Thomas
Malcomson
*a and
Martin J.
Paterson
b
aDepartment of Chemistry, Lancaster University, Lancaster, LA1 4YB, UK. E-mail: t.malcomson@lancaster.ac.uk
bSchool of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 4AS, UK. E-mail: m.j.paterson@hw.ac.uk
First published on 24th September 2020
Given the prevalence of fluorescence spectroscopy in biological systems, and the prevalence of pterin derivatives throughout biological systems, presented here is an assessment of the two-photon absorption spectroscopy as it applies to a range of the most commonly studied pterin derivatives. QR-CAMB3LYP//ccpVTZ calculations suggest that the use of two-photon spectroscopic methods would enable a more capable differentiation between closely related derivatives in comparison to the one-photon spectra, which show minimal qualitative deviation. Study of short tail derivatives shows that, in most cases, two-photon accessible states solely involve the π* LUMO as the particle orbital, with biopterin, neopterin, and 6-(hydroxymethyl)pterin presenting exceptional potential for targetting. Investigation of derivatives in which the tail contains an aromatic ring resulted in the observation of a series of two-photon accessible states involving charge transfer from the tail to the pterin moiety, the cross sections of which are highly dependent on the adoption of a planar geometry. The observation of these states presents a novel method for tracking the substitution of biologically important molecules such as folic acid and 5-methenyltetrahydrofolylpolyglutamate.
Behaving as weak acids in aqueous solution, pterins form a dominant equilibrium at pH > 5 consisting of the acidic (amino) and basic (phenolate) form (Fig. 1)2 with the pKa of this equilibrium, centred around the protonation state of the N3 proton, is ca. 83 for the derivatives shown in Table 1. The pKa of other pterin-based protons, specifically those located on the N1 amino group, are <2.2
State | Acid | Base | ||
---|---|---|---|---|
Energy | σ TP | Energy | σ TP | |
1 | 3.8466 | 0.000 | 3.6378 | 0.000 |
2 | 4.3353 | 0.058 | 3.7888 | 0.077 |
3 | 4.7731 | 0.001 | 4.2833 | 0.000 |
4 | 4.9175 | 0.000 | 4.6724 | 0.026 |
5 | 5.0735 | 0.164 | 4.7912 | 0.002 |
6 | 5.4911 | 0.006 | 4.9873 | 0.003 |
7 | 6.0768 | 0.006 | 5.3212 | 0.286 |
8 | 6.2832 | 0.008 | 5.6911 | 0.007 |
9 | 6.3615 | 0.260 | 5.8665 | 0.010 |
10 | 6.5497 | 0.332 | 5.9080 | 0.002 |
Throughout this work, for the ease of discussion, the acidic and basic forms of each molecule, when appropriate, will be marked by a subscript letter (PTa and PTb representing the acidic and basic forms of the unsubstituted pterin moiety, respectively).
