counterparts are 
 highly ph - sensitive and prone to rapid decomposition .
primary amine diazeniumdiolate ions can be stabilized by 
 o - derivatization , however , so we decided 
 to pursue o - benzylated derivative h2n 
n(o)=nobn 
 ( 1 ) as our initial synthetic target . as starting 
 material , we chose known compound 2 , already 
 available from a previous study .
early 
 attempts at hydrolyzing 2 under either basic or acidic 
 conditions were unsuccessful , so we resorted to aminolysis by n - butylamine as a possible means of breaking the c = n 
 bond to free the desired ammonia derivative .
this reaction was chosen 
 on the basis of a previous report on the 
 aminolysis of benzamidine with n - butylamine to produce n - butylbenzamidine and n , n-dibutylbenzamidine , which were fully characterized .
our 
 expectation was that 2 would follow a similar reaction 
 course to produce 1 in addition to the alkylated benzamidines .
accordingly , 2 was dissolved in n - butylamine to make a 3.7 mm 
 solution and heated at 70 c .
after 1 h , significant amounts of one 
 expected product , n - butylbenzamidine 3 ( along with its further aminolysis product n , n-dibutylbenzamidine 4 ) , were seen , 
 but there was no trace of the desired product 1 .
after 
 30 h , the starting material was almost gone , the same two butylated 
 amidines were the major products , and a tiny amount of mono - n - butylated 2 ( structure 5 in scheme 1 ) was detected , again without any evidence of the presence of 1 . to probe the origins of the significant 
 signal associated with the void volume in these chromatograms ,
a different 
 chromatographic system was used to identify the remaining products 
 and establish a material balance .
, a near - quantitative yield of o - benzylhydroxylamine ( 6 ) was found by lc 
ms , 
 along with a high recovery of nitrous oxide as determined by gas chromatography 
 ( figure s1 , supporting information ) .
the 
 overall transformation is shown in scheme 1 , while its time course is shown in figure 1 .
total 
 ion chromatograms show the rates of starting material ( 2 ) consumption and product ( 3 , 4 , and 5 ) formation .
time points were taken at 1 , 10 , and 30 h for 
 panels a , b , and c , respectively .
product 6 ( not shown 
 in these chromatograms ) was identified in a separate chromatographic 
 treatment of the same reaction .
products were identified as follows : 3 , m / z obsd = 177.1382 m / z , calcd = 177.1386 ; 4 , m / z obsd = 233.2018 , m / z calcd = 233.2012 ; 5 , m / z obsd = 327.1815 , m / z calcd = 327.1816 . 

product 6 , m / z = 124.0764 , 
 separated with 
 distinct chromatographic conditions , was identified and quantified 
 by lc 
the structures of product 3 and product 4 were confirmed via purification and h nmr ( figures s2 and s3 , supporting information , respectively ) .
while 
 various mechanisms 
 can be conceived , we rationalize the above results as follows .
the 
 first step is postulated to be attack by the amine at the c = n 
 carbon of 2 to generate tetrahedral intermediate 7 , shown in scheme 2 . of its three 
 c 
n bonds , cleavage of the nh2 group of 7 to form 5 was very slow , with only a small yield being 
 observed after many hours .
the fastest 
 nondegenerate reaction was loss of the diazeniumdiolated nitrogen 
 to release 3 and 1 , with the latter undergoing 
 a series of deprotonation / tautomerization steps as represented in 
 scheme 1 , ultimately leading to nitrous oxide 
 and hydroxylamine 6 in good yields .
total ion chromatograms showing the rates of 
 starting material ( benzamidine ) consumption and product ( 3 and 4 ) formation .
time points were taken at 1 , 3 , and 
 18 h ( panels a , b , and c , respectively ) . to probe the effect of n - diazeniumdiolating an amidine group on its susceptibility 
 to nucleophilic attack at the c = n carbon , we reacted benzamidine 
 itself with butylamine under the conditions used in the aminolysis 
 of 2 .
the time courses of reacting benzamidine 
 vs 2 with butylamine are compared in figure 3 .
it is apparent that the presence of the diazeniumdiolate 
 substituent has a pronounced dampening effect on the electrophilicity 
 of the imine carbon , since a significant quantity of 2 remains even after a 30-h reaction .
relative rates of aminolysis of 2 ( red line ) vs benzamidine 
 ( blue ) in n - butylamine at 70 c . apparently , 

