Use of a Dihydrogen Osmium Complex as Versatile ' NMR Recognition Probe
a
Zai-Wei Li and Henry Taube*
= - 13.45 ppm) diminishes in intensity and a doublet grows in at 8 = - 13.83 and -13.86 ppm (Fig. 1A). This phase of the reaction is complete in 10 min. We conclude that the probe is attached to the phosphate (3JPH = 5.46 Hz) and, because
r-
I
C')
cyD
C14 U')
H-H A
CH (en)
NH2'1/ |NH2
L
NH2 (en)
When a salt of 1 with PF- or CF3SO- as counterion is dissolved in water the variable ligand L is water. Water (or any other oxygen donor solvent) is rapidly replaced by other ligands in great variety. For each new composition, a characteristic value of 8 for the dihydrogen unit is registered in the NMR spectrum. All of the values of 8 are negative and are clearly separated from the shifts arising from protons of organic molecules. This property provides a convenient identification of the variable ligand L and also provides ready access to JH-D and T1 (H2). The latter property can be the source of important information about the dynamics of proteins and other molecules (4). The results obtained with DGMP (2'-
Downloaded from on March 26, 2016
X
A new recognition probe for biomolecules, [en2Os(.q2-H2)]2+ (1; en, ethylenediamine), is reported. In aqueous solution, 1 binds readily to a variety of biomolecules, including nucleotides, RNA, amino acids, peptides, and phospholipids. In each case, binding leads to a characteristic proton nuclear magnetic resonance (1 H NMR) for the dihydrogen that appears in a spectral window in the range 8 = 0 to -20 parts per million, and as well to characteristic values of the coupling JHD and of the relaxation time T1. Small structural differences in molecules such as DGMP (2'-deoxyguanosine 5'-monophosphate) and IMP (inosine 5'-monophosphate) or Asp and Glu can readily be distinguished, such as when 1 binds to the N-7 position of the nucleobase of DGMP or IMP and when 1 binds to the carboxylate of Asp or Glu. Upon one-electron oxidation of the metal center, diamagnetic 1 is converted to a paramagnetic probe.
Proton NMR has become one of the most powerful tools for investigating biomolecules (1, 2). Although higher field NMR instruments continue to be developed, the congestion of resonance signals in a small spectral range, usually between 8 = 0 and 10 ppm, complicates the interpretation of the observations. The species [trans-en2Os(ri-H2) 12+, 1, depicted below, has been described (3). Herein we report the results of experiments undertaken to explore its application as a recognition probe for biomolecules.
the reaction is not complete at equilibrium, that the affinity for the dinegative phosphate is not high. The equilibrium quotient, Ka (6), for the formation of the probe-RPO` complex is 3 102. Moreover, this attachment is labile, as appears to be the case for all a oxygen-donor ligands. A peak at 8 = -9.99 ppm, already discernible after 10 min, continues to grow at the expense of the others, and after 24 hours the conversion to the most stable form is almost complete (Fig. 1B). Accompanying the growth of the peak at 8 = -9.99 ppm, H-8 and H-1' shift from 7.96 to 8.65 ppm and from 6.09 to 6.32 ppm, respectively, indicating binding of the probe at N-7. Addition of a small amount of acid catalyzes exchange between rl-H2 and D20. For binding at N-7, JHD is 16.54 Hz, a coupling constant in the range characteristic of X2H2 (7). For this case only, the 1H NMR spectra covering the usual range of values of 8 are included and the NMR resonances for the NH2 and CH2 protons are identified. The former show structure corresponding to the three species shown in Fig. 1A.
. % a
_ _ L1D 13
A ik
-4iIlA
B
deoxyguanosine 5'-monophosphate) illustrate several important facets of the properties of the probe. When DGMP is added to a solution of 1 (as the PF- salt) in D20, each solute at 0.010 M, the original peak (8
Department of Chemistry, Stanford University, Stanford, CA 94305. *To whom correspondence should be addressed.
