Abstract: A general and unambiguous approach was developed for structural elucidation of 5-substituted-4-thiopyrimidine ribo- and 2'-deoxy-nucleosides using NMR spectroscopy. Systematic assignment of proton and carbon signals of5-bromo-4-thio-uridine and other nucleosides was firmly established by COSY. The NMR data of various 4-thiopyrimidine nucleosides and deoxyribonucleosides are compared and the key contributing factors are discussed. The approach presented here is applicable to other modified nucleosides and nucleotides, as well as nucleobases.
Keywords: NMR; 1H; 13C; pyrimidine; 5-substituted pyrimidine; 4-thiothymidine; base; nucleoside; DNA; RNA
Nucleic acids (DNA and RNA) are the fundamental biomolecules, playing crucial roles in all forms of life. DNA is the genetic material for almost all living species except some virus while RNA's primary biological functions lie with the transmission (transcription and translation) of the genetic information. Chemically, both DNA and RNA are composed of bases, sugar and phosphate, but the subtle difference between the two types of nucleic acids is that in RNA ribose is used as the sugar block while in DNA deoxyribose is used instead. The moiety containing the base and sugar is termed as ribonucleoside and deoxyribonucleoside respectively, however for convenience the term of nucleoside is generally used for the both types of nucleosides in the literature and in this paper unless it is spefified. . There are only four (normal) deoxynucleosides used for the storage of genetic information, however, DNA bases and deoxynucleosides are susceptible to damage by chemical, physical and biological actions, much more base-modified deoxynucleosides have been documented.2-4 As to RNA, four (normal) ribonucleosides are used for the transcription of the genetic information, the number of other naturally occurring bases are huge due to RNA's wide of biological functions (Ref here). Therefore the large number of modified nucleosides offers an attractive playfield for chemists to prepare and for other scientists to explore their properties.
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Nucleosides can be modified on either the sugar or the base. Both types of modified nucleosides are of biological interest. For instance, sugar-modified nucleosides, such as azidothymidine (AZT), acyclovir (Zovirax) and famciclovir (Famvir) are primarily used as antiviral agents2. The alteration in the sugar moiety is designed to prevent further DNA elongation, thus inhibiting viral production. However, as genetic information is encoded in the bases in DNA and transmitted by the bases in RNA, base-modified nucleosides would be certainly of greater biological significance in various research fields including cancer study and have been subjected to extensive studies (see a recent review).5
Cancer incidence is rising in the modern society and its fundamental cause can be ascribed to DNA damages and subsequent mutations [1, Lindahl, Nature 193]. Any agent or treatment that can lead to DNA damage could be used to reduce and remove cancerous cells. This, in fact, is the principle underlying chemotherapy and radiotherapy. However, often such treatments are often toxic and indiscriminating, thus unsatisfactory. Obviously, better treatments are urgently required. Recently we reported that 4-thiothymidine (2b), an analogue of the naturally occurring nucleoside thymidine (1b), can be readily incorporated into DNA of prolific cells (such as cancerous cells) and activated by UVA light to kill cells [2,3]. To systemically exploit other 4-thionucleosides as possible anti-cancer drugs, we synthesized 5-bromo-4-thio-2'-deoxyuridine (2e) , more recently 5-iodo-4-thio-2-deoxyuridine (2f) and their ribonucleosides (4a-f).
As many of these 5-substituted-4-thionucleosides are synthesized in the first time, a full characterization of these compounds is essential. Among all analytical methods, nuclear magnetic resonance (NMR) spectroscopy is often the primary tool since it can uncover structural details, such as stereochemistry. X-ray crystallography could also accomplish this aim only if a diffraction quality crystalline form of the nucleoside is obtainable.
NMR spectroscopy has been extensively used for studies of naturally occurring nucleoside, however, the data on modified nucleosides in literature are patchy and sometimes contradicting due to the rarity of some of modified nucleosides. There are few papers systematically examining nucleosides by sole use of NMR.11This has prompted us to carry out a systematic NMR studies on nucleosides and base-modified nucleosides. Previously we reported a general and unambiguous approach to determine the structures of purine-modified nucleosides (Raman, MRC-2008). Recently we extended our work to some 4-thiopyrimidine deoxynucleosides. (Zhang and Xu 2011, Molecules) With the availability of its ribonucleosides, we were able to carry out a systematic and comparable NMR study of both types of nucleosides. Here we use 5-bromo-4-thio-pyrimidne ribonucleoside as examples to illustrate a general approach to NMR study of pyrimidine-modified nucleosides. The approach presented here would be applicable for many other modified nucleosides.
