the second generation polyamidoamine ( pamam ) dendrimer with amidoethanol surface groups and ethylenediamine core was purchased from sigma - aldrich ( poole , dorset , uk ) and used without further purification .
the metal salts : agno3 , cucl22h2o , zncl2 , feso4 , and fecl3 were purchased from fisher scientific ( loughborough , uk ) , sigma - aldrich ( poole , dorset , uk ) , sigma chemical co. ( st . louis , mo ) , sigma chemical c. ( st . louis , mo ) , and bdh ltd .
the pamam dendrimer was diluted to a concentration of 1 mm in methanol ( fisher scientific , loughborough , uk ) ; metal salts were dissolved in water ( j. t. baker , deventer , holland ) to a concentration of 1 mm .
analytical solutions of pamam dendrimer and metal salts were prepared ( 1:1 ) in methanol : water ( 50:50 , vol / vol ) .
a portion of this solution was diluted in methanol : water : acetic acid ( fisher scientific , loughborough , uk ) ( 49:49:2 , vol / vol ) to give a final concentration of 10 m pamam dendrimer and 10 m metal salt .
all mass spectrometry experiments were performed on a thermo finnigan ltq ft mass spectrometer ( thermo fisher scientific , bremen , germany ) .
samples were introduced to the mass spectrometer by an advion biosciences triversa nanomate electrospray source ( advion biosciences , ithaca , ny ) .
xcalibur 2.0 software ( thermo fisher scientific ) was used for data acquisition and analysis .
all mass spectra were acquired at a resolution of 100,000 at m / z 400 .
automated gain control ( agc ) was used to accumulate sufficient precursor ions ( target value 1  10 ) .
electrons were generated on the surface of an indirectly heated barium tungsten cylindrical dispenser cathode ( 5.1 mm diameter , situated 154 mm from the cell , 1 mm off - axis ) , ( heat wave labs , inc , watsonville , ca ) .
the current across the electrode was 1.1 a. precursor ions were irradiated with electrons for 70 ms .
all cid experiments were performed in the front - end linear ion trap and the fragments transferred to the icr cell for detection .
cid experiments were performed with helium gas at normalized collision energy of 35% . each ecd and cid scan consisted of five co - added microscans .
electrospray ionization of solutions containing equimolar concentrations of the second generation pamam dendrimer ( ethylenediamine core , 16 amidoethanol surface groups ; pamamg2oh ) and metal ions leads to the formation of multiply - charged metal - containing ions with the general formula [ pd + m + mh ] , where pd is the pamam dendrimer , m is the metal ion , n is the oxidation state of metal ion , and m is the number of protons . at higher concentrations of metal salt , complexes with the general formula
[ pd + 2 m + mh ] and [ pd + 3 m + mh ] were observed , however the relative abundances of these complexes were much lower than those containing a single metal ion .
the results presented here were obtained for [ pd + m + mh ] precursor ions , however similar fragmentation behavior ( both ecd and cid ) was observed for all charge states .
( for comparison , the ecd and cid mass spectra of [ pd + cu + 2h ] and [ pd + ag + 2h ] ions are shown in supplementary figures 14 , which can be found in the electronic version of this article ) .
the notation system for representing fragmentation sites along the backbone of the pamam dendrimer is shown in scheme 1b .
the system is based on that proposed by oh and coworkers , and follows the conventional nomenclature for peptide and protein fragment ions .
assignments are given in the form gn(m ) , where subscript n refers to the generation in which fragmentation takes place and m denotes the type of fragmentation : ax , b / y , c / z .
gn(in ) and gn(out ) refer to fragmentation that takes place core - side of the tertiary amines .
l and j concern cleavages between carbon atoms in the amidoamine branches , and k refers to cleavage surface - side of the tertiary amines .
fragment assignments were made on the basis of accurate mass measurement and verified by comparison of observed and expected isotope patterns .
