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Abstraction: A general and unambiguous attack was developed for structural elucidation of 5-substituted-4-thiopyrimidine ribo- and 2′-deoxy-nucleosides utilizing NMR spectrometry. Systematic assignment of proton and C signals of5-bromo-4-thio-uridine and other nucleosides was steadfastly established by COSY. The NMR informations of assorted 4-thiopyrimidine nucleosides and deoxyribonucleosides are compared and the cardinal contributing factors are discussed. The attack presented here is applicable to other modified nucleosides and bases, every bit good as nucleobases.

Keywords: NMR ; 1H ; 13C ; pyrimidine ; 5-substituted pyrimidine ; 4-thiothymidine ; base ; nucleoside ; DNA ; RNA

Introduction

Nucleic acids ( DNA and RNA ) are the cardinal biomolecules, playing important functions in all signifiers of life. Deoxyribonucleic acid is the familial stuff for about all life species except some virus while RNA ‘s primary biological maps lie with the transmittal ( written text and interlingual rendition ) of the familial information. Chemically, both DNA and RNA are composed of bases, sugar and phosphate, but the elusive 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 alternatively. The mediety incorporating the base and sugar is termed as ribonucleoside and deoxyribonucleoside severally, nevertheless for convenience the term of nucleoside is by and large used for the both types of nucleosides in the literature and in this paper unless it is spefified. . There are merely four ( normal ) deoxynucleosides used for the storage of familial information, nevertheless, 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 written text of the familial information, the figure of other of course happening bases are immense due to RNA ‘s broad of biological maps ( Ref here ) . Therefore the big figure of modified nucleosides offers an attractive playfield for chemists to fix and for other scientists to research their belongingss.

Nucleosides can be modified on either the sugar or the base. Both types of modified nucleosides are of biological involvement. For case, sugar-modified nucleosides, such as azidothymidine ( AZT ) , acyclovir ( Zovirax ) and famciclovir ( Famvir ) are chiefly used as antiviral agents2. The change in the sugar mediety is designed to forestall farther DNA elongation, therefore suppressing viral production. However, as familial information is encoded in the bases in DNA and transmitted by the bases in RNA, base-modified nucleosides would be surely of greater biological significance in assorted research Fieldss including malignant neoplastic disease survey and have been subjected to extended surveies ( see a recent reappraisal ) .5

Cancer incidence is lifting in the modern society and its cardinal cause can be ascribed to DNA amendss and subsequent mutants [ 1, Lindahl, Nature 193 ] . Any agent or intervention that can take to DNA harm could be used to cut down and take cancerous cells. This, in fact, is the rule implicit in chemotherapy and radiation therapy. However, frequently such interventions are frequently toxic and undiscriminating, therefore unsatisfactory. Obviously, better interventions are desperately required. Recently we reported that 4-thiothymidine ( 2b ) , an parallel of the of course happening nucleoside thymidine ( 1b ) , can be readily incorporated into DNA of fecund cells ( such as cancerous cells ) and activated by UVA visible radiation to kill cells [ 2,3 ] . To systemically work other 4-thionucleosides as possible anti-cancer drugs, we synthesized 5-bromo-4-thio-2′-deoxyuridine ( 2e ) [ 4 ] , more late 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 clip, a full word picture of these compounds is indispensable. Among all analytical methods, atomic magnetic resonance ( NMR ) spectrometry is frequently the primary tool since it can bring out structural inside informations, such as stereochemistry. X-ray crystallography could besides carry through this purpose merely if a diffraction quality crystalline signifier of the nucleoside is gettable.

NMR spectrometry has been extensively used for surveies of of course happening nucleoside, nevertheless, the informations on modified nucleosides in literature are patchy and sometimes beliing due to the rareness of some of modified nucleosides. There are few documents consistently analyzing nucleosides by exclusive usage of NMR.11This has prompted us to transport out a systematic NMR surveies on nucleosides and base-modified nucleosides. Previously we reported a general and unambiguous attack to find the constructions of purine-modified nucleosides ( Raman, MRC-2008 ) . Recently we extended our work to some 4-thiopyrimidine deoxynucleosides. ( Zhang and Xu 2011, Molecules ) With the handiness of its ribonucleosides, we were able to transport out a systematic and comparable NMR survey of both types of nucleosides. Here we use 5-bromo-4-thio-pyrimidne ribonucleoside as illustrations to exemplify a general attack to NMR survey of pyrimidine-modified nucleosides. The attack presented here would be applicable for many other modified nucleosides.