Pterins, and their derivatives, are found throughout biology. While their oxidised states have been found extensive medical use, acting as markers for a range of conditions, including: vitiligo,4 cardiovascular diseases,5,6 along with activation of immune responses and synthesis of neurotransmitters,7,8 pterins are also found naturally within human biology; folic acid (vit B9) acts as a coenzyme during reactions involved in the synthesis of both purine and pyridine DNA bases.9 Biopterin (BPT) has been found taking the role of a coenzyme in hydroxylation reactions in the metabolism of both amino acids and nitric oxide10,11 while neopterin (NPT), a BPT metabolite, is synthesised predominantly in activated macrophages with high levels of NPT found in response to infections caused by viruses, parasites and intracellular bacteria.12–17 Despite the role of 5,10-methenyltetrahydrofolylpolyglutamate, a derivative of folic acid (FA), in DNA repair as a light-harvesting antenna in DNA photolyases,18–20 oxidised pterins have also been shown to cause photo-activated DNA damage when exposed to UV light.21–25
The ability of pterins to produce singlet oxygen (O2(1Δg), denoted as 1O2 for simplicity)26,27 has garnered particular interest due to its role in photodynamic therapy (PDT).28,29 This metastable state of molecular oxygen, produced primarily through photosensitisation, is significantly more reactive than the triplet ground state (O2(3∑−g)). The pterin-induced production of 1O2 is brought about due to ready access of the pterin molecules to a triplet excited state through intersystem crossing,30,31 enabling a spin crossover with the 3O2 state of molecular oxygen in place of the commonly observed pterin phosphorescence.26,32
However, the ideal wavelength of light needed to achieve the production of singlet oxygen is in the region of 350 nm which has a very poor tissue penetration value as, due to scattering from the cell nuclei, mitochondria, the Golgi apparatus and the cellular surface itself,33,34 the optical penetration of biological tissue is low, usually measured in millimetres.35 Tissue penetration varies greatly depending on the tissue type and the wavelength used, with longer wavelengths appearing to penetrate to a greater degree, with Stolik et al. reporting a depth of 4.23 ± 0.03 mm.35
While 1O2 has been shown to be a dominant mediator of phototoxic effects, it is a short lived species (<200 ns in vitro).36–38 As a result of this, the diffusion of 1O2 is limited to short distances (≈1 μm),39–41 limiting the cytotoxicity to immediate area around the production location. The low diffusion of 1O2 through a biological medium does, however, limit its use as a photoactivated target due to the tissue depth penetration of the UV light required to excite the pterin molecules.42,43 This limitation can be reduced through with the use of near IR light (λ = 600–100 nm) via two-photon absorption (TPA) which, in addition to the increased tissue penetration of higher wavelength light, increases the spatial resolution of photo-activation.
Despite this promising avenue of investigation, coupled with the pterin molecule acting as a design precursor for a number of fluorescent DNA analogues44,45 and the otherwise extensive detail to which the photochemistry of these molecules has been investigated,26,27,30–32,46 studies of the potential two-photon activation of pterin derivatives is poorly explored. In response to this, we present here a study of the susceptibility of a series of well studied, biochemically relevant pterin derivatives to two-photon activation through the use of quadratic response (QR) density functional theory (DFT) methodologies. The use of these methodologies for the accurate determination of TPA spectra has been well established across a range of molecules of both biological and photochemical importance.47–53
The structures studied here are presented in three groups: the first involves small functional group alterations to the core pterin moiety at the C6 (Table 1); the second involves the inclusion of extended side chains (Fig. 2) centred around the folic acid structure (FA); the final grouping of structures (Fig. 3), centred around 5-methenyltetrahydrofolylpolyglutamate (MTHFG), denote a series of biological, pterin derived, cofactors. These groups are selected to provide a thorough representation of the TPA viability of the pterin family of molecules throughout their biological function.
The successful determination of the TPA spectra for these derivatives opens up a number of avenues for further investigation, from TPA access to the triplet states of these derivatives and their potential for 1O2 production, to the ability to track the presence of specific derivatives despite the one-photon absorption (OPA) spectra of these structures showing little qualitative variation.
Two-photon cross sections (σTP), as defined through QR-DFT implemented in Dalton,84 are determined by:
![]() | (1) |
![]() | (2) |
δTP = FδF + GδG + HδH | (3) |
![]() | (4) |
![]() | (5) |
![]() | (6) |
![]() | (7) |
The inclusion of substituents containing an aromatic group, such as those highlighted in Fig. 2, results in similar trends to those of the smaller, non-aromatic, substituents in that large cross-section values relating to the higher energy peak of the OPA spectra (Fig. 8 and 9) remaining accessible in both acid and base forms, while the high values observed in lower states are quenching when adopting the basic form (Tables 5 and 6).
Overall, despite the structures with tails inclusive of aromatic groups showing greater similarity between the acid and base forms than their small tail counterparts, the TPA spectra mimics the properties of the OPA spectra closely with similar shifts in state energies. The TPA differs from the OPA spectra, however, in the emergence of additional accessible states upon adoption of the base form of a number of derivatives; this, adding additional possibilities for the identification of the form present in vitro through the use of fluorescent spectroscopy.
