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2.1C: Isotopes - Biology

2.1C: Isotopes - Biology


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Isotopes are various forms of an element that have the same number of protons, but a different number of neutrons.

Learning Objectives

  • Discuss the properties of isotopes and their use in radiometric dating

Key Points

  • Isotopes are atoms of the same element that contain an identical number of protons, but a different number of neutrons.
  • Despite having different numbers of neutrons, isotopes of the same element have very similar physical properties.
  • Some isotopes are unstable and will undergo radioactive decay to become other elements.
  • The predictable half-life of different decaying isotopes allows scientists to date material based on its isotopic composition, such as with Carbon-14 dating.

Key Terms

  • isotope: Any of two or more forms of an element where the atoms have the same number of protons, but a different number of neutrons within their nuclei.
  • half-life: The time it takes for half of the original concentration of an isotope to decay back to its more stable form.
  • radioactive isotopes: an atom with an unstable nucleus, characterized by excess energy available that undergoes radioactive decay and creates most commonly gamma rays, alpha or beta particles.
  • radiocarbon dating: Determining the age of an object by comparing the ratio of the 14C concentration found in it to the amount of 14C in the atmosphere.

What is an Isotope?

Isotopes are various forms of an element that have the same number of protons but a different number of neutrons. Some elements, such as carbon, potassium, and uranium, have multiple naturally-occurring isotopes. Isotopes are defined first by their element and then by the sum of the protons and neutrons present.

  • Carbon-12 (or 12C) contains six protons, six neutrons, and six electrons; therefore, it has a mass number of 12 amu (six protons and six neutrons).
  • Carbon-14 (or 14C) contains six protons, eight neutrons, and six electrons; its atomic mass is 14 amu (six protons and eight neutrons).

While the mass of individual isotopes is different, their physical and chemical properties remain mostly unchanged.

Isotopes do differ in their stability. Carbon-12 (12C) is the most abundant of the carbon isotopes, accounting for 98.89% of carbon on Earth. Carbon-14 (14C) is unstable and only occurs in trace amounts. Unstable isotopes most commonly emit alpha particles (He2+) and electrons. Neutrons, protons, and positrons can also be emitted and electrons can be captured to attain a more stable atomic configuration (lower level of potential energy ) through a process called radioactive decay. The new atoms created may be in a high energy state and emit gamma rays which lowers the energy but alone does not change the atom into another isotope. These atoms are called radioactive isotopes or radioisotopes.

Radiocarbon Dating

Carbon is normally present in the atmosphere in the form of gaseous compounds like carbon dioxide and methane. Carbon-14 (14C) is a naturally-occurring radioisotope that is created from atmospheric 14N (nitrogen) by the addition of a neutron and the loss of a proton, which is caused by cosmic rays. This is a continuous process so more 14C is always being created in the atmosphere. Once produced, the 14C often combines with the oxygen in the atmosphere to form carbon dioxide. Carbon dioxide produced in this way diffuses in the atmosphere, is dissolved in the ocean, and is incorporated by plants via photosynthesis. Animals eat the plants and, ultimately, the radiocarbon is distributed throughout the biosphere.

In living organisms, the relative amount of 14C in their body is approximately equal to the concentration of 14C in the atmosphere. When an organism dies, it is no longer ingesting 14C, so the ratio between 14C and 12C will decline as 14C gradually decays back to 14N. This slow process, which is called beta decay, releases energy through the emission of electrons from the nucleus or positrons.

After approximately 5,730 years, half of the starting concentration of 14C will have been converted back to 14N. This is referred to as its half-life, or the time it takes for half of the original concentration of an isotope to decay back to its more stable form. Because the half-life of 14C is long, it is used to date formerly-living objects such as old bones or wood. Comparing the ratio of the 14C concentration found in an object to the amount of 14C in the atmosphere, the amount of the isotope that has not yet decayed can be determined. On the basis of this amount, the age of the material can be accurately calculated, as long as the material is believed to be less than 50,000 years old. This technique is called radiocarbon dating, or carbon dating for short.

Other elements have isotopes with different half lives. For example, 40K (potassium-40) has a half-life of 1.25 billion years, and 235U (uranium-235) has a half-life of about 700 million years. Scientists often use these other radioactive elements to date objects that are older than 50,000 years (the limit of carbon dating). Through the use of radiometric dating, scientists can study the age of fossils or other remains of extinct organisms.


Characterization of new mesomeric betaines arising from methylation of imidazo[2,1-c][1,2,4]triazin-4(1H)-one, pyrazolo[5,1-c][1,2,4]triazin-4(1H)-one, and 1,2,4-triazolo[5,1-c][1,2,4]triazin-4(1H)-one

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Introduction

The terpenoid, small-compound strigolactones (SLs) are plant hormones that regulate plant shoot branching 1,2 , which is an important agronomic trait that determines crop yields. In addition, SLs stimulate the germination of root parasitic weeds 3,4 that cause devastating crop damage 5 and induce hyphal branching in symbiotic arbuscular mycorrhizal fungi that promote the growth of host plants by capturing essential inorganic nutrients from the soil 6,7,8 . Owing to the importance of SLs in agriculture, the mechanism and signal transduction pathway of SLs have been extensively researched to provide a perspective on the broader SL functions. However, the mechanisms responsible for SL recognition by plants are poorly understood.