the dominant fragmentation channel for ecd of protonated pamam dendrimers is cleavage at the tertiary amines in the interior of the dendrimer . pronounced amide bond cleavage in the interior of the dendrimer is also observed .
typically , the charge - reduced species [ m + nh ] constitutes a minor peak .
the ecd ms / ms spectrum of [ pd + cu + 3h ] is shown in figure 1a and the fragments are detailed in supplementary table 1 .
the results suggest that electron capture occurs either by the metal ion or by the dendrimer ligand .
the base peak in this mass spectrum can be assigned to the charge - reduced complex , [ pd + cu + 3h ] , formed as a result of electron capture by the divalent copper ion . a series of peaks corresponding to fragment ions that arise from dissociation of this charge - reduced complex were also observed .
for example , fragments formed as a result of cleavage within generation g0 of the dendrimer ligand , [ pd + cu  g0(kout ) + h ] and [ pd + cu  g0(x ) + h ] , were found .
all of the cu - containing fragments derived from cleavages surface - side of the tertiary amines ( k cleavages , marked red on the mass spectrum ) or core - side of the amide ( a / x cleavage , marked blue on the mass spectrum ) .
those cleavages are minor or non - existent in the ecd of protonated dendrimers . a series of fragment ions containing cu , e.g. , [ pd + cu  g1(out ) ] and [ pd + cu  g1(out ) + h ] , was also observed .
the presence of cu - containing fragments resulting from cleavage surface - side of the tertiary amines ( k cleavages ) , e.g. , [ pd + cu  g2(kout ) + h ] and [ pd + cu  g2(kout ) ] , demonstrates the influence of the metal ion on fragmentation behavior : those cleavages are rare in the ecd of protonated pamam dendrimer ions . in addition , nonmetal - containing fragments resulting from cleavage at tertiary amine or amide bonds were observed .
it is not possible to say whether these fragments are the result of electron capture by the metal or ligand , however , as they reflect the patterns observed for protonated species , we speculate they arise following electron capture by the ligand .
the following fragments fall within this category : g1(out ) , g0(out ) , gcore(out ) , gcore(in ) , g1(y ) and g0(y ) . for comparison ,
the ecd mass spectrum of [ pd + cu + 2h ] is shown in supplemental figure 1 .
cu - containing fragments deriving from cleavage surface - side of the tertiary amines ( k cleavage ) and core - side of the amide ( a / x cleavage ) , and cu - containing fragments deriving from k cleavage are observed .
the ecd ms / ms spectrum of [ pd + fe + 2h ] complex is shown in figure 1b .
intense peaks corresponding to the charge - reduced species formed following metal ion electron capture , and fragments thereof , are observed .
the fe - containing fragments include [ pd + fe  g0(x ) ] , [ pd + fe  g0(kout ) ] , [ pd + fe  g1(out ) ] , [ pd + fe g1(x ) ] , [ pd + fe  g0(x ) + h ] , [ pd + fe  g1(out ) + h ] , and [ pd + fe  g1(x ) + h ] .
no fe - containing fragments were observed , suggesting that the competition between electron capture by the metal ion and the ligand is weighted in the former 's favor .
again , cleavages core - side of the amide ( a / x ) are prevalent ( marked blue on the mass spectrum ) .
cleavage surface - side of the tertiary amine ( k ) was also observed ( marked red on the mass spectrum ) . unlike the cu complex
, the ecd mass spectrum of the fe complex is dominated by fragments arising from cleavage at amide bonds : g0(y ) and g1(y ) , and at tertiary amines of dendrimer ligand : g0(out ) , g1(out ) , and g0(kout ) .
it is not clear whether these fragments are the result of electron capture by the metal ion or the dendrimer ligand .
the ecd ms / ms spectra ( see figure 2 ) for complexes of ag , zn , and fe with pamamg2oh dendrimer ligands are very similar . as seen for protonated pamamg2oh dendrimer ions , ecd of [ pd + ag + 4h ] , [ pd + zn + 3h ] , and [ pd + fe + 3h ] complexes are dominated by cleavage at the tertiary amines and amide bonds of pamamg2oh ligand .