Abbrv. | Name | R1 | R2 |
---|---|---|---|
a R1 groups for pterin derivatives with extended side chains are shown in Fig. 2. b Structures for derivatives involving biological alterations to the central pterin moiety are shown in Fig. 3. | |||
PT | Pterin | –H | H |
BPT | Biopterin | –(CHOH)2CH3 | H |
CPT | 6-Carboxypterin | –COOH | H |
DPT | 6,7-Dimethylpterin | –CH3 | –CH3 |
FPT | 6-Formylpterin | –CHO | H |
HPT | 6-(Hydroxymethyl)pterin | –CH2OH | H |
MPT | 6-Methylpterin | –CH3 | H |
NPT | Neopterin | –(CHOH)2CH2OH | H |
RPT | Rhamnopterin | –(CHOH)3CH3 | H |
PA | Pteroic acid | H | |
FA | Folic acid | H | |
MFA | 10-Methylfolic acid | H | |
FN | Folinic acid | H | |
MTHF | L-5-Methyltetrahydrofolate | H | |
MTHFG | 5,10-Methenyltetrahydrofolyl-polyglutamate | H |
State | BPT | CPT | DPT | FPT | HPT | MPT | NPT | RPT | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
E | σ TP | E | σ TP | E | σ TP | E | σ TP | E | σ TP | E | σ TP | E | σ TP | E | σ TP | |
1 | 3.89 | 0.007 | 3.80 | 0.000 | 3.99 | 0.002 | 3.46 | 0.000 | 3.85 | 0.000 | 3.87 | 0.001 | 4.00 | 0.014 | 3.82 | 0.009 |
2 | 4.29 | 0.646 | 4.28 | 0.186 | 4.32 | 0.616 | 3.80 | 0.000 | 4.24 | 0.057 | 4.25 | 0.489 | 4.24 | 0.497 | 4.19 | 0.723 |
3 | 4.75 | 0.031 | 4.39 | 0.002 | 4.85 | 0.037 | 4.16 | 0.343 | 4.83 | 0.002 | 4.83 | 0.024 | 4.92 | 0.120 | 4.83 | 0.115 |
4 | 4.95 | 0.099 | 4.72 | 0.613 | 5.00 | 1.070 | 4.38 | 0.002 | 4.96 | 0.002 | 4.95 | 0.024 | 5.00 | 1.150 | 4.96 | 0.190 |
5 | 4.98 | 3.010 | 4.85 | 0.134 | 5.01 | 1.220 | 4.67 | 0.660 | 5.05 | 0.128 | 5.03 | 2.030 | 5.03 | 0.037 | 5.02 | 0.869 |
6 | 5.47 | 0.181 | 4.94 | 0.015 | 5.52 | 0.049 | 4.76 | 0.002 | 5.46 | 0.071 | 5.48 | 0.680 | 5.42 | 10.900 | 5.32 | 4.780 |
7 | 6.06 | 0.307 | 5.41 | 0.050 | 6.13 | 0.060 | 5.22 | 0.027 | 6.11 | 0.099 | 6.14 | 0.060 | 5.50 | 0.663 | 5.50 | 0.122 |
8 | 6.22 | 6.630 | 5.77 | 0.076 | 6.28 | 9.460 | 5.58 | 0.036 | 6.24 | 8.920 | 6.30 | 0.321 | 6.10 | 0.101 | 6.11 | 0.830 |
9 | 6.32 | 4.640 | 6.09 | 0.091 | 6.38 | 0.598 | 5.74 | 0.114 | 6.29 | 0.273 | 6.34 | 4.700 | 6.31 | 4.650 | 6.21 | 3.000 |
10 | 6.42 | 0.282 | 6.21 | 9.490 | 6.43 | 5.140 | 6.07 | 0.659 | 6.44 | 0.723 | 6.46 | 5.990 | 6.35 | 2.740 | 6.25 | 3.370 |
State | BPT | CPT | DPT | FPT | HPT | MPT | NPT | RPT | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
E | σ TP | E | σ TP | E | σ TP | E | σ TP | E | σ TP | E | σ TP | E | σ TP | E | σ TP | |
1 | 3.57 | 0.424 | 3.45 | 0.001 | 3.75 | 0.116 | 3.30 | 0.001 | 3.64 | 0.003 | 3.60 | 0.022 | 3.65 | 1.070 | 3.64 | 0.134 |