SLs contain a structural core that consists of a tricyclic-lactone (ABC-ring) and a butenolide group (D-ring) that are connected via an enol ether linkage (compounds 14 Supplementary Fig. S1a). In plants, SLs are synthesized from carotenoids through carlactone (4) (ref. 9), a butenolide-containing compound with SL-like biological activity, by a sequential reaction of several enzymes, including an iron-binding protein D27 (ref. 9), the carotenoid cleavage dioxygenases 7 and 8, and a cytochrome P450 protein MAX1 (refs 10, 11, 12) (Supplementary Fig. S1b). In addition, studies on a set of branching mutants of some plant species have indicated that the d14 and d3/max2/rms4 genes are closely related to a pathway downstream of SLs 1,2,13,14 (Supplementary Fig. S1b).

D14 genes encode α/β-hydrolase family proteins 13,14,15,16 and their mutants exhibit a highly branched phenotype and are insensitive to SLs 13,14 . The d3/max2/rms4 mutants are also SL insensitive 1,2 . D3/MAX2/RMS4 genes encode members of the F-box-family protein 17,18,19,20 that participate in an Skp-cullin-F box (SCF) complex 18 and have diverse roles in the plant lifecycle 14,21,22,23,24,25,26,27,28,29,30 . Recently, it has been reported that petunia DAD2, an orthologue of rice DWARF14 (D14) protein, can hydrolyse the enol ether linkage of GR24 and interacts with PhMAX2A, a petunia orthologue of Arabidopsis MAX2 protein, indicating that the formation of the DAD2–PhMAX2A complex initiates an SCF-mediated signal transduction pathway 31 . However, it still remains unclear how the hydrolyzation of SLs induces the formation of the DAD2–PhMAX2 complex and what is/are the target(s) of the SCF complex.

The Arabidopsis gibberellin (GA) biosynthetic mutant ga1-3 exhibits enhanced shoot branching, and the overexpression of GA 2-oxidase genes in rice, in which GA levels are reduced, promotes tillering 32 . These results suggest that GAs regulate branching together with or without SLs. The DELLA-family proteins are key negative regulators of GA signalling through interaction with growth promoting transcription factors, such as PIFs. DELLA proteins have recently been reported to upregulate jasmonate (JA) signalling by physically interacting with JAZs, repressors of JA signalling 33 . Further, JAZs reportedly inhibit DELLAs−PIFs interactions. Thus, JA inhibits plant growth by inducing the degradation of JAZs and restoring the interaction of DELLAs with PIFs 34 (Supplementary Fig. S1c). In addition, recent reports suggest that a transcription factor, which broadly regulates brassinosteroid signalling, BZR1, mediates growth response to GA via direct interaction with DELLAs 35,36 . These reports indicate that DELLAs have a role in regulating multiple signals.

To investigate whether DELLAs mediate crosstalk between GA and SL signalling pathways, we explored the interaction between the rice D14 and the DELLA protein SLR1. Here we show that the rice D14 interacts with SLR1 in an SL-dependent manner, thus contributing to the negative regulation of GA signalling 37 (Supplementary Fig. S1d). Moreover, a series of crystallographic and biochemical studies suggest an advanced molecular mechanism of D14 in SL perception and signal transduction. Our results provide new insight into the crosstalk between the GA and SL signalling pathways.


Primary Sjögren’s Syndrome and Epigenetics

Amandine Charras , . Yves Renaudineau , in The Epigenetics of Autoimmunity , 2018

11.6 Histones in pSS

Global histone H3 and H4 hyperacetylation, and histone H3 demethylation, at lysine 4 characterize CD4 + T cells from SLE patients [87] . In addition to positively affecting transcription, these modifications can also lead to the development of specific auto-Ab. This was demonstrated with the lupus-derived monoclonal Ab BT-164 and KM-2, which recognize histone H3 trimethylated lysine 27 and histone H4 acetylated lysine 8, 12, and 16, respectively [88–90] . Blocking histone hyperacetylation represents an interesting therapeutic alternative as two histone deacetylase inhibitors (Trichostatin A and suberoylanilide hydroxamic acid) can revert histone modifications and improve SLE symptoms in mice without affecting auto-Ab titers [91,92] . These results provide arguments to suggest that the DNA methylation modifications that characterize pSS patients are associated with histone modifications, and that some of the antihistone auto-Abs detected in pSS target posttranslational histone modifications [93] . TNFα inhibition of AQP5 expression in the NS-SV-AC cell line was tested, and demonstrated that such inhibition was independent of DNA methylation but controlled by histone H4 acetylation [56] .