electron capture by the metal ions in the zn and fe complexes was not observed .
a peak corresponding to [ pd + 4h ] was observed following ecd of [ pd + ag + 4h ] . that species must be the result of electron capture by ag . nevertheless , no dendrimer fragments containing ag(0 ) were observed . for comparison ,
the ecd mass spectrum of [ pd + ag + 2h ] is shown in supplemental figure 2 .
peaks corresponding to fragments deriving from cleavage at tertiary amines and amide bonds in the inner generations dominate the spectrum . a peak corresponding to [ pd + 2h ] was also observed .
the results suggest that the ecd behavior of these complexes depends strongly on the properties of the metal ions .
these findings are supported by previous work , which showed that the nature of the metal cation dictates the ecd of peptide   and lipid complexes [ 24 , 25 ] . in the present case
, the electron can be captured either by the metal ion or by the pamamg2oh ligand .
when the electron is captured by the pamamg2oh ligand , the ecd is due to protonation of the tertiary amines and the presence of amide bonds in the dendrimer backbone and can be explained on the basis of a charge solvation model [ 20 , 21 ] . when the electron is captured by the metal ( fe and cu ) , two main fragmentation channels
are observed : a / x cleavage ( core - side of the amide ) and k cleavage ( surface side of the tertiary amine ) . on the basis of electron paramagnetic resonance ( epr ) experiments , tomalia and
coworkers   reported that in copper - dendrimer complexes the metal ion could be coordinated at various internal or external locations of the dendrimer .
crooks and coworkers , however , suggested that dendrimers with amidoethanol surface groups form metal ion complexes with interior tertiary amines .
a study of cu(ii ) complexes of a generation g0 pamam dendrimer by nolan et al .   showed that for [ pd + cu + 4h ] , the metal was coordinated via the two core tertiary amines , and three amide oxygens ( two equatorial and one axial ) . the complex [ pd + cu + 2h ] was coordinated via the two core tertiary amines , one axial amide oxygen and two equatorial amide nitrogens .
it is not clear which , if not both , coordination chemistries exist for the [ pd + cu + 3h ] complex studied here . as fe(iii ) also shows octahedral stereochemistry , it is reasonable to assume that [ pd + fe + 2h ] is also coordinated via the two core tertiary amines , one axial amide oxygen and either two amide nitrogens or two further amide oxygens .
for both the fe and cu complexes , the pronounced a / x cleavage can be explained by coordination of the metal by amide nitrogens and/or oxygens . although the metal - containing fragments reveal a / x cleavage , complementary fragments are not observed .
those fragments may arise from capture of the electron by the ligand ( see below ) or they may be the result of secondary cleavage of x ions via loss of co. in addition to the coordination chemistry described above , we hypothesize that for larger pamam dendrimers , the outer tertiary amines ( g0 , g1 , g2 ) may be involved in metal complexation , replacing the amide nitrogens / oxygens .
no fragments were observed that contained the dendrimer core but not the metal ion , suggesting that the core is essential in coordination of the metal .
in addition to the metal ion , the complexes studied also varied in the number of protons attached : the fe complex has two protons , the fe , zn , and cu complexes have three protons and the ag complex has four protons .
we have observed previously that for the pamamg2oh dendrimer ecd behavior is independent of the number of protons attached .
that finding was echoed by oh and coworkers   in their work on the ecd of pamamg3oh .
their results showed that dominant cleavage at the tertiary amines and production of y fragments was observed consistently over all charge states studied ( 4 + through 8 + ) .
the results here suggest that ecd of metal complexes of pamamg2oh is independent of the number of the protons attached : ecd of [ pd + ag + 2h ] ( supplemental figure 2 ) reflects that of [ pd + ag + 4h ] and not that of [ pd + fe + 2h ] .