2 | 3.61 | 0.859 | 3.73 | 0.268 | 3.79 | 1.330 | 3.61 | 0.327 | 3.73 | 0.107 | 3.69 | 1.340 | 3.68 | 0.157 | 3.66 | 1.280 |
3 | 4.26 | 0.026 | 4.10 | 0.000 | 4.37 | 0.009 | 3.83 | 0.001 | 4.30 | 0.001 | 4.26 | 0.011 | 4.36 | 0.013 | 4.31 | 0.007 |
4 | 4.56 | 0.701 | 4.28 | 0.326 | 4.64 | 0.882 | 4.06 | 0.001 | 4.59 | 0.232 | 4.65 | 0.703 | 4.60 | 0.772 | 4.62 | 0.758 |
5 | 4.76 | 0.071 | 4.31 | 0.029 | 4.85 | 0.026 | 4.28 | 0.110 | 4.78 | 0.003 | 4.83 | 0.532 | 4.82 | 0.062 | 4.81 | 0.055 |
6 | 4.90 | 0.021 | 4.81 | 0.123 | 5.07 | 0.040 | 4.29 | 2.730 | 4.96 | 0.042 | 4.97 | 0.032 | 4.95 | 0.014 | 4.96 | 0.058 |
7 | 5.11 | 4.590 | 5.05 | 10.700 | 5.30 | 4.180 | 4.77 | 0.139 | 5.22 | 9.770 | 5.24 | 4.440 | 5.17 | 4.030 | 5.18 | 5.440 |
8 | 5.71 | 0.566 | 5.28 | 0.016 | 5.63 | 0.848 | 4.91 | 0.001 | 5.61 | 39.600 | 5.67 | 0.078 | 5.78 | 0.200 | 5.74 | 0.110 |
9 | 5.85 | 0.178 | 5.39 | 0.048 | 5.69 | 0.064 | 5.30 | 0.040 | 5.66 | 0.068 | 5.90 | 0.209 | 5.89 | 0.214 | 5.75 | 20.200 |
10 | 5.92 | 0.100 | 5.44 | 0.055 | 5.87 | 0.151 | 5.43 | 0.102 | 5.85 | 0.048 | 5.94 | 0.035 | 5.91 | 1.800 | 5.93 | 0.104 |
With the exception of FPT, which consists of a predominantly HOMO → LUMO+1 transition, the dominant state for each derivative (Tables 2 and 4) involve the same π–π* transition (Fig. 6); the presence of this state represents a transition that, while energetically effected by the tail, remains accessible.
![]() | ||
Fig. 6 Dominant orbital transition describing the two-photon accessible state showing high cross sections across small tail pterin derivatives, as shown for the DPTa structure. |
In contrast, the TPA spectra appears to consist of states which can be characterised in one of three ways: charge transfer states, in which density moves from the aromatic ring of the tail to the pterin moiety; or π–π* states isolated to either the pterin moiety or the tail; representations of these states are shown in Fig. 7.
For the acid form (Table 5) shows that, for both PAa and FAa, the S1 state situated at 3.87 and 3.84 eV, respectively, is significantly more accessible than its OPA counterpart with respect to the rest of their corresponding spectra; this state is, notably, quenched upon adopting the base form (Table 6). Unlike the S1 state, the second TPA accessible region, represented by S5 of each derivative in the acid form remains relatively intact in the base form with an average shift from 4.80 eV to 4.40 eV (noting that the TPA accessible S5 state of the acid form (Table 5) migrates to the S4 position when in the base form (Table 6)).