Mechanism and transition state structure of aryl methylphosphonate esters doubly coordinated to a dinuclear cobalt(III) center

Reactivities of five phosphonate esters each coordinated to a dinuclear Co(III) complex were investigated ([Co(2)(tacn)(2)(OH)(2)](3+) tacn = 1,4,7-triazacyclononane substituent = m-F, p-NO(2) (1a) p-NO(2) (1b) m-NO(2) (1c) p-Cl (1d) unsubstituted (1e)). Hydrolysis of the phosphonate esters in 1a to 1e is specific base catalyzed and takes place by intramolecular oxide attack on the bridging phosphonate. These data define a Brønsted beta(lg) of -1.12, considerably more negative than that of the hydrolysis of the uncomplexed phosphonates (-0.69). For 1b, the kinetic isotope effects in the leaving group are (18)k(lg) = 1.0228 and (15)k = 1.0014, at the nonbridging phosphoryl oxygens (18)k(nonbridge) = 0.9954, and at the nucleophilic oxygen(18)k(nuc) = 1.0105. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 1b that is similar to that of a phosphate monoester complex with the same leaving group, rather than the isoelectronic diester complex. The data from these model systems parallel the observation that in protein phosphatase-1, which has an active site that resembles the structures of these complexes, the catalyzed hydrolysis of aryl methylphosphonates and aryl phosphates are much more similar to one another than the uncomplexed hydrolysis reactions of the two substrates.

Figures

(A) Reaction scheme for kinetic…

(A) Reaction scheme for kinetic studies on the hydrolysis of TACN-Co(III) complex-bound aryl…

Crystal structures of the cations…

Crystal structures of the cations of 1d and 1e .

pH-rate profiles for the hydrolysis…

pH-rate profiles for the hydrolysis of complexes la-e The solid lines were fitted…

Linear free energy relationship between…

Linear free energy relationship between leaving group p K a and second-order rate…

The isotopic isomers of p…

The isotopic isomers of p -nitrophenyl methylphosphonate used for isotope effects measurements.


AQA CHEM1

I've created a bit more finished mark scheme, but allocated marks quite generally. Currently, the mark scheme is out of 74. This mark scheme is not conclusive and may contain errors.

  • Attempt at working e.g. (206 + 207 + 2*208)/4
  • Correct answer - 207.3
  • To 1 decimal place
  • Silicon is in a (3)p subshell
  • While Aluminium is in a (3)s subshell
  • The 3p subshell is more shielded (ora)
  • Sodium/Na
  • Second Ionisation Energy removes an electron from the second principal energy level (as opposed to third)
  • Which has more shielding
  • tendency / strength / ability / power of an atom / element / nucleus to attract / pull / withdraw electrons / e - density / bonding pair / shared pair
  • in a covalent bond
  • (Electronegativity) increases
  • Due to more protons/higher nuclear charge
  • No significant increase in shielding
  • Decreasing atomic radii
  • Ionic Bond
  • Strong Electrostatic attraction
  • Between positive metal ion and negative non-metal ion
  • Which requires a lot of energy to overcome
  • Ionic bond between metal and non-metal/Oxygen and Nitrogen not metals
  • Oxygen and Nitrogen both form negative ions
  • Attempt at calculation
  • Ratio of 1:1 for empirical formula, OF
  • Multiplied by 2 for molecular formula, O2F2
  • C4H10
  • Higher boiling point/closer to normal temperature
  • Easier to reach (-1°C) boiling point of C4H10
  • Stating the ideal gas equation - nRT = PV
  • (100000*4.31)/(8.31*298)
  • = 174 moles
  • 174*(4/10) = 69.6 moles
  • 3 bonding pairs and 1 lone pair from As
  • (Trigonal) Pyramidal
  • 2 bonding pairs and 2 lone pairs from Cl
  • Bent/V-shaped
  • As + has 4 valence electrons
  • Each forms a bonding pair/no lone pairs
  • Equal repulsion/pairs furthest apart
  • Tetrahedral shape
  • 1CH 3 CH 2 CH2CH3 + 3.5O2 -> 1C(something)(CO)2O + 4H2O
    or
  • 2CH 3 CH 2 CH2CH3 + 7O2 -> 2C(something)(CO)2O + 8H2O
  • Attempt at using concentration = moles/volume: 0.543/0.25
  • Answer multiplied by 2/3: 2.17*(2/3) = 1.45 mol dm -3
(Original post by S.S2303)
thanks for that!! ))

(Original post by Last Chance)
Post a thread for unlimited positive rep? Awesome man.

"You have reached the limit of how many posts you can rate today! "

Not done as bad as I thought if this is correct. 60/70 - what do you think this will get me?

That'll be a comfortable A, in the region of 90-95 UMS.

I appeared to have missed a question, the one requiring the use of Concentration = Moles/Volume. The values were (0.543/0.25)*(2/3) to get 1.448 mol dm -3 and was worth 2 marks. Anyone remember the exact equation used in the question?

(Original post by SomePotential)
I've created a bit more finished mark scheme which seems to account for 69 out of 70 marks. This mark scheme is not conclusive and may contain errors.