RESULTS AND DISCUSSION

Scheme 1: Chemical transmutation of 5-substituted nucleosides to its 4-thio-anaologues

A fable to the strategy here: ? ? ? ? ? ?

The criterion nucleosides fall into two types: purine and pyrimidine nucleosides. In our old paper ( Raman-MRC-2008 ) we have extensively studied on NMR of purine nucleoside. In this one our focal point will be on pyrimidine nucleosides, viz. thymidine ( 1 ) ( of DNA ) and uridine ( 3 ) ( of RNA ) and their base-modified parallels, although the rules discussed here should be applicable to cytidine, another pyrimidine nucleoside.

NMR peak assignment

Sugar protons

The first measure in NMR structural elucidation is to delegate all the NMR signals ( extremums ) to their corresponding atoms ( e.g. H or C ) . Proton assignment is ever the initial measure as it is comparatively easy to get 1H NMR spectra due to the copiousness of NMR-sensitive protons in most organic molecules ( including pyrimidine nucleosides ) . As the sugar medieties are unchanged 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 path to delegating 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 construction and enumerations of 4e.

Although 5-bromo-4-thiouridine is a double modified nucleoside, nevertheless their alterations are happening at the 4-and 5-positions of the base and should non impact much of the sugar protons in footings of their NMR belongingss. By comparing with the reported informations for normal nucleosides ( REF here, Colin B. Reese Tetrahedron, and MRC-Raman ) , we can be confident that the chemical displacements of all the sugar protons would be below 6 ppm. The protons of the sugar OHs can be readily identified by the agencies of D2O exchanges. Thus we can tentatively delegate the extremums below 6ppm to their corresponding sugar protons as shown in Figure 1. Our above assignments are farther supported by the COSY spectrum as shown Figure 2

Figure 2: The sugar subdivision of H-H COSY spectrum of 4e ( 5-bromo-4-thiouridine ) , ( Inset: chemical construction and enumerations of 4e ) . Top out on the diagonal line are the NMR signals of the protons on the sugar. The off-diagonal extremums ( cross-peaks ) straight correlate the conjugate protons. The arrows indicate paths to the conjugate spouses.

Figure 2 shows the sugar portion of the 1H-1H Correlation Spectroscopy ( COSY ) spectrum of 4e ( 5-bromo-4-thiouridine ) . COSY generates a 2D spectrum and normally used to place nuclei ( such as protons in the instance of 1H-1H COSY ) that exhibit a scalar ( J ) yoke. The presence of off-diagonal extremums ( cross-peaks ) in the spectrum straight correlates the conjugate spouses ( the protons in the instance of 1H-1H COSY ) . It has long been established ( ref, C.B. Reese, Tetrahedron ) that 1aˆ?-H has the highest chemical displacement value among all sugar protons due to the deshielding consequence of negatively charged N atom ( at the glycosidic place ) in the base and O atom ( at 4aˆ?-position ) in the sugar, therefore the extremums at I? 5.68 ppm can be confidently assigned to 1aˆ?-H. Subsequently, we can get down from the assigned 1aˆ?-H on the diagonal line ( indicated by a flecked line in Figure 2 ) to follow its cross-peak with 2aˆ?-H signal so following the pointers to place 2aˆ?-H ( I? 4.07 ) on the diagonal line. From 2′-H signal on the diagonal line, we can easy happen its cross-peak with 2′-OH ( I? 5.48 ) . Using the same pointer attack, all other sugar protons can be identified ( viz. 3aˆ?-H, 3aˆ?-OH, 4aˆ?-H, 5aˆ?-OH, 5aˆ?-Ha and 5aˆ?-Hb ) . The 1H-1H COSY spectrum besides clearly show that the two chemically tantamount 5aˆ?-Ha and 5aˆ?-Hb atoms are non wholly the same in NMR as evidenced by the presence of two sets of signals for the two 5aˆ?-H atoms

The above assignment of NMR extremums of 5-bromo-4-thiouridine ( 4e ) is in good understanding 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 farther support to our attack to delegating nucleoside sugar protons. The same attack of the consecutive assignment has been used for all sugar protons of 5-substituted-4-thiopyrimidne deoxynucleoside ( 2a-f ) and ribonucleoside ( 4a-f ) . Their chemical displacements are summarized in Table 1.