the ecd of [ pd + cu + 2h ] ( supplemental figure 1 ) reflects that of [ pd + cu + 3h ] .
the ecd of [ pd + cu + 3h ] ( figure 1a ) does not mirror that of [ pd + zn + 3h ] and [ pd + fe + 3h ] ( figure 2b and c ) .
a study on the ecd of complexes of divalent metal ions and diacylglycerophosphocholine by o'hair and coworkers   revealed that those metals with the highest second ionization energies stabilized the charge - reduced complexes via change in the oxidation state , whereas those metals with lowest second ionization energies promoted electron capture by ( and fragmentation of ) the ligand . we have found that , for those metals with 3d valence electrons , the ecd behavior of metal ion 
pamam complexes also tracks with the highest ionization energy ( ie ) : the fe ( ie3 30.6 ev ) and cu ( ie2 20.3 ev ) stabilize the charge - reduced complexes , via change in the oxidation state of the metal ion .
( in fact , no fe - containing fragments were observed ) . for metal ions with lower ionization energies , zn ( ie2 17.9 ev ) , and fe ( ie2 16.1 ev ) , formation of charge - reduced complexes and change in oxidation state of metal ion is not observed . instead ,
electron capture by , and fragmentation of , the pamamg2oh dendrimer ligand is promoted . for the ag complexes ( ie1 7.6 ev ; 4d valence electrons ) ,
the presence of [ pd + nh]species suggests that electron capture by the metal ion does occur .
the cid mass spectrum of the [ pd + cu + 3h ] complex , shown in figure 3a , reveals four intense signals , which can be assigned to [ 1/2pd + cu ] , [ 1/2pd + cu + h ] , [ 1/2pd + 2h ] , and [ 1/2pd + 3h ] , respectively ( fragments are detailed in supplementary table 3 ) .
these fragment ions are the result of cleavage between the two carbon atoms of the ethylenediamine core of the dendrimer ligand .
other abundant fragments , including [ 1/2pd  g2(lout ) + 3h ] , [ 1/2pd  g0(z ) + h ] and [ 1/2pd  g1(z ) + h ] also involve cleavage in the core of dendrimer ( marked red on the mass spectrum ) .
( for comparison , supplemental figure 3 shows the cid mass spectrum of [ pd + cu + 2h ] .
similar fragmentation behavior was observed ) . that fragmentation behavior is particularly surprising considering the cid of protonated pamamg2oh dendrimers [ 19 , 21 ] , in which l and k cleavages , and combinations of the two cleavage types in various stoichiometries , from the outermost generation were the dominant processes .
no core c  c bond cleavage was observed in the cid of protonated pamam precursor ions .
the results suggest that the cid of the [ pd + cu + 3h ] complex is linked with the coordination of the divalent copper ion to the pamamg2oh dendrimer ligand .
as described above , it has been suggested that coordination of the cu ion involves the tertiary amines of the dendrimer core .
that idea is supported by the fact that the cu - containing fragment [ pd + cu  g0(kout ) + 2h ] , which arises through the loss of g0(kout ) from the innermost generation of the complex , is observed , but cu - containing fragments from the outer generations , e.g. , [ cu+ g2(kout ) + nh ] ) or [ cu + g1(kout ) + nh ] ) , are not .
secondly , the abundance of fragments resulting from the cleavage of the core c  c bond suggests that both core tertiary amines coordinate the metal ion , and as a result the c  c bond is weakened .
the cid ms / ms spectrum of [ pd + fe + 2h ] precursor ions is shown in figure 3b .
the most abundant peaks correspond to [ 1/2pd + fe  h ] , [ 1/2pd + fe ] , [ 1/2pd + 3h ] , and [ 1/2pd + 2h ] , respectively , and are formed as a result of the cleavage between carbon atoms in the ethylenediamine core ( marked red on the mass spectrum ) .