  • Attempt at working e.g. (206 + 207 + 2*208)/4
  • Correct answer - 207.3
  • To 1 decimal place
  • Silicon is in a (3)p subshell
  • While Aluminium is in a (3)s subshell
  • The 3p subshell is more shielded (ora)
  • Sodium/Na
  • Second Ionisation Energy removes an electron from the second principal energy level (as opposed to third)
  • Which has more shielding
  • tendency / strength / ability / power of an atom / element / nucleus to attract / pull / withdraw electrons / e - density / bonding pair / shared pair
  • in a covalent bond
  • (Electronegativity) increases
  • Due to more protons/higher nuclear charge
  • No significant increase in shielding
  • Decreasing atomic radii
  • Ionic Bond
  • Strong Electrostatic attraction
  • Between positive metal ion and negative non-metal ion
  • Which requires a lot of energy to overcome
  • Ionic bond between metal and non-metal/Oxygen and Nitrogen not metals
  • Oxygen and Nitrogen both form negative ions


f. Calculate the empirical and molecular formula of FO, where fluorine is 54.7% of mass and has a relative molecular mass of 70. [3]
[LIST][*]Attempt at calculation[*]Ratio of 1:1 for empirical formula, OF[*]Multiplied by 2 for molecular formula, O2F2

  • Branched chain isomers have less surface area/volume (ora)
  • So more/stronger Van der Waals
  • C4H10
  • Higher boiling point/closer to normal temperature
  • Easier to reach (-1°C) boiling point of C4H10
  • Stating the ideal gas equation - nRT = PV
  • (100000*4.31)/(8.31*298)
  • = 174 moles
  • 174*(4/10) = 69.6 moles
  1. 4NH3(g) + 5O2(g) -> 4NO(g) + 6H2O(g)
  2. 2NO(g) + O2(g) -> 2NO2(g)
  3. 3NO2(g) + H2O(l) -> 2HNO3(aq) + NO(g)
  • Moles = 3000/17
  • Moles = 176
  • 3 bonding pairs and 1 lone pair from As
  • (Trigonal) Pyramidal
  • 2 bonding pairs and 2 lone pairs from Cl
  • Bent/V-shaped
  • As + has 4 valence electrons
  • Each forms a bonding pair/no lone pairs
  • Equal repulsion/pairs furthest apart
  • Tetrahedral shape

I think the Q you didn't include was the balancing equation one:

From this, I've got 66/70. Not bad

(Original post by SomePotential)
That'll be a comfortable A, in the region of 90-95 UMS.

I appeared to have missed a question, the one requiring the use of Concentration = Moles/Volume. The values were (0.543/0.25)*(2/3) to get 1.448 mol dm -3 and was worth 2 marks. Anyone remember the exact equation used in the question?

(Original post by TheGirlNextDoor)
I think the Q you didn't include was the balancing equation one:

(Original post by TheGirlNextDoor)
I think the Q you didn't include was the balancing equation one:

I reckon by that mark scheme I got 48 - 50/ 70, hopefully a B!

I think you need to correct a few:

3d) the first part is right but if you are going to mention ions, it must refer to the fact that ionic bonding is impossible as both elements require the gaining of electrons
4b) less surface area weaker/less vdw
4c)this was ambiguous- it was only worth 1 mark for sure, and it can be argues either way but if you think about it, less energy is required if the boiling point is lower, so I believe it to be c3h8

a question has been missed out on the products of a reaction (CO2 and H2O)- both are greenhouse gases that ansorb infr-red radiation

(Original post by SomePotential)
I've created a bit more finished mark scheme which seems to account for 69 out of 70 marks. This mark scheme is not conclusive and may contain errors.

  • Attempt at working e.g. (206 + 207 + 2*208)/4
  • Correct answer - 207.3
  • To 1 decimal place
  • Silicon is in a (3)p subshell
  • While Aluminium is in a (3)s subshell
  • The 3p subshell is more shielded (ora)
  • Sodium/Na
  • Second Ionisation Energy removes an electron from the second principal energy level (as opposed to third)
  • Which has more shielding
  • tendency / strength / ability / power of an atom / element / nucleus to attract / pull / withdraw electrons / e - density / bonding pair / shared pair
  • in a covalent bond
  • (Electronegativity) increases
  • Due to more protons/higher nuclear charge
  • No significant increase in shielding
  • Decreasing atomic radii
  • Ionic Bond
  • Strong Electrostatic attraction
  • Between positive metal ion and negative non-metal ion
  • Which requires a lot of energy to overcome
  • Ionic bond between metal and non-metal/Oxygen and Nitrogen not metals
  • Oxygen and Nitrogen both form negative ions

f. Calculate the empirical and molecular formula of FO, where fluorine is 54.7% of mass and has a relative molecular mass of 70. [3]
[LIST][*]Attempt at calculation[*]Ratio of 1:1 for empirical formula, OF[*]Multiplied by 2 for molecular formula, O2F2