the presence of the diazeniumdiolate group on a benzamidine nitrogen 
 greatly diminishes the electrophilicity of the carbon atom of the 
 adjacent c = n double bond . in an effort to explain the unexpectedly 
 sluggish aminolysis rate of 2
, we performed a computational 
 chemistry study comparing rate - determining aminolysis reaction energetics 
 of 2 and benzamidine .
the theoretical methodology we 
 have used has been shown to provide very reliable predictions of chemical 
 properties for these types of systems when compared against experimental 
 observations .
for facility we have used methylamine 
 ( instead of n - butylamine ) and the o - methyl 
 ( instead of benzyl ) derivatives of 2 ( 2a ) and 1 ( 1a ) , as we expect that the results 
 will be commensurate .
lowest energy structures were located for all 
 species occurring in reactions 4 and 5 , with the only structural constraint being a z conformation for the n(o)no fragment in 2a and 1a.45 the resultant 
 computed 
 changes in free energies , g310(h2o solvent ) , for the aminolysis reactions of 2a and benzamidine are 3.78 and 5.45 kcal / mol , respectively . 

therefore , the slow aminolysis rate of 2 ( assuming a 
 similar behavior to 2a ) can be simply attributed to the 
 endothermicity of the first reaction step while the exothermicity 
 of benzamidine aminolysis gives rise to a comparably faster rate of 
 reaction .
experimental confirmation of these predicted free energy 
 changes should be possible in principle ; however , the essentially 
 immediate decomposition of 1 via release of n2o would make such a determination for 2 difficult .
in 
 fact , it may be that this quick degradation helps drive the aminolysis 
 of 2 forward , albeit very slowly ; i.e. , the reaction 
 is akin to a slow leak . although the observed slow aminolysis 
 rate of 2 relative 
 to that of benzamidine can be rationalized via reaction energetics , 
 it is also instructive to investigate the electrophilicity of the 
 imine carbon center in the two systems .
figure 4 shows the optimized structures of 2a and benzamidine 
 together with selected bond lengths and mulliken atom charges . optimized mp2/adz 
 [ pcm ] structures of 2a ( a ) and benzamidine 
 ( b ) together with selected bond lengths (  ) and mulliken charges 
 ( red ) where the value of 0.06 corresponds to the o - methyldiazenium diolate group ( sum over all of its atoms ) .
when comparing 2a against benzamidine we find that 
 the imine c = n bond length in the former ( 1.323  ) is noticeably 
 longer than that of the latter ( 1.304  ) while the formally designated 
 single c 
n bond length of 2a ( 1.352  ) is 
 shorter than its benzamidine counterpart ( 1.382  ) .
nh2 group is more nearly planar in 2a as indicated by the dihedral angle between the two planes defined 
 by atoms h 
c and hn  c (  
 = 159 ) which is noticeably larger than that for benzamidine 
 (  = 139 ) .
the results suggest that in 2/2a there is some resonance of the type shown in scheme 3 that results in delocalization of the  bond . 

the 60:40 ratio of the two resonance forms was crudely estimated by 
 comparing the c 
indeed , the computed mulliken atomic charges support this 
 notion 
 whereby the imine nitrogen in 2a has a more negative 
 charge ( 0.73 ) than in benzamidine ( 0.53 ) .
additionally , 
 the imine carbon is less positive in 2a ( + 0.18 ) than 
 in benzamidine ( + 0.29 ) .
the comparative bond lengths and charges 
 indicate that nucleophilic 
 attack on the imine double bond is more difficult for 2/2a than benzamidine since ( i ) the  electrons 
 are delocalized over three centers in 2/2a and ( ii ) the electrophilicity of the 2/2a imine carbon is subdued , relative to benzamidine , due to its lower 
 positive charge . therefore
, we must conclude that the diazeniumiolate 
 group acts to greatly suppress the reactivity of the imine c = n 
 group .
our results show that 
 the presence of a diazeniumdiolated amine 
 group attached to an imine nitrogen of an amidine dramatically slows 
 the rate - determining step of its aminolysis , namely nucleophilic attack 
 at the imine carbon , while also playing a dominant role in the subsequent 
 product - determining step .
thus , presumed tetrahedral intermediate 7 formed on attack of butylamine on the c = n carbon 
 of 2 eliminated the elements of 1 far more 
 rapidly than it expelled ammonia .
theoretical calculations confirmed 
 that the presence of a diazeniumdiolate group increases the electron 
 density at the imine carbon of 2a , rendering the initial 
 nucleophilic attack endothermic
. the results also point to base 
 sensitivity on the part of the desired 
 o - protected diazeniumdiolate 1 , whose nh2 group s acidity allows the two protons to be ionized 
 or to tautomerize readily to neighboring basic sites en route to complete 
 removal .
loss of n2o from the resulting intermediate leaves o - benzylhydroxylamine in near - quantitative yield . while the desired diazeniumdiolated ammonia derivative 1
was neither isolated from the above - described experiments nor directly 
 observed as an intermediate , our data strongly suggest that it was 
 generated in essentially theoretical yield , only to be rapidly destroyed 
 through a series of prototropic and base - induced reactions to form 
 n2o and o - benzylhydroxylamine 6 , both of which were also recovered in high yields . as for 
 attempts to isolate diazeniumdiolated ammonia derivatives 
 for studies of their physicochemical properties and possible pharmacological 
 utility , future work will seek to avoid the basic conditions that 
 proved problematic in our aminolysis reactions , focusing instead on 
 neutral and acidic conditions in an effort to minimize the influence 
 of the prototropic equilibria that lead to fragmentation of the h2n  n(o)=nor .
all other compounds 
 used here including authentic compound 6 were obtained 
 from commercial sources . in their respective 
 reactions 