RESULTS AND DISCUSSION
Scheme 1: Chemical transformation of 5-substituted nucleosides to its 4-thio-anaologues
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Marked to Standard
The standard nucleosides fall into two types: purine and pyrimidine nucleosides. In our previous paper (Raman-MRC-2008) we have extensively studied on NMR of purine nucleoside. In this one our focus will be on pyrimidine nucleosides, namely thymidine (1) (of DNA) and uridine (3) (of RNA) and their base-modified analogues, although the principles discussed here should be applicable to cytidine, another pyrimidine nucleoside.
NMR peak assignment
The first step in NMR structural elucidation is to assign all the NMR signals (peaks) to their corresponding atoms (e.g. H or C). Proton assignment is always the initial step as it is relatively easy to acquire 1H NMR spectra due to the abundance of NMR-sensitive protons in most organic molecules (including pyrimidine nucleosides). As the sugar moieties are unaltered in 4-thiopyrimidine nucleosides, the assignment of sugar protons of a nucleoside should be applicable to the sugar protons in any other base-modified nucleosides. An unambiguous route to assigning all the sugar protons is exemplified by 4-thio-5-bromuridine as shown below.
Figure 1: 1H-NMR spectrum of 4e (5-bromo-4-thiouridine). Inset: Chemical structure and numberings of 4e.
Although 5-bromo-4-thiouridine is a doubly modified nucleoside, however their modifications are occurring at the 4-and 5-positions of the base and should not affect much of the sugar protons in terms of their NMR properties. By comparison with the reported data for normal nucleosides (REF here, Colin B. Reese Tetrahedron, and MRC-Raman), we can be confident that the chemical shifts of all the sugar protons would be below 6 ppm. The protons of the sugar OHs can be readily identified by the means of D2O exchanges. Thus we can tentatively assign the peaks below 6ppm to their corresponding sugar protons as shown in Figure 1. Our above assignments are further supported by the COSY spectrum as shown Figure 2
Figure 2: The sugar section of H-H COSY spectrum of 4e (5-bromo-4-thiouridine), (Inset: chemical structure and numberings of 4e). Peaks on the diagonal line are the NMR signals of the protons on the sugar. The off-diagonal peaks (cross-peaks) directly correlate the coupled protons. The arrows indicate routes to the coupled partners.
Figure 2 shows the sugar part of the 1H-1H Correlation Spectroscopy (COSY) spectrum of 4e (5-bromo-4-thiouridine). COSY generates a 2D spectrum and commonly used to identify nuclei (such as protons in the case of 1H-1H COSY) that exhibit a scalar (J) coupling. The presence of off-diagonal peaks (cross-peaks) in the spectrum directly correlates the coupled partners (the protons in the case of 1H-1H COSY). It has long been established (ref, C.B. Reese, Tetrahedron) that 1â€²-H has the highest chemical shift value among all sugar protons due to the deshielding effect of electronegative N atom (at the glycosidic position) in the base and O atom (at 4â€²-position) in the sugar, thus the peaks at Î´ 5.68 ppm can be confidently assigned to 1â€²-H. Subsequently, we can start from the assigned 1â€²-H on the diagonal line (indicated by a dotted line in Figure 2) to trace its cross-peak with 2â€²-H signal then following the arrows to identify 2â€²-H (Î´ 4.07) on the diagonal line. From 2'-H signal on the diagonal line, we can easily find its cross-peak with 2'-OH (Î´ 5.48 ). Using the same arrow approach, all other sugar protons can be identified (namely 3â€²-H, 3â€²-OH, 4â€²-H, 5â€²-OH, 5â€²-Ha and 5â€²-Hb). The 1H-1H COSY spectrum also clearly show that the two chemically equivalent 5â€²-Ha and 5â€²-Hb atoms are not completely the same in NMR as evidenced by the presence of two sets of signals for the two 5â€²-H atoms
The above assignment of NMR peaks of 5-bromo-4-thiouridine (4e) is in good agreement with those of the related 5-bromo-urindine (3e) (see Table 1b) and 4-thio-5-bromo-2'-deoxyuridine (2e) (see Table 1a). This offers a further support to our approach to assigning nucleoside sugar protons. The same approach of the sequential assignment has been used for all sugar protons of 5-substituted-4-thiopyrimidne deoxynucleoside (2a-f) and ribonucleoside (4a-f). Their chemical shifts are summarized in Table 1.