the presence of metal - containing fragments such as [ pd + fe  g2(lout ) + 2h ] suggest that the metal ion is coordinated to the interior of the dendrimer .
it was postulated earlier that coordination of fe(iii ) also involves the core tertiary amines .
we hypothesize that coordination of the fe ion weakens the core c  c bond thus promoting its cleavage following vibrational excitation .
the cid mass spectrum of the [ pd + ag + 4h]complex , shown in figure 4a , is dominated by peaks corresponding to fragments arising from l and k cleavages in the outer g1 and g2 generations ( i.e. , [ pd + ag  g2(lout ) + 4h ] , [ pd + ag  2g2(lout ) + 4h ] , and [ pd  g1(lout )  g1(kout ) + 4h ] ) .
in addition , there are abundant peaks corresponding to [ gcore(out ) + 2h ] , [ gcore(out ) + ag+ h ] and g0(kout ) .
most of the fragments observed for the [ pd + ag+ 4h]complex were also observed for the protonated [ pd + 6h ] ions in our earlier study .
( similar fragmentation was recorded following cid of [ pd + ag + 2h ] , see supplemental figure 4 ) . in all cases ,
similar fragmentation behavior has been reported for doubly charged [ pd + ag + h ] ions .
thus , apparently , the cid of silver ion / pamamg2oh dendrimer complexes does not depend on the number of mobile protons .
the presence of the silver - containing fragments [ gcore(out ) + ag + h ] and [ ag + g0(y ) ] , and absence of silver - containing fragments from generations g2 or g1 , suggest that the silver ion is bound close to the core of the dendrimer .
however , cid of [ pd + ag + 4h ] does not result in any fragments deriving from cleavage of the core c  c bond . according to our hypothesis that observation suggests that coordination of the ag ion does not involve both core tertiary amines .
that idea is further corroborated by the presence in the mass spectrum of [ gcore(out ) + 2h ] and [ gcore(out ) + ag + h ] . in their study of ag(i ) complexes of a generation g0 pamam dendrimer , nolan et al
the first species was [ pd + ag + 3h ] and the second was [ pd + 2ag ] .
the structure proposed for the mononuclear complex involved trigonal planar coordination via the two core tertiary amines and an amide nitrogen ; and for the dinuclear complex , linear coordination of one of the core tertiary amines and a second , outer , tertiary amine , possibly accompanied by additional interaction of an amide oxygen .
the cid results for [ pd + ag + 4h ] suggest that of the two possibilities , the latter coordination structure is more likely . the cid behavior of [ pd + zn+ 3h ] complex , shown in figure 4b , also reflects that observed for the protonated dendrimer .
the majority of the fragments derive from l and k cleavages in generations g2 , g1 , and g0 .
peaks corresponding to [ pd  g0(kout ) + 3h ] , [ pd  g0(kout )  g2(lout ) + 3h ] , [ g0(kout ) + zn ] , and the lack of zn - containing core fragments suggest that the zn is either not , or weakly , bound to the core of the dendrimer . exclusive loss of the zinc ion from the [ pd + zn + 3h]complex is not observed .
fragments deriving from cleavage in generations g0 and g1 are observed both with and without the metal ion , e.g. , [ pd  g1(lout )  g2(kout ) + h ] and [ pd + zn  g1(lout )  g2(kout ) + h ] ; g0(kout ) and [ g0(kout ) + zn ] ; [ pd  g0(kout ) + 3h ] and [ pd + zn  g0(kout ) + h ] .
the same is not true for fragments originating from cleavage in the outermost generation ( g2 ) : the fragments [ pd + zn  g2(lout ) + 3h ] and [ pd + zn  2g2(lout ) + 3h ] are not accompanied by nonmetal - containing partners , suggesting the metal ion is not bound to g2 .
similar overall cid behavior was observed for the [ pd + fe + 3h ] complex ( figure 4c , supplementary table 4 ) .