210
I I tlI
10
1U lIIIIIIIIIIIIIIIIIIAlIIIIIIIIIIaIIIIIII I1111111 1111111 AII 6
8
2
4
0
-2 ppm
-4
j-lIIII.I-10pII1lIII;1IIII -12
j I IIII
-6
-8
Fig. 1. Proton NMR spectra (200 MHz) of 1 with DGMP in D20 (both 0.010 M, pD min. (B) After 24 hours. SCIENCE
*
VOL. 256
*
10 APRIL 1992
=
II lgIIII
-14
7). (A) After 10
0~ UMMUM
In many cases, partial isomerization to a cis form accompanies substitution. This secondary process does not interfere with many applications of the probe and may in fact enhance its power when the effect is further investigated. The small peak at 8 = -7.99 ppm (Fig. 1B) is assignable to the cis form. The equilibrium quotient for binding to the two forms is 2.9 x 103. The 'H NMR traces for several nucleotides are compared in Fig. 2, and data collected for them are summarized in Table 1. The observations with IMP (inosine 5' -monophosphate) are similar to those with DGMP. Binding to nucleobases is observed also for AMP (adenosine 5'-
monophosphate), as well as for the simpler bases UMP (uridine 5'-monophosphate) and TMP (thymidine 5'-monophosphate), but in these cases the affinity is considerably less than it is for DGMP or IMP. The value of Ka for binding to cytidine 5'-monophosphate is
a
Zai-Wei Li and Henry Taube*
= - 13.45 ppm) diminishes in intensity and a doublet grows in at 8 = - 13.83 and -13.86 ppm (Fig. 1A). This phase of the reaction is complete in 10 min. We conclude that the probe is attached to the phosphate (3JPH = 5.46 Hz) and, because
r-
I
C')
cyD
C14 U')
H-H A
CH (en)
NH2'1/ |NH2
L
NH2 (en)
When a salt of 1 with PF- or CF3SO- as counterion is dissolved in water the variable ligand L is water. Water (or any other oxygen donor solvent) is rapidly replaced by other ligands in great variety. For each new composition, a characteristic value of 8 for the dihydrogen unit is registered in the NMR spectrum. All of the values of 8 are negative and are clearly separated from the shifts arising from protons of organic molecules. This property provides a convenient identification of the variable ligand L and also provides ready access to JH-D and T1 (H2). The latter property can be the source of important information about the dynamics of proteins and other molecules (4). The results obtained with DGMP (2'-
Downloaded from on March 26, 2016
X
A new recognition probe for biomolecules, [en2Os(.q2-H2)]2+ (1; en, ethylenediamine), is reported. In aqueous solution, 1 binds readily to a variety of biomolecules, including nucleotides, RNA, amino acids, peptides, and phospholipids. In each case, binding leads to a characteristic proton nuclear magnetic resonance (1 H NMR) for the dihydrogen that appears in a spectral window in the range 8 = 0 to -20 parts per million, and as well to characteristic values of the coupling JHD and of the relaxation time T1. Small structural differences in molecules such as DGMP (2'-deoxyguanosine 5'-monophosphate) and IMP (inosine 5'-monophosphate) or Asp and Glu can readily be distinguished, such as when 1 binds to the N-7 position of the nucleobase of DGMP or IMP and when 1 binds to the carboxylate of Asp or Glu. Upon one-electron oxidation of the metal center, diamagnetic 1 is converted to a paramagnetic probe.
Proton NMR has become one of the most powerful tools for investigating biomolecules (1, 2). Although higher field NMR instruments continue to be developed, the congestion of resonance signals in a small spectral range, usually between 8 = 0 and 10 ppm, complicates the interpretation of the observations. The species [trans-en2Os(ri-H2) 12+, 1, depicted below, has been described (3). Herein we report the results of experiments undertaken to explore its application as a recognition probe for biomolecules.
the reaction is not complete at equilibrium, that the affinity for the dinegative phosphate is not high. The equilibrium quotient, Ka (6), for the formation of the probe-RPO` complex is 3 102. Moreover, this attachment is labile, as appears to be the case for all a oxygen-donor ligands. A peak at 8 = -9.99 ppm, already discernible after 10 min, continues to grow at the expense of the others, and after 24 hours the conversion to the most stable form is almost complete (Fig. 1B). Accompanying the growth of the peak at 8 = -9.99 ppm, H-8 and H-1' shift from 7.96 to 8.65 ppm and from 6.09 to 6.32 ppm, respectively, indicating binding of the probe at N-7. Addition of a small amount of acid catalyzes exchange between rl-H2 and D20. For binding at N-7, JHD is 16.54 Hz, a coupling constant in the range characteristic of X2H2 (7). For this case only, the 1H NMR spectra covering the usual range of values of 8 are included and the NMR resonances for the NH2 and CH2 protons are identified. The former show structure corresponding to the three species shown in Fig. 1A.
. % a
_ _ L1D 13
A ik
-4iIlA
B
deoxyguanosine 5'-monophosphate) illustrate several important facets of the properties of the probe. When DGMP is added to a solution of 1 (as the PF- salt) in D20, each solute at 0.010 M, the original peak (8
Department of Chemistry, Stanford University, Stanford, CA 94305. *To whom correspondence should be addressed.
210
I I tlI
10
1U lIIIIIIIIIIIIIIIIIIAlIIIIIIIIIIaIIIIIII I1111111 1111111 AII 6
8
2
4
0
-2 ppm
-4
j-lIIII.I-10pII1lIII;1IIII -12
j I IIII
-6
-8
Fig. 1. Proton NMR spectra (200 MHz) of 1 with DGMP in D20 (both 0.010 M, pD min. (B) After 24 hours. SCIENCE
*
VOL. 256
*
10 APRIL 1992
=
II lgIIII
-14
7). (A) After 10
0~ UMMUM
In many cases, partial isomerization to a cis form accompanies substitution. This secondary process does not interfere with many applications of the probe and may in fact enhance its power when the effect is further investigated. The small peak at 8 = -7.99 ppm (Fig. 1B) is assignable to the cis form. The equilibrium quotient for binding to the two forms is 2.9 x 103. The 'H NMR traces for several nucleotides are compared in Fig. 2, and data collected for them are summarized in Table 1. The observations with IMP (inosine 5' -monophosphate) are similar to those with DGMP. Binding to nucleobases is observed also for AMP (adenosine 5'-
monophosphate), as well as for the simpler bases UMP (uridine 5'-monophosphate) and TMP (thymidine 5'-monophosphate), but in these cases the affinity is considerably less than it is for DGMP or IMP. The value of Ka for binding to cytidine 5'-monophosphate is