State | PA | FA | MFA | |||
---|---|---|---|---|---|---|
E | σ TP | E | σ TP | E | σ TP | |
1 | 3.87 | 51.100 | 3.84 | 60.600 | 3.58 | 0.078 |
2 | 3.95 | 0.040 | 3.95 | 0.027 | 4.02 | 0.086 |
3 | 4.22 | 9.930 | 4.22 | 10.200 | 4.20 | 0.977 |
4 | 4.69 | 6.830 | 4.67 | 0.987 | 4.54 | 0.825 |
5 | 4.85 | 41.900 | 4.82 | 55.700 | 4.74 | 23.100 |
6 | 4.89 | 1.090 | 4.86 | 13.900 | 4.81 | 5.770 |
7 | 4.90 | 6.360 | 4.89 | 0.944 | 4.89 | 0.012 |
8 | 4.99 | 0.197 | 4.99 | 0.268 | 4.99 | 2.800 |
9 | 5.09 | 10.000 | 5.08 | 12.900 | 5.09 | 1.680 |
10 | 5.43 | 0.000 | 5.25 | 3.320 | 5.23 | 0.950 |
State | PA | FA | MFA | |||
---|---|---|---|---|---|---|
E | σ TP | E | σ TP | E | σ TP | |
1 | 3.64 | 0.765 | 3.64 | 0.822 | 3.56 | 0.125 |
2 | 3.66 | 0.193 | 3.66 | 0.206 | 3.65 | 3.020 |
3 | 4.32 | 0.005 | 4.32 | 0.006 | 4.22 | 0.237 |
4 | 4.48 | 16.700 | 4.47 | 34.000 | 4.31 | 64.000 |
5 | 4.54 | 6.220 | 4.54 | 8.490 | 4.49 | 3.500 |
6 | 4.69 | 1.390 | 4.59 | 1.880 | 4.57 | 3.860 |
7 | 4.76 | 5.890 | 4.77 | 3.360 | 4.61 | 7.790 |
8 | 4.78 | 0.078 | 4.78 | 0.091 | 4.72 | 0.131 |
9 | 4.92 | 0.027 | 4.92 | 0.036 | 4.87 | 1.200 |
10 | 5.16 | 2.560 | 5.15 | 3.380 | 5.10 | 3.660 |
The S1 state of these derivatives, which show a large cross sectional value for both PAa and FAa (Table 5), is characterised by a charge transfer from the π HOMO, isolated on the pterin moiety, to the π* LUMO on the aromatic ring of the tail, as shown in Fig. 7. The methylation of the bridging nitrogen in MFA (Fig. 2), which causes the structure to fold so that the phenyl ring of the tail is orientated perpendicular to the plane of the pterin moiety, may explain the near zero cross section observed for this derivative through the breaking of any π overlap that would aid in the transfer of electron density. The reduction in cross section as these derivatives move to their base form can be explained in a similar manner, in which the change in electronic structure of the pterin moiety, including the development of a formal negative charge on the oxygen, would reduce the ability of this moiety to accommodate charge from the tail. In contrast, the higher energy state (S5 in the acid form; S4 in the base form) is characterised by a π–π* transition isolated to the aromatic ring located in the tail which explains the observation that, while the cross sections of the states to change from the acid to base form, the state remains accessible across both forms.