Pyrimidine bases have few protons, i.e. 3-H (i.e. N3-H) and 5-H and 6-H. When 5-position is substituted, for instance by a bromo group in the case of 4e, there are only two protons for assignment, namely the imino proton at N3 position (exchangeable) and 6-H proton (non-exchangeable). The exchangeable proton, N3-H, can be readily identified by using D2O exchange. Furthermore due to the strong deshielding effect of N atom (in the form of two amide groups), the imino proton usually appears at lower field (with higher d value). Thus the peak at around 13 ppm can be undoubtedly assigned as the N3-H. It is worth noting that when the oxygen at the 4-position is replaced by sulfur atom, N-3 proton shifts further downfield from 11.8 (such as 5-bromouridine, 3e) to 13.02 (such as 5-bromo-4-uridineas 4e). This also holds true for base-modified deoxynucoesides (see Table 1a). For example, N3-H proton shifts from 11.78 (1e) to 13.08 (2e). However, it is also worth pointing out that the signals of exchangeable protons are often broad and their chemical shifts can vary somewhat depending upon the solvents and conditions used.
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Pyrimidine-modified nucleoside 4e has one non-exchangeable pyrimine proton appearing 8.65 (See Figures 1 and Table 1b), the peak is readily assigned to the proton at 6-position. Other pyrimidine-modified nucleosides (2a-f and 4a-f) have also been examined and their proton chemical shifts are listed in Table 1, which offers the following common features:
a) All the sugar protons appear between Î´ 2.0 to 6.0 ppm; b) 1'-H has highest Î´ value (around 6 ppm) among all sugar protons and is in the form of doublet for ribonucleosides and of double-doublet for deoxyribonucleosides. The rest of the sugar protons always show multiplets; c) 6-H always appears as a singlet and shows the highest Î´ value among all non-exchangeable protons derived from the sugar and base; d) 2'-OH (doublet, only in ribose) and 3'-OH (doublet) and 5'-OH (triplet) protons are exchangeable as well as the imino proton which would have the highest Î´ value in the molecule of nucleoside. These exchangeable protons are distinguishable and easily singled out by D2O exchange.
13C peaks of the sugar and pyrimidine
Figure 3 13C-NMR spectrum of 4e (5-bromo-4-thiouridine).
A 13C NMR spectrum of 4-thio-5-bromo-uridine is shown in Figure 3. The 13C NMR signals are initially assigned. This assignment has been verified by using a Heteronuclear Multiple-Quantum Correlation (HMQC) technique. HMQC is used to correlate directly bonded nuclei (in this case carbon-proton nuclei) and offer structural information on the bonded atoms. This approach has the same principle as that employed in the assignment of 1H signals by COSY. Since each of the sugar carbons has at least one proton attached, the 13C peaks, for example of 4e, can be readily assigned to their carbon atoms from the known protons by HMQC as demonstrated in Figure 4 below.
Assignment of sugar carbons
Figure 4: H-C COSY spectra of 4e: the top right shows the sugar part and the low left covers the base part.
In the previous section, we have unambiguously assigned 1H signals to their corresponding protons in the sugar. Now we can easily trace from 1H protons to their coupled 13C partners. For instance, the 1H signal located at Î´ 5.67ppm has been confirmed to be the 1'-H of 4e (see Figure 1 and Table 1b). We can find a cross-peak from which we can trace to its coupled 13C partner, i.e. 1'-C (see the dashing line in Figure 4). In a similar vein, we can allocate 2'-C, 3'-C, 4'-C and 5'-C. It is also reassuring that there is no cross-peak for the sugar OHs as the protons in the OH groups are not directly linked to any carbon.
Assignment of pyrimidine carbons
The pyrimidine has two types of carbons, ones with proton attached (e.g. 6-C in 4e) and the others without (e.g. 2-C, 4-C and 5-C in 4e). The former can be readily identified by using the same NMR technique of HMQC. Taking 4e again as an example, as the peak of singlet (Î´ 8.65ppm) has been previously assigned as 6-H (cf. Figure 1 and Table 1b), the 6-H peak can be traced in the HMQC spectrum (Figure 4) to identify a cross-peak leading (via a broken line) to its coupled carbon (Î´ 137.37 ppm).