Pyrimidine protons

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 case by a bromo group in the instance of 4e, there are merely two protons for assignment, viz. the imino proton at N3 place ( exchangeable ) and 6-H proton ( non-exchangeable ) . The exchangeable proton, N3-H, can be readily identified by utilizing D2O exchange. Furthermore due to the strong deshielding consequence of N atom ( in the signifier of two amide groups ) , the imino proton normally appears at lower field ( with higher vitamin D value ) . Thus the extremum at around 13 ppm can be doubtless assigned as the N3-H. It is deserving observing that when the O at the 4-position is replaced by sulfur atom, N-3 proton displacements farther downfield from 11.8 ( such as 5-bromouridine, 3e ) to 13.02 ( such as 5-bromo-4-uridineas 4e ) . This besides holds true for base-modified deoxynucoesides ( see Table 1a ) . For illustration, N3-H proton displacements from 11.78 ( 1e ) to 13.08 ( 2e ) . However, it is besides deserving indicating out that the signals of exchangeable protons are frequently wide and their chemical displacements can change slightly depending upon the dissolvers and conditions used.

Pyrimidine-modified nucleoside 4e has one non-exchangeable pyrimine proton looking 8.65 ( See Figures 1 and Table 1b ) , the extremum is readily assigned to the proton at 6-position. Other pyrimidine-modified nucleosides ( 2a-f and 4a-f ) have besides been examined and their proton chemical displacements are listed in Table 1, which offers the undermentioned common characteristics:

a ) All the sugar protons appear between I? 2.0 to 6.0 ppm ; B ) 1′-H has highest I? value ( around 6 ppm ) among all sugar protons and is in the signifier of doublet for ribonucleosides and of double-doublet for deoxyribonucleosides. The remainder of the sugar protons ever show multiplets ; degree Celsiuss ) 6-H ever appears as a vest and shows the highest I? value among all non-exchangeable protons derived from the sugar and base ; vitamin D ) 2′-OH ( doublet, merely in ribose ) and 3′-OH ( doublet ) and 5′-OH ( three ) protons are exchangeable every bit good as the imino proton which would hold the highest I? value in the molecule of nucleoside. These exchangeable protons are distinguishable and easy singled out by D2O exchange.

13C extremums 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 ab initio assigned. This assignment has been verified by utilizing a Heteronuclear Multiple-Quantum Correlation ( HMQC ) technique. HMQC is used to correlate straight bonded karyon ( in this instance carbon-proton karyon ) and offer structural information on the bonded atoms. This attack has the same rule as that employed in the assignment of 1H signals by COSY. Since each of the sugar Cs has at least one proton attached, the 13C extremums, for illustration of 4e, can be readily assigned to their C atoms from the known protons by HMQC as demonstrated in Figure 4 below.

Assignment of sugar Cs

Figure 4: H-C COSY spectra of 4e: the top right shows the sugar portion and the low left covers the basal portion.

In the old subdivision, we have unequivocally assigned 1H signals to their corresponding protons in the sugar. Now we can easy follow from 1H protons to their coupled 13C spouses. For case, the 1H signal located at I? 5.67ppm has been confirmed to be the 1′-H of 4e ( see Figure 1 and Table 1b ) . We can happen a cross-peak from which we can follow to its conjugate 13C spouse, i.e. 1′-C ( see the darting line in Figure 4 ) . In a similar vena, we can apportion 2′-C, 3′-C, 4′-C and 5′-C. It is besides reassuring that there is no cross-peak for the sugar OHs as the protons in the OH groups are non straight linked to any C.

Assignment of pyrimidine Cs

The pyrimidine has two types of Cs, 1s 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 utilizing the same NMR technique of HMQC. Taking 4e once more as an illustration, as the extremum of vest ( I? 8.65ppm ) has been antecedently assigned as 6-H ( cf. Figure 1 and Table 1b ) , the 6-H extremum can be traced in the HMQC spectrum ( Figure 4 ) to place a cross-peak leading ( via a broken line ) to its coupled C ( I? 137.37 ppm ) .