the dominant pathways involved k and l cleavages in the outermost generation ( g2 ) : ( [ pd + fe  g2(lout ) + 3h ] and [ pd + fe  2g2(lout ) + 3h ] are formed .
fragments from g1 are observed both with and without fe , e.g. , [ pd  g1(kout ) + 3h ] , and [ pd + fe  g1(kout ) + h ] .
it is not clear which is true in the present example , however the results suggest that the fe is at best weakly bound to only one of the core tertiary amines .
note the marked difference between the cid of the fe complex and the fe complex ( figure 3b ) .
we have investigated the electron capture dissociation ( ecd ) and collision - induced dissociation ( cid ) of complexes of the pamamg2oh dendrimer with different metal ions ( ag , cu , zn , fe , and fe ) . complementary information regarding the site and coordination of the metal ions can be obtained from the two techniques . for the cu complex and the fe complex , electron capture by the metal resulted in pronounced a / x cleavage suggesting that amide nitrogen atoms and/or oxygen atoms were involved in metal coordination .
in addition , the pronounced k cleavage suggested that outer tertiary amines were also involved in metal coordination .
the cid of these two complexes indicates that both the core tertiary amines are involved in metal coordination .
the ecd of the zn and fe complexes mimicked that of protonated pamam dendrimer ions , i.e. , capture of the electron by the ligand was observed .
fragments observed following ecd of the ag complexes were the result of electron capture by the ligand . for the fourth period metals ( 3d valence shells ) ,
the ecd behavior of the metal complexes tracked with the highest ionization energy : electron capture by the metal ion was observed for those with the highest ionization energy ( fe and cu ) and by the ligand for those with the lowest ( zn and fe ) .
the cid behavior of the ag complex suggests that the metal is coordinated by one , but not both , of the core tertiary amines , while the cid of the zn and fe suggests that the metal is located in the interior of the dendrimer ( g0 , g1 ) but is not necessarily coordinated to the core .
supplemental figure 1ecd ft - icr mass spectrum of [ pd + cu + 2h ] ions .
k cleavages are marked in red ; a / x cleavages are marked in blue.supplemental figure 2ecd ft - icr mass spectrum of [ pd + ag + 2h ] ions .
pd = pamam dendrimer.supplemental figure 3cid ft - icr mass spectrum of [ pd + cu + 2h ] ions .
cleavages between carbon atoms in the ethylenediamine core are marked in red on the spectrum.supplemental figure 4cid ft - icr mass spectrum of [ pd + ag + 2h ] ions .
pd = pamam dendrimer.supplementary table 1supplementary table 2supplementary table 3supplementary table 4 ecd ft - icr mass spectrum of [ pd + cu + 2h ] ions .
k cleavages are marked in red ; a / x cleavages are marked in blue .
ecd ft - icr mass spectrum of [ pd + ag + 2h ] ions .
cid ft - icr mass spectrum of [ pd + cu + 2h ] ions .
cleavages between carbon atoms in the ethylenediamine core are marked in red on the spectrum .
cid ft - icr mass spectrum of [ pd + ag + 2h ] ions .
 the electron capture dissociation ( ecd ) and collision - induced dissociation ( cid ) of complexes of polyamidoamine ( pamam ) dendrimers with metal ions ag+ , cu2 + , zn2 + , fe2 + , and fe3 + were determined by fourier transform ion cyclotron resonance mass spectrometry . 
 complexes were of the form [ pd + m + mh]5 + where pd = generation two pamam dendrimer with amidoethanol surface groups , m = metal ion , m = 24 . 
 complementary information regarding the site and coordination chemistry of the metal ions can be obtained from the two techniques . 
 the results suggest that complexes of fe3 + and cu2 + are coordinated via both core tertiary amines , whereas coordination of ag+ involves a single core tertiary amine . 
 the zn2 + and fe2 + complexes do not appear to involve coordination by the dendrimer core . 
Experimental
Results and Discussion
Conclusions
Supplementary data