Assignment of the remaining pyrimidine carbons (i.e. 2-C, 4-C and 5-C) is more challenging as these carbon atoms do not bear any protons, thus HMQC is no use in this instance. However, chemical shifts of any atoms (13C in this case) are influenced by their surrounding chemical environments. 5-bromo-4-thiouridine (4e) is doubly modified nucleoside at C-4 and C-5 positions. Comparison with its related compounds should indicate how different modifications affect chemical shifts and would also offer a clear clue to the assignment of these three 13C NMR peaks.
First we prepared 5-fluoro-4-thiouridine (4c) and examined its 13C spectrum. Its pyrimidine 13C peaks is tentatively assigned and summarized in Table 2. Fluorine is an NMR sensitive atom and can couple with 13C signals through its bonding with carbon atoms. The 13C signal at Î´ 151.05 has the largest coupling constant, so this carbon must be the carbon directed bonded with fluorine atom and can be assigned as 5-C. As 6-C has a proton attached with it, the 13C signal at Î´ 127.37 can be readily assigned as 6-C by using 1H-13C COSY technique as discussed above. The other coupled peak at Î´ 127.37 with a lower coupling constant (J=30Hz) can be confidently assigned as 4-C. The 13C signal at Î´146.85 and without any coupling with fluorine will certainly be the remaining pyrimidine carbon atom, namely 2-C.
Table 2 Comparison of 13C NMR Chemical shifts of carbons on bases
Chemical structure of 5-bromo-4-thiouridine (4e) is very similar to that of 5-fluoro-4-thiouridine. The only difference lies with the substituent at 5-position, thus this difference would be reflected in the chemical shifts of 5-C, not much of other carbon atoms. Therefore we can tentatively assign 2-C, 4-C and 5-C of 4e (see Table 2). This tentative assignment is well supported with the data from its deoxy analogue, namely 5-bromo-4-thio-2'deoxynucleoside (2e).
Using the above-described approach all 13C signals for a number of pyrimidine-modified nucleosides have been unambiguously assigned and are listed in Table 3, from which the following conclusions can be drawn: a) all of the sugar carbons have d values lower than 100 ppm. 12-C has higher value than the rest sugar carbons. 22-C (in deoxynucleoside) has the lowest d value and its signals are located around 40 ppm, thus often buried within the signals of DMSO-d6 when used as the solvent; b) all of the pyrimidine carbons have d values higher than 90 ppm and higher than the sugar carbons except in 5-iodo analogues which is due to the heavy atom effect (discussed below). The carbon at 4-position has highest Î´ value among all the carbons. The sulfur atom at 4-position makes the Î´ value of 4-C ever higher. In all the cases, 4-C of 4-thiournidines has a higher Î´ value than those of 4-oxy-uridine analogues. The following order: C-4> C2> C-6> C5 have been noted except in the case of fluorine-substituted nucleosides.
Comparison between ribo and deoxyribo: Tables 1 and 2 list 1H and 13C chemical shifts of base-modified deoxy- and ribo-nucleosides. There is little difference in the chemical shifts of the bases between these two types of nucleosides, however, some interesting differences was noted in chemical shifts of the sugar part. As expected, the major difference is at the 2-position. 2'-H and 2'-C in all of the dexoynucleosides appear in higher fields (i.e with lower d values) than those in the ribonucleosides. The orders of chemical shifts for the sugar protons are the same for both types of nucleosides, namely: 1 -H>3'-H> 4'-H >5'-H since the effects on the sugar protons (except 2'-H) is minimum. However, the effects on sugar carbons (besides 2-C) are unexpectedly different. For the deoxynucleosides, the order of the chemical shift is 4'-C> 1'-C > 3'-C >5'-C while in the ribo it is 1'-C> 4'-C> 3'-C >5'-C. The increased d value of 1 -C in the ribo can be ascribed to the strong negativity of 2 -oxygen atom. It is interesting to note that the effects of the presence of 2 -oxygen atom in the ribo are substantially different on the two neighboring carbons (1 -C and 3 -C) (see Table 5). The influence on 1'-C is noticeably high while the influence 3'-C is minimum and at the scale similar to the far located 5'-C. This indicates the orientation of 2'-OH points towards to 1'-C and carbon away from 3'-C to avoid the potential electron repulsion from its 3'-OH. The NMR differences also suggest 5-substituted-4-thiouuridines adopt the 3'-C endo form as other ribonucleosides (ref here).