  • Branched chain isomers have less surface area/volume (ora)
  • So more/stronger Van der Waals
  • C4H10
  • Higher boiling point/closer to normal temperature
  • Easier to reach (-1°C) boiling point of C4H10
  • Stating the ideal gas equation - nRT = PV
  • (100000*4.31)/(8.31*298)
  • = 174 moles
  • 174*(4/10) = 69.6 moles
  • 3 bonding pairs and 1 lone pair from As
  • (Trigonal) Pyramidal
  • 2 bonding pairs and 2 lone pairs from Cl
  • Bent/V-shaped
  • As + has 4 valence electrons
  • Each forms a bonding pair/no lone pairs
  • Equal repulsion/pairs furthest apart
  • Tetrahedral shape

i thought that in the exam but when I looked at it again, it was only 1 mark ( i am 100% sure)

(Original post by Mr.Suhk)
I think you need to correct a few:

3d) the first part is right but if you are going to mention ions, it must refer to the fact that ionic bonding is impossible as both elements require the gaining of electrons
4b) less surface area weaker/less vdw
4c)this was ambiguous- it was only worth 1 mark for sure, and it can be argues either way but if you think about it, less energy is required if the boiling point is lower, so I believe it to be c3h8

a question has been missed out on the products of a reaction (CO2 and H2O)- both are greenhouse gases that ansorb infr-red radiation

3d) There will be a range of points to get the marks on, I only included a few. The actual mark schemes will have different wordings.
4b) Another typo >_>
4c) The question didn't ask which required more/less energy, but which was easier. I'd argue getting below -1°C would be easier than getting below -42°C. However, you could be right and they might accept both arguments.

Seem to have missed a lot of questions, must have been quite generous with the marks allocated.


Abstract

Reactivities of five phosphonate esters each coordinated to a dinuclear Co(III) complex were investigated ([Co2(tacn)2(OH)2<>2P(Me)OAr>] 3+ tacn = 1,4,7-triazacyclononane substituent = m-F, p-NO2 (1a) p-NO2 (1b) m-NO2 (1c) p-Cl (1d) unsubstituted (1e)). Hydrolysis of the phosphonate esters in 1a to 1e is specific base catalyzed and takes place by intramolecular oxide attack on the bridging phosphonate. These data define a Brønsted βlg of −1.12, considerably more negative than that of the hydrolysis of the uncomplexed phosphonates (−0.69). For 1b, the kinetic isotope effects in the leaving group are 18 klg = 1.0228 and 15 k = 1.0014, at the nonbridging phosphoryl oxygens 18 knonbridge = 0.9954, and at the nucleophilic oxygen 18 knuc = 1.0105. The KIEs and the βlg data point to a transition state for the alkaline hydrolysis of 1b that is similar to that of a phosphate monoester complex with the same leaving group, rather than the isoelectronic diester complex. The data from these model systems parallel the observation that in protein phosphatase-1, which has an active site that resembles the structures of these complexes, the catalyzed hydrolysis of aryl methylphosphonates and aryl phosphates are much more similar to one another than the uncomplexed hydrolysis reactions of the two substrates.


Discussion

Kinetic isotope effects reflect the difference in bonding to the labeled atom between the ground state and transition state of the rate-limiting step of a chemical reaction. 20 Consequently they serve as a useful tool in probing transition state structures and reaction mechanisms of both catalyzed and uncatalyzed reactions, including phosphoryl transfer. A primary kinetic isotope effect at an atom undergoing bond cleavage will be normal, due to the preference of the heavier isotope for the lower energy (more tightly bonded) position. Secondary kinetic isotope effects are normal (ϡ) when the labeled atom becomes more loosely bonded in the transition state, or inverse (ρ) when bonding becomes tighter. A large number of KIEs has been reported for enzymatic, uncatalyzed, and metal-catalyzed phosphate ester reactions, which provides a background for the interpretation of the KIE data measured in this study.

The magnitude of 15 k reflects the amount of negative charge developed on the leaving group in the transition state. It results from delocalization of charge arising from the P-O bond fission, which involves the nitrogen via resonance. This IE is increasingly normal as more charge is delocalized, reaching an observed maximum of 1.003 for a transition state with extensive leaving group bond fission and no charge neutralization. 21 The 18 klg KIE is a measure of the extent of P-O fission in the transition state. A large normal value for this KIE indicates extensive P-O bond weakening in the transition state, and can reach 1.03 for a loose transition state with extensive bond fission. 21 The secondary isotope effect 18 knonbridge reveals changes in bonding to the phosphoryl group (metaphosphate-like or phosphorane-like) in the transition state. While bond order considerations may be dominant in primary kinetic isotope effects, bending, torsional and vibrational modes could be more dominant for an atom bonded to a site undergoing a change in hybridization or steric considerations in the transition state. An increase in bond order to bridge atoms would result in an inverse KIE, while a decrease would result in a normal KIE. An increase in steric congestion in the transition state results in “stiffening” of bending modes giving rise to an inverse KIE and vice versa. A recent density functional theory analysis of the uncatalyzed hydrolysis of pNPP obtained a loose transition state with computed 22 leaving group and nonbridge KIEs close to the experimental 23 ones. The 18 knuc is a measure of nucleophilic participation in the transition. Nucleophile KIEs are generally normal and large for an early transition state and diminishes, as the degree of bond formation increases, to inverse if the bond to the nucleophile is completely formed in the transition state. 24 – 26 The later is observed when nucleophilic attack results in an intermediate, whose breakdown is rate-limiting. 16 , 17 Further discussion of nucleophile isotope effects can be found in Supporting Information.