State | FNA | MTHF | MTHFG | |||
---|---|---|---|---|---|---|
E | σ TP | E | σ TP | E | σ TP | |
1 | 4.73 | 9.220 | 4.73 | 5.680 | 4.74 | 12.800 |
2 | 4.84 | 0.575 | 4.74 | 6.310 | 4.76 | 3.330 |
3 | 4.88 | 3.020 | 4.82 | 27.200 | 4.91 | 23.000 |
4 | 4.95 | 19.500 | 5.02 | 1.240 | 4.92 | 0.570 |
5 | 5.21 | 2.900 | 5.25 | 4.190 | 5.09 | 4.870 |
6 | 5.24 | 1.730 | 5.26 | 5.340 | 5.17 | 7.310 |
7 | 5.27 | 1.600 | 5.28 | 3.100 | 5.25 | 1.790 |
8 | 5.51 | 0.374 | 5.74 | 0.498 | 5.76 | 22.400 |
9 | 5.60 | 4.390 | 5.86 | 3.130 | 5.82 | 3.310 |
10 | 5.88 | 1.340 | 5.94 | 3.750 | 5.85 | 0.028 |
11 | 5.92 | 0.175 | 6.00 | 6.640 | 5.97 | 0.173 |
12 | 6.01 | 1.030 | 6.02 | 0.012 | 6.05 | 0.159 |
13 | 6.01 | 0.011 | 6.06 | 0.113 | 6.11 | 0.782 |
14 | 6.07 | 0.079 | 6.13 | 1.090 | 6.18 | 16.400 |
15 | 6.12 | 23.200 | 6.16 | 2.370 | 6.27 | 4.120 |
16 | 6.16 | 7.360 | 6.22 | 7.850 | 6.33 | 1.830 |
17 | 6.25 | 10.100 | 6.33 | 2.870 | 6.36 | 0.262 |
18 | 6.29 | 1.010 | 6.36 | 0.084 | 6.41 | 1.180 |
19 | 6.34 | 7.400 | 6.47 | 6.050 | 6.44 | 2.210 |
20 | 6.46 | 5.200 | 6.53 | 4.100 | 6.52 | 9.670 |
21 | 6.46 | 2.300 | 6.57 | 2.280 | 6.53 | 4.410 |
22 | 6.52 | 12.600 | 6.57 | 6.940 | 6.61 | 1.450 |
23 | 6.61 | 0.817 | 6.68 | 18.900 | 6.63 | 1.160 |
24 | 6.68 | 2.290 | 6.71 | 5.680 | 6.73 | 2.400 |
25 | 6.73 | 4.490 | 6.73 | 2.980 | 6.78 | 27.200 |
26 | 6.75 | 2.490 | 6.76 | 41.100 | 6.80 | 4.940 |
27 | 6.78 | 13.200 | 6.81 | 9.400 | 6.82 | 4.260 |
28 | 6.84 | 0.972 | 6.86 | 4.900 | 6.83 | 2.830 |
29 | 6.86 | 1.300 | 6.92 | 1.280 | 6.86 | 0.421 |
30 | 6.92 | 0.757 | 6.95 | 2.460 | 6.90 | 12.000 |
In a similar manner to the pterins involving tails which contain an aromatic moiety, the oxidised derivatives studied here present a bright π–π* state that is shared across the derivatives (Table 7). This state can be found at S4 for FNA (E = 4.95 eV; σTP = 19.5 GM) and S3 for MTHF and MTHFG (E = 4.82 eV; σTP = 27.2 GM and E = 4.91 eV; σTP = 23.0 GM, respectively). However, while this is the only bright state on the TPA spectra of FNA and MTHF that overlaps with the lower band of the OPA spectra, this is not the case with MTHFG. The cyclisation between the tail and the oxidised pterin moiety results in the structure of MTHFG adopting a more planar geometry which, as seen with the acidic form of both PAa and FAa (Table 5), results in additional bright states. The first of these states is S1 (E = 4.74 eV; σTP = 12.8 GM), which is characterised as a π–π* transition from a hole orbital with density on the substituted ring of the oxidised pterin moiety, the 5-membered ring linking it to the tail, and the aromatic ring of the tail itself, to a particle π* orbital isolated on the oxidised pterin moiety. The second of these states is S8 (E = 5.76 eV; σTP = 22.4 GM) which is characterised by a charge transfer from a π hole orbital isolated on the oxidised pterin moiety to a particle π* orbital on the aromatic ring of the tail (Fig. 3; bottom); the charge transfer nature of this transition makes the high cross section associated to it dependent on the planarity of the geometry compared to the twisted geometry of MTHF in an analogous manner as seen when comparing the acid forms of FAa and MFAa (Table 5).