Figure 5: The presence of 2'-oxygen atom in ribonucleosides has a different effects on its neighbouring 1'-C and 3'-C. The differences are calculated from the chemical shifts between the ribo and deoxynucleisides containing 5-bromo-4-thiopyrimidine (4e-2e) and 5-iodo-4-thiopyrimidine(4f-2f).
Modification at 4-position The replacement of the oxygen at 4-psotion the sulphur atom give rise to a thiocarbonyl group which is evident from the shift of N3-H and thiocarbonyl carbon signals in the 1H and 13C-NMR spectra respectively. Exchangeable signals in the Î´12.68-13.10 ppm range in the low-field part of 1H-NMR spectra, attributable to the N-H protons, support the structures of molecules 4. The appearance of only one carbon signal in the Î´C 185.30-190.70 ppm region (characteristic for a thiocarbonyl group), confirm the presence of the thiocarbonyl moieties in compounds 4. It is interesting to note that the 1H chemical shifts of the imino proton (NH) in all the thionucleosides 4 are substantially higher (at around 13 ppm) than those of the parent nucleosides 3 that resonate at Î´ 11.2-11.83 ppm (Table 1). This difference offers a valuable NMR window to detect the imino proton of thionucleosides, as in general there are no signals from normal nucleosides appearing at such a low field. In addition these NH-proton signals are exchangeable and readily identifiable by D2O exchange experiments. Therefore these would also be a good marker in NMR studies of 4-thionucleosides and their corresponding DNAs and RNA.
Modification at 5-position: The presence of a substituent at the 5-position affects chemical shifts of the bases, in particular the bonded carbon (i.e. 5-C) (see Tables 3a and 3b). As expected, fluorine atom has the strongest effect on the 5-C signals. Figure 6 plots 13C chemical shifts of 5-C against with the electronegativity of the 5-substitutents. The greater the electronegativity of atom or group, the lower the electron density around C-X and the further downfield the chemical shift. In all cases, when the hydrogen atom at 5-position is replaced by fluorine atom, the 5-C has the highest values, shifting from around 100 ppm to 140 ppm.
This is due to the extremely high electronegativity of fluorine. F, Cl, Br and I are each more electronegative than the H atom, it would be anticipated that, when the H atom of 5-C are replaced by these substituents, the 13C resonance should be progressively shifted to much lower field. This prediction hold true for F and Cl group, not for Br and I group. The 13C resonance of 5-C for 5-bromonucleosides (1e, 2e, 3e and 4e) and 5-iodo-nucleosides (1f, 2f, 3f and 4f) is shifted to higher field relative to that of the non-modified uracil nucleosides. Clearly the electron-withdrawing effect alone is not enough to explain those. This unusual effect could be explained by the "heavy atom effect" that is when a carbon atom is attached by heavy halogen atom (such as Br or I), the diamagnetic interactions arising from numerous electrons of bromo or iodine atom increase the shielding effect of the substituted carbon atom, so that the NMR resonances shift upï¬eld.
Confusing data in the literature
Potential usefulness of the paper
5-Substituted-4-thio-2'-deoxyuridines can be effectively prepared from its parent nucleosides and have distinctive NMR and UV properties that can be used for easy monitoring and exploited as potential UVA-induced anticancer agents.
NMR instruments: 300 MHz from JOEL (JNM-LA 300, FT NMR) and 400MHz from JOEL (JNM-EX 400, FT NMR). NOE spectra were acquired with a 90Â° pulse, relaxation delay of 3s, a relatively short interpulse delay of 13 micro seconds with 64 scans. The NOESY spectra (DMSO-d6) were recorded using a 512Ã-512 data matrix and 256 time increments of each 16 scans (mixing time 0.5 s, pulse: 13 microseconds @ 90 deg) with a relaxation delay of 1.5sec. The COSY spectra (DMSO-d6) were obtained in the magnitude mode with 512 points in the F2 dimension and 256 increments in the F1 dimension. Each increment FID was obtained with 4 scans with a relaxation delay of 1.5 s.
Materials and methods: All chemicals and solvents, unless stated otherwise, were from either Aldrich or Sigma. All chemicals and solvents were used directly without further purification. Nucleosides on TLC were identified using p-anisaldehyde/ethanol/H2SO4 (5:90:5) solution that converted the nucleosides into black spots on heating. (_
Preparation of 4-thio-5-substituted pyrimidine- nucleosides:
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