Assignment of the staying pyrimidine Cs ( i.e. 2-C, 4-C and 5-C ) is more ambitious as these C atoms do non bear any protons, therefore HMQC is no usage in this case. However, chemical displacements of any atoms ( 13C in this instance ) are influenced by their encompassing chemical environments. 5-bromo-4-thiouridine ( 4e ) is double modified nucleoside at C-4 and C-5 places. Comparison with its related compounds should bespeak how different alterations affect chemical displacements and would besides offer a clear hint to the assignment of these three 13C NMR extremums.

First we prepared 5-fluoro-4-thiouridine ( 4c ) and examined its 13C spectrum. Its pyrimidine 13C extremums is tentatively assigned and summarized in Table 2. Fluorine is an NMR sensitive atom and can match with 13C signals through its bonding with C atoms. The 13C signal at I? 151.05 has the largest yoke invariable, so this C must be the C directed bonded with fluorine atom and can be assigned every bit 5-C. As 6-C has a proton attached with it, the 13C signal at I? 127.37 can be readily assigned as 6-C by utilizing 1H-13C COSY technique as discussed above. The other conjugate extremum at I? 127.37 with a lower yoke changeless ( J=30Hz ) can be confidently assigned as 4-C. The 13C signal at I?146.85 and without any matching with F will surely be the staying pyrimidine C atom, viz. 2-C.

Table 2 Comparison of 13C NMR Chemical displacements of Cs on bases

2-C

4-C

5-C

6-C

S4-FU ( 4c )

146.85

180.37

2J=30 Hz

151.05

1J=217Hz

127.37

2J=41Hz

S4-BrU ( 4e )

147.37

186.45

106.70

137.37

S4-BrdU ( 2e )

147.19

186.39

106.67

137.29

Chemical construction of 5-bromo-4-thiouridine ( 4e ) is really similar to that of 5-fluoro-4-thiouridine. The lone difference lies with the substituent at 5-position, therefore this difference would be reflected in the chemical displacements of 5-C, non much of other C atoms. Therefore we can tentatively delegate 2-C, 4-C and 5-C of 4e ( see Table 2 ) . This probationary assignment is good supported with the informations from its deoxy parallel, viz. 5-bromo-4-thio-2’deoxynucleoside ( 2e ) .

Using the above-described attack all 13C signals for a figure of pyrimidine-modified nucleosides have been unequivocally assigned and are listed in Table 3, from which the following decisions can be drawn: a ) all of the sugar Cs have d values lower than 100 ppm. 12-C has higher value than the remainder sugar Cs. 22-C ( in deoxynucleoside ) has the lowest vitamin D value and its signals are located around 40 ppm, therefore frequently buried within the signals of DMSO-d6 when used as the dissolver ; b ) all of the pyrimidine Cs have d values higher than 90 ppm and higher than the sugar Cs except in 5-iodo parallels which is due to the heavy atom consequence ( discussed below ) . The C at 4-position has highest I? value among all the Cs. The sulfur atom at 4-position makes the I? value of 4-C of all time higher. In all the instances, 4-C of 4-thiournidines has a higher I? value than those of 4-oxy-uridine parallels. The undermentioned order: C-4 & gt ; C2 & gt ; C-6 & gt ; C5 have been noted except in the instance of fluorine-substituted nucleosides.

Further treatment

Comparison between ribo and deoxyribo: Tables 1 and 2 list 1H and 13C chemical displacements of base-modified deoxy- and ribo-nucleosides. There is small difference in the chemical displacements of the bases between these two types of nucleosides, nevertheless, some interesting differences was noted in chemical displacements of the sugar portion. As expected, the major difference is at the 2-position. 2′-H and 2′-C in all of the dexoynucleosides appear in higher Fieldss ( i.e with lower vitamin D values ) than those in the ribonucleosides. The orders of chemical displacements for the sugar protons are the same for both types of nucleosides, viz. : 1 -H & gt ; 3′-H & gt ; 4′-H & gt ; 5′-H since the effects on the sugar protons ( except 2′-H ) is minimal. However, the effects on sugar Cs ( besides 2-C ) are out of the blue different. For the deoxynucleosides, the order of the chemical displacement is 4′-C & gt ; 1′-C & gt ; 3′-C & gt ; 5′-C while in the ribo it is 1′-C & gt ; 4′-C & gt ; 3′-C & gt ; 5′-C. The increased vitamin D value of 1 -C in the ribo can be ascribed to the strong negativeness of 2 -oxygen atom. It is interesting to observe that the effects of the presence of 2 -oxygen atom in the ribo are well different on the two adjacent Cs ( 1 -C and 3 -C ) ( see Table 5 ) . The influence on 1′-C is perceptibly high while the influence 3′-C is minimal and at the graduated table similar to the far located 5′-C. This indicates the orientation of 2′-OH points towards to 1′-C and carbon off from 3′-C to avoid the possible negatron repulsive force from its 3′-OH. The NMR differences besides suggest 5-substituted-4-thiouuridines follow the 3′-C endo signifier 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 displacements between the ribo and deoxynucleisides incorporating 5-bromo-4-thiopyrimidine ( 4e-2e ) and 5-iodo-4-thiopyrimidine ( 4f-2f ) .