The nucleophile

The nucleophile in the hydrolysis of TACN Co(III) complex-bound phosphate esters is an oxo species formed from initial deprotonation of one of the bridging hydroxides ( scheme 1 ), as demonstrated by the specific base catalysis observed ( figure 3 ). 14 No evidence for pH independent cleavage is observed, showing that the bridging hydroxide is too poor a nucleophile to promote departure of the leaving group. The X-ray structures show that the bridging oxygen atoms are within van der Waals contact of the phosphorus atom, and in the structure of 1d one of these atoms is almost collinear with the P-O bond of the leaving group (12° from linearity) the reaction only requires the phosphonate to pivot around the Co(III) coordinated non-bridging oxygens to induce bond formation. Consistent with a structure that requires very little movement to occur to progress from the ground state to the transition state, the entropy of activation of 1b is very close to zero (𢄣 J mol 𢄡 K 𢄡 ), in contrast to the intermolecular reaction between the uncomplexed phosphonate and hydroxide that has a value (� J mol 𢄡 K 𢄡 ) typical of a bimolecular reaction.

Proposed mechanism and transition state structure.

Since the labeled complex used for the determination of the nucleophile isotope effect has an 18 O label at both of the bridging positions (see Figure 1 ). In our analysis, we assume that the KIE arises from the nucleophilic atom, and that the second oxygen contributes negligibly to the observed isotope effect. The normal 18 knuc value of 1.011 ± 0.002 for complex 1b indicates the presence of nucleophilic participation in the rate-limiting step. This value reflects the fractionation of isotopes on the equilibrium deprotonation of the bridging hydroxide to form the oxo nucleophile, as well as the kinetic isotope effect on nucleophilic attack. The equilibrium isotope effect (EIE) for deprotonation of hydroxide to yield a metal-bound oxide, either singly or doubly coordinated to metal ions, has not been reported. The EIE for deprotonation of a water molecule coordinated to a single Co(III) ion is 1.012, obtained from the EIEs between (NH3)5Co(III)-OH2 and water (1.0196) and (NH3)5Co(III)-OH and water (1.008). 27 Coordination to a second Co(III) should further reduce the EIE. This trend arises from the fact that the force constant for the coordination of oxygen to the metal ions will be greater for hydroxide than for water, and in turn, greater for the oxide than for hydroxide. Thus, the fractionation arising from loss of the O-H bond is partly compensated for by enhanced coordination to the Co(III) ions. As a result, the EIE for deprotonation in this reaction should be smaller than the observed 18 knuc of 1.0105 ± 0.0005. If so, this implies that the KIE on nucleophilic attack is small and normal, an indication of nucleophilic bond formation in the rate-limiting step. This assumption is further supported by an earlier observation by Cassano and co-workers that the 18 knuc for the hydrolysis of a diester substrate changes from 1.068 ± 0.007 for free hydroxide catalyzed hydrolysis 24 to 1.027 ± 0.013 for Mg 2+ -hydroxide coordinated hydrolysis. 25 This difference was also attributed to a reduction in the fractionation factor for nucleophile deprotonation arising from coordination to magnesium ion.

The leaving group

Complementing KIE data, Brønsted β values provide information on the changes occurring at an atom if it is electronically varied in a series of kinetic or equilibrium experiments. The impact of varying the pKa of the leaving group will depend on the degree of bonding to the leaving atom in the transition state, but also on its solvation and electronic environment, so does not provide a simple measure of bond order. If the equilibrium effect of the perturbation (e.g. leaving group variation) is known, the kinetic effect can be normalized to provide a measure of the position of the transition state in terms of effective charge at the leaving atom. 28

For the bound phosphonates, the Brønsted plot ( figure 4 ) reveals that βlg = 𢄡.12 ± 0.03 for hydroxide catalysed hydrolysis of the phosphonates coordinated to the Co(III) complex. If we assume that the effective charge on the phenolic oxygen of bound aryl methylphosphonate is about the same as that in a doubly protonated (neutral) aryl phosphate, we can normalize the Brønsted data. The amount of charge on the phenolic oxygen in phenolate anion and in doubly protonated aryl phosphate are reported as 𢄡, and +0.83, respectively. 28 The total change in the charge on hydrolysis is thus estimated to be 𢄡 – 0.83 = 𢄡.83. Since the value of βlg is 𢄡.12, the extent of P _ O bond cleavage at the transition state for the reactions is estimated to be about 1.12/1.83 = 0.61 and the effective charge on the phenolic oxygen at the transition state is expected to be about 0.83 – 1.12 = 𢄠.29. 29 By way of comparison, the same analysis for the uncomplexed phosphonates gives βlg = 𢄠.69 ± 0.02 (close to the value of 𢄠.64 ± 0.03 reported for hydrolysis of an analogous group of methoxy aryl diesters under the same reaction conditions 15 , suggesting a later transition state for the more reactive complexed species. If we assume that the uncomplexed phosphonate has a starting effective charge that is similar to the isoelectronic monoprotonated aryl phosphate (+0.74), then the extent of cleavage can be estimated as 0.69/1.74 = 0.40 and the effective charge on the phenolic oxygen at the transition state is about 0.74 – 0.69 = +0.05.