either 7 mg of compound 2 or 3 mg of benzamidine was 
 dissolved in 7 ml of neat butylamine , for a final concentration of 
 3.7 mm , in a tightly sealed reaction vessel and heated to 70 c . 
 at each reported time point
the reaction was allowed to cool to room 
 temperature before aliquots were removed for dilution and analysis . 

all compounds were separated and identified with 
 an hplc coupled with a quadrupole time - of - flight ( q - tof ) mass spectrometer . 

separations of all compounds , with the exception of compound 6 , were performed on a reversed - phase c18 column , 3 m , 
 2.1  150 mm , at a flow rate of 0.2 ml / min under h2o / acetonitrile/0.1% formic acid gradient conditions .
separation of 
 compound 6 was performed on a polar end - capped c18 column , 
 3-m , 2.1  150 mm , at a flow rate of 0.2 ml / min under 
 h2o / acetonitrile gradient conditions .
positive ions were 
 generated by a dual electrospray source with a 150 v fragmenter voltage .
reactions were 
 run according to the conditions presented above with the following 
 exceptions . increased quantities of compound 2 ( 14 mg ) 
 and butylamine ( 14 ml ) were used .
each reaction was run under a constant 
 stream of n2 , which flowed out of a condensing column and 
 into a gas chromatography system with an electron capture detector . 

a high surface area carbon molecular sieve column ( 2 m  2.0 
 mm i.d . , 80/100 mesh ) was used with helium as carrier gas .
the gc 
 operation conditions were as follows : injector and detector temperatures 
 were at 250 c , oven temperature was programmed from 90 to 200 
 c at 20 c / min and held at 200 c for 1.1 min .
helium 
 flow was 30 ml / min , and nitrogen was used as makeup gas at 2 ml / min .
plesset perturbation theory 
 ( mp2 ) method together with the correlation - consistent aug - cc - pvdz 
 ( adz ) basis set .
water solvent effects were modeled 
 by use of the polarized continuum model ( pcm ) , whereby the 
 density of tesserae on the cavity surface was set to 240 .
geometries 
 were optimized using analytical gradients , and hessians were computed seminumerically with analytic gradients 
 so that gibbs free energies at 310 k could be determined .
 recent theoretical studies have suggested 
 that the parent diazeniumdiolate 
 ion , h2n  n(o)=no (  diazeniumdiolated 
 ammonia  ) , might be stable enough to be isolated and that it 
 could potentially serve as a uniquely advantageous prodrug form of 
 bioactive nitroxyl ( hno ) . 
 here , we report on an attempt to isolate 
 its o2-benzylated derivative by aminolysis of the c = n 
 bond in phc(nh2)=n  n(o)=nobn . 
 the 
 reaction proved remarkably sluggish in comparison to aminolysis of 
 unsubstituted benzamidine , and the desired product could not be isolated , 
 apparently because of base sensitivity of the nh2 group . 
 
 consistent with this interpretation , o - benzylhydroxylamine 
 and n2o were recovered from the reaction mixture in high 
 yields , along with n , n-dibutylbenzamidine . 
 
 theoretical calculations rationalize the observed slow aminolysis 
 by demonstrating that the diazeniumdiolate group greatly suppresses 
 the electrophilicity of the adjacent c = n carbon center , rendering 
 attack at that position endothermic . 
 the data provide significant 
 insights into the challenges inherent to the pursuit of diazeniumdiolated 
 ammonia . 
Introduction
Results
and Discussion
Conclusion
Experimental
Section