Alteration at 4-position The replacing of the O at 4-psotion the sulfur atom give rise to a thiocarbonyl group which is apparent from the displacement of N3-H and thiocarbonyl C signals in the 1H and 13C-NMR spectra severally. Exchangeable signals in the I?12.68-13.10 ppm scope in the low-field portion of 1H-NMR spectra, attributable to the N-H protons, back up the constructions of molecules 4. The visual aspect of merely one C signal in the I?C 185.30-190.70 ppm part ( characteristic for a thiocarbonyl group ) , confirm the presence of the thiocarbonyl medieties in compounds 4. It is interesting to observe that the 1H chemical displacements of the imino proton ( NH ) in all the thionucleosides 4 are well higher ( at around 13 ppm ) than those of the parent nucleosides 3 that resonate at I? 11.2-11.83 ppm ( Table 1 ) . This difference offers a valuable NMR window to observe the imino proton of thionucleosides, as in general there are no signals from normal nucleosides looking at such a low field. In add-on these NH-proton signals are exchangeable and readily identifiable by D2O exchange experiments. Therefore these would besides be a good marker in NMR surveies of 4-thionucleosides and their corresponding DNAs and RNA.

Alteration at 5-position: The presence of a substituent at the 5-position affects chemical displacements of the bases, in peculiar the bonded C ( i.e. 5-C ) ( see Tables 3a and 3b ) . As expected, fluorine atom has the strongest consequence on the 5-C signals. Figure 6 secret plans 13C chemical displacements of 5-C against with the electronegativity of the 5-substitutents. The greater the electronegativity of atom or group, the lower the negatron denseness around C-X and the farther downfield the chemical displacement. In all instances, when the H atom at 5-position is replaced by fluorine atom, the 5-C has the highest values, switching from around 100 ppm to 140 ppm.

This is due to the highly high electronegativity of F. F, Cl, Br and I are each more negatively charged 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 increasingly shifted to much lower field. This anticipation keep true for F and Cl group, non 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 U nucleosides. Clearly the electron-withdrawing consequence entirely is non plenty to explicate those. This unusual consequence could be explained by the “ heavy atom consequence ” that is when a C atom is attached by heavy halogen atom ( such as Br or I ) , the diamagnetic interactions originating from legion negatrons of bromo or I atom increase the screening consequence of the substituted C atom, so that the NMR resonances shift upi¬?eld.

Confusing informations in the literature

Potential utility of the paper

Decision

5-Substituted-4-thio-2′-deoxyuridines can be efficaciously prepared from its parent nucleosides and have typical NMR and UV belongingss that can be used for easy monitoring and exploited as possible UVA-induced anticancer agents.

EXPERIMENTAL

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 90A° pulsation, relaxation hold of 3s, a comparatively short interpulse hold of 13 micro seconds with 64 scans. The NOESY spectra ( DMSO-d6 ) were recorded utilizing a 512A-512 information matrix and 256 clip increases of each 16 scans ( blending clip 0.5 s, pulsation: 13 microseconds @ 90 deg ) with a relaxation hold of 1.5sec. The COSY spectra ( DMSO-d6 ) were obtained in the magnitude manner with 512 points in the F2 dimension and 256 increases in the F1 dimension. Each increase FID was obtained with 4 scans with a relaxation hold of 1.5 s.

Materials and methods: All chemicals and dissolvers, unless stated otherwise, were from either Aldrich or Sigma. All chemicals and dissolvers were used straight without farther purification. Nucleosides on TLC were identified utilizing p-anisaldehyde/ethanol/H2SO4 ( 5:90:5 ) solution that converted the nucleosides into black musca volitanss on warming. ( _

Preparation of 4-thio-5-substituted pyrimidine- nucleosides:

Recognitions

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