Linear free energy relationship between leaving group pKa and second-order rate constant for hydroxide catalysed hydrolysis of 1a𠄾 (filled circles: βlg = 𢄡.12 ± 0.03 intercept = 13.4 ± 0.3) and corresponding aryl methyl phosphonates (open circles: βlg = 𢄠.69 ± 0.02 intercept = 𢄠.1 ± 0.2). The solid lines were fitted by linear least-squares regression.

These analyses use the assumption that a neutral OH or Me substituent on the transferring phosphorus will have a similar effect on the βeq value, but it is likely that the effect of changing an oxygen to a carbon substituent will be to reduce the βeq value for hydrolysis as the less electronegative methyl group moderates the electrophilicity of the phosphorus centre. For example, βeq decreases from 1.87 for diethylphosphate transfer to 1.30 for diphenylphosphate transfer to 1.22 for diphenylphosphinate transfer. 30 This would suggest that the transition states are more advanced than indicated above. If the value for βeq is reduced to an estimate of 1.3, the extent of reaction for the complexed and uncomplexed phosphonates would be 0.86 and 0.53 respectively. By way of further comparison, the overall picture is of a synchronous transition state for the uncomplexed phosphonate with little charge development at the leaving oxygen atom, but a rather more advanced transition state for the complexed phosphonate with more substantial charge development.

The estimate of leaving group departure in the transition state from LFER analysis of the phosphonate complexes is in reasonable agreement with both the 18 klg and the 15 k for 1b. The primary 18 klg of 1.0228 is 2/3 of 1.0340, the largest magnitude that has been observed for this isotope effect with the p-nitrophenyl leaving group. 31 The magnitude of 15 k, 1.0014, is about one-half of its maximum of 1.0030, 21 indicating about a half of a formal negative charge on the leaving group. This is reasonable agreement given the small magnitude of this secondary KIE. Also, 15 k is sensitive only to the extent of charge delocalization, which will be affected by the neighbouring negative charge on the phosphoryl group in the transition state. The maximal values of 15 k have been observed in reactions of the pNPP dianion, in which charge repulsion between the proximal anionic phosphoryl group and the leaving group enhances delocalization. In the present complex, coordination may lessen the negative charge on the phosphoryl, and hence its effect on delocalization.

Interestingly, the LFER and KIE data for the phosphate diester complex differ significantly from those of the phosphonate complex 1b despite the isoelectronic nature of the diester and the methylphosphonate. The data for the diester complex, particularly the inverse 18 knuc, are more consistent with a two-step addition-elimination mechanism with rate-limiting expulsion of the leaving group despite the low basicity of nitrophenolate this unusual outcome may result from strain associated with formation of a coordinated phosphorane intermediate. 16 In acidic solution, diphenyl methylphosphonate incorporates isotopically labeled water at about 8% the rate of hydrolysis, 32 implicating a stable phosphorane intermediate, whereas neutral phosphate esters such as triphenyl phosphate do not show any observable incorporation of solvent label into the starting ester, and incorporation is concomitant with hydrolysis. 33 These observations would suggest that the substitution of a methoxy with a methyl group would favor the associative pathway, so it is not clear why the methylphosphonate should not also form a phosphorane intermediate when constrained in the context of the Co(III) complex.

A combination of the normal 18 knuc and a significantly large normal 18 klg indicate that both bond formation and bond fission occur in the rate-limiting step, consistent with a concerted mechanism. The similarity of the LFER and KIE data for complex 1b with the corresponding data for the phosphate monoester complex make evident a similar transition state for the two reactions.

This implies that when bound to the dinuclear metal center, the phosphonate ester reacts more like a phosphate monoester than the diester for which it is assumed as an analogue. It is interesting to note that a similar observation is apparent when examining the effect that catalysis by PP1 has on phosphate monoester and methylphosphonate ester hydrolysis. It appears that the dinuclear metal ion reaction centre stabilises transition states that are altered from their uncomplexed counterparts to present very similar kinetic parameters for both phosphate monoesters and methylphosphonate esters. A distinction with the PP1 catalysed reaction lies in the observation that a much higher sensitivity to the leaving group is observed for the TACN Co(III) complexes for both substrates. However, it is likely that the enzyme utilizes general acid catalysis at the leaving group as part of its catalytic machinery, which would suppress this sensitivity, whereas in the model complex no additional functionality is available. With similar effective charges at the leaving group in the transition state, it could be expected that such interactions will have a similar effect on both reactants. A second contrast is that the model complexes show � fold higher reactivity when the methylphosphonate is bound compared to the phosphate monoester, whereas in the PP1 reactions, the monoesters are �-fold more reactive. However, the PP1 parameters also include substrate binding from solution, whereas the complexes are analogous to the Michaelis complex. The higher charge of the monoester dianion would suggest that enhanced ground state binding would be expected, offsetting potential differences in intrinsic reactivity when bound. We also note that the methylphosphonate complexes are about 10-fold more reactive than the corresponding aryl methyl phosphate diesters, which reflects the differences also observed in the PP1 catalysed reactions of these substrates. Here, the ground state binding may be expected to be less of a differential factor and so may represent subtle differences in the stabilization of the two functional groups. This difference is matched by the differential in reactivity of the uncomplexed substrates, where the diester is also about 10-fold less reactive than the methylphosphonate.