In addition to the TPA accessibility of the low energy shoulder of the OPA spectra (Fig. 10), all three structures studied also show significant accessibility for the high energy OPA peak. Each structure presents a constantly accessible π–π* state isolated to the tail such as that seen in S5 of FAa (Fig. 7); this state remains at ≈6.80 eV across the structures. In comparison, the second accessible state of this band more structurally specific; S22 of FNA (E = 6.52 eV; σTP = 12.6 GM) shows a π–π* isolated to the head in which the particle orbital presents significant Rydberg character, whereas S23 (E = 6.68 eV; σTP = 18.9 GM) in MTHF and S30 (E = 6.90 eV; σTP = 12.0 GM) in MTHFG present charge transfer from tail to head and π–π* character isolated to the head, respectively.
![]() | ||
Fig. 10 One-photon absorption spectra of structures derived from modifications to the pterin moiety. |
Of particular interest in the comparison of the OPA and TPA spectra is the emergence of TPA accessible states in both FNA and MTHFG which lie in the dark region between the OPA spectra (≈6.0 eV; Fig. 10) in the form of a charge transfer state in which density shifts from the pterin moiety to the aromatic ring of the tail; these states are S15 in FNA (E = 6.12 eV; σTP = 23.2 GM) and S14 in MTHFG (E = 6.18 eV; σTP = 16.4 GM). While these states present similar character to that of S3 in MTHF (E = 4.82 eV; σTP = 27.2 GM), these states are not accessible in the OPA spectra.
The accessibility of these two states for MTHFG (S1 & S8) is a particularly promising find as, in comparison to the OPA spectra (Fig. 10) which shows minimal differentiation between MTHFG and MTHF, the TPA spectra does enable the differentiation of these structures. This presents a novel route for investigating the activity of the methylenetetrahydrofolate reductase enzyme (MTHFR)85–87 which catalyses the reduction and decyclisation of MTHFG to MTHF, a cofactor in the conversion of homocysteine to methionine via the methionine synthase enzyme, a process vital for DNA reproduction as part of the cysteine cycle.88,89
Due to the lack of symmetry present throughout the pterin systems, the parity rules which act to limit the accessible states of symmetric systems do not apply to the structures studied here; this has the effect of, theoretically, rendering the entire singlet manifold assessable, with the cross section of a given state determined predominantly by the overlap of the particle and hole orbitals.
Across the pterin structures, TPA accessible states can be isolated into three different groups of π–π* transitions: those where both the hole and particle orbitals are located on the pterin moiety; those where both are orbitals are isolated to the tail; and those involving charge transfer from the tail to the pterin moiety. The later two types of transitions are only present in derivatives containing an aromatic ring in the tail; derivates without an aromatic ring present TPA accessible states that are depicted solely by transitions from various hole orbitals to the same particle orbital (with the exception of FPT, this particle orbital is shown to be the LUMO). The analysis of the TPA spectra, when compared to its OPA counterpart, has shown the potential for targetting specific derivatives despite the minimal qualitative variation evident in the OPA spectra; this is particularly relevant for NPTa, BPTb, and HPTb.
Of particular interest are the third category of states, those involving charge transfer from tail to pterin moiety where the nature of these states means that access to them is heavily dependent on the molecular geometry. This geometry dependence enables photochemical investigation of methylation and substitution events such as the conversion of FA to MFA, or the ring opening of MTHFG to form MTHF; the utilisation of TPA in these situations presents the possibility for structural differentiation that is not observable in the OPA spectra, due to the density of accessible states, potentially lending new insight into these biologically important processes.
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