The small inverse 18 knonbridge KIE, 0.9954 ± 0.0001, implies little change in bond order to the nonbridge oxygen atoms. This is not inconsistent with the concerted mechanism implied by the other data. The interpretation of 18 knonbridge is difficult due to multiple contributions not only differences in P-O bond order, but accompanying changes in the interactions between these oxygen atoms and the Co atoms will also contribute. The trigonal bipyramidal transition state will also result in changes in the bending modes of the latter bonds. In uncatalyzed reactions, the loose, metaphosphate-like transition states of monoester reactions result in very small, inverse 18 knonbridge values, while the more symmetric transition states in diester reactions give rise to small, normal values, which become still larger in the more associative reactions of triesters. A concerted reaction with synchronous bond formation and bond fission should result in minimal bond order changes between phosphorus and the nonbridge oxygen atoms, but in a reaction involving a metal complex, the bending modes may well stiffen resulting in an inverse effect. For comparison, the hydrolysis of the complex of the diester ethyl p-nitrophenyl phosphate is accompanied by a small normal 18 knonbridge of 1.0006 ± 0.0004. 19 The analogous KIE for the phosphate monoester complex cannot be measured due to nonequivalence of the three nonbridging oxygens in the complex.


2.1C: Isotopes - Biology

a Department of Chemistry, Imperial College London, UK
E-mail: [email protected], [email protected]

b Department of Surgery & Cancer, Imperial College London, UK
E-mail: [email protected]

c Department of Bioengineering, Imperial College London, UK

Abstract

Microbubble (MB) contrast agents have revolutionalised the way ultrasound (US) imaging can be used clinically and pre-clinically. Contrast-enhanced US offers improvements in soft-tissue contrast, as well as the ability to visualise disease processes at the molecular level. However, its inability to provide in vivo whole-body imaging can hamper the development of new MB formulations. Herein, we describe a fast and efficient method for achieving 68 Ga-labelling of MBs after a direct comparison of two different strategies. The optimised approach produces 68 Ga-labelled MBs in good yields through the bioorthogonal inverse-electron-demand Diel–Alder reaction between a trans-cyclooctene-modified phospholipid and a new tetrazine-bearing HBED-CC chelator. The ability to noninvasively study the whole-body distribution of 68 Ga-labelled MBs was demonstrated in vivo using positron emission tomography (PET). This method could be broadly applicable to other phospholipid-based formulations, providing accessible solutions for in vivo tracking of MBs.


There are regional differences to climate change including within Australia

Over the past 100 years, temperature has increased over almost the entire globe the rate of increase has been largest in continental interiors (Figure 2.2). The average surface temperatures over the Australian continent and its surrounding oceans have increased by nearly 1°C since the beginning of the 20th century (Figure 2.3). Seven of the ten warmest years on record in Australia have occurred since 2002. However there are differences across Australia with some regions having warmed faster and others showing relatively little warming (Figure 2.3 right).

Since the mid 1990s there have been significant increases in wet season rainfall over northwest Australia (Figure 2.4 left), a declining trend in southwest Australia, and a 15% decline in late autumn and early winter rainfall in the southeast (Figure 2.4 right).

Figure 2.2: Surface temperature has increased across most of the world since 1901. This map shows the distribution of the average temperature change between 1901 and 2012. Adapted from IPCC (2013), Fifth Assessment Report, Working Group 1, Figure 2.21.

Figure 2.3: Temperature has risen over Australia and in the surrounding ocean since the beginning of the 20th century, although there are regional differences. Plot on left shows deviations from the 1961–1990 average of sea surface temperature and temperatures over land in the Australian region map on right shows distribution of annual average temperature change across Australia since 1910. Adapted from BOM/CSIRO State of the Climate 2014.

Figure 2.4: Recent rainfall in northern Australia has been higher than average during the northern wet season, and in southern Australia it has been drier during the southern wet season. The maps show the relative ranking (in 10% increments) of rainfall from July 1995 to June 2014 compared with the average since 1900 for (left) northern Australian wet season (Oct–Apr) and (right) southern Australian wet season (Apr–Nov). Adapted from BOM/CSIRO State of the Climate 2014.


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