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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nucleosides Nucleotides Nucleic Acids. Author manuscript; available in PMC 2010 May 1.
Published in final edited form as:
Nucleosides Nucleotides Nucleic Acids. 2009 May; 28(5): 352–378.
doi:  10.1080/15257770903044523
PMCID: PMC2829739
NIHMSID: NIHMS158371

Thiophosphate Analogs of c-di-GMP: Impact on Polymorphism

Abstract

Seven phosphorothioate analogs of c-di-GMP (all diastereomers of mono-, di-, and trithiophosphates) were prepared to assess the impact of the thioate substitutions on c-di-GMP polymorphism using 1D 1H and 31P NMR, along with 2D NOESY and DOSY, for both the Na+ and K+ salts. The K+ salts display more extensive higher order complex formation than the Na+ salts, resulting primarily in octamolecular complexes with K+, but tetramolecular complexes with Na+. Further, the presence of one or two [SP] sulfurs specifically stabilizes anti complexes and/or destabilizes syn complexes, while the presence of two [SP] sulfurs promotes extensive aggregation.

Keywords: c-di-GMP, thiophosphates, polymorphism, guanine quartets

INTRODUCTION

The bacterial signaling molecule, cyclic diguanosine monophosphate (c-di-GMP, Figure 1), is increasingly recognized as having widespread consequences for human health through its multiple roles.14 Not only is c-di-GMP a major factor in the activation of bacterial biofilm formation and repression of motility, it also helps to regulate virulence. Further, although c-di-GMP is not a signaling molecule in Eukarya, it has been shown to be an immunostimulatory agent that can trigger the innate immune response in mice.5,6 Yet the mechanisms for how c-di-GMP functions remain unclear. Studies using a series of analogs of c-di-GMP7 should facilitate elucidation of its modes of action, and would also allow evaluation of their potential use in the design of new therapeutic agents.

We have recently reported a new synthetic procedure for c-di-GMP,8 as well as a description of its concentration and metal dependent polymorphism.9 In addition to a self-intercalated bimolecular structure, the molecule can adopt four different but related higher order guanine quartet structures, all in slow equilibrium. We also demonstrated that the analog in which one guanine residue is replaced with hypoxanthine (c-GMP-IMP) does not adopt these higher order complexes, presumably because the absence of even one guanine amino group precludes quartet formation.9

We now present results for a new family of analogs of c-di-GMP: a series of seven phosphorothioate derivatives that includes all diastereomers of mono-, di-, and trithiophosphates. The [RP] and [SP] monothiophosphates of c-di-GMP had been synthesized some years ago by a triester method as part of a study of the regulatory system of bacterial cellulose synthesis.10 Both diastereomers proved to be effective activators of cellulose synthase,10 while showing resistance to hydrolysis by a specific phosphodiesterase. A more recent synthesis of these monothiophosphates of c-di-GMP has been reported that used an initial amidite coupling, followed by a triester cyclization, without separation of the diastereomers.11 In view of these promising results, all of the phosphorothioates described here might well show significant activity and enhanced resistance to hydrolysis in a wide range of bacterial pathways that use c-di-GMP for signaling, with some diastereomers likely more effective than others. Because such phosphorothioates are attractive candidates, synthetic methods for making them are needed, and an understanding of their behavior in solution is essential.

RESULTS

Synthesis

The syntheses of all diastereomers of the mono-, di-, and trithiophosphate analogs of c-di-GMP, 5–7, were carried out as shown in Scheme 1. This approach is based on our earlier synthesis of unmodified c-di-GMP.8,9 The first step is coupling of the standard commercially available guanosine phosphoramidite, 1, with the guanosine H-phosphonate monoester, 2a, or the H-thiophosphonate monoester, 2b. After coupling, the new phosphite triester is selectively oxidized using tert-butyl hydroperoxide, or sulfurized using elemental sulfur, in either case without affecting the H-phosphonate or H-thiophosphonate monoester. Detritylation is carried out using sodium bisulfate adsorbed to silica gel.12 Approximately equal amounts of the diastereomers of 3b or 3c are produced by the sulfurization. The linear dimers 3a–c are then ready for cyclization by treatment with pivaloyl chloride or diphenylchlorophosphate. Oxidation using iodine and water or sulfurization using elemental sulfur gives the cyclic derivatives 4a–e. Sulfurization of the H-phosphonate diester produced by cyclization gives only the [RP] diastereomer, as had been shown by Battistini for c-di-CMP.13 Purification of the linear and cyclic dimers 3a–c and 4a–e was carried out on silica gel, normally without attempting to separate the diastereomers at this stage. The polar groups present, along with the mixtures of diastereomers, hampered the silica purification and lowered the yields. Deprotection of 4a–e was carried out by treatment with methylamine in H2O to remove the isobutyryl and cyanoethyl protecting groups, followed by desilylation using triethylamine trihydrogen fluoride. Purification of 5–7 was carried out by RP HPLC to isolate each of the diastereomers 5a,b, 6a,b,c, and 7a,b. The separations were difficult, requiring repeated chromatography to get pure diastereomers, with concomitant loss of material that in some cases was significant, such that the overall yields from 2 to 5–7 did not exceed 10%.

Configuration Assignments

The only other work describing synthesis and characterization of stereochemistry of c-di-GMP monothiophosphates10 made assignments based on a comparison of the 31P NMR chemical shifts to those of the c-AMP phosphorothioates (c-AMPS), whose configuration had been established.14 The 31P NMR chemical shifts that we have found are nearly identical to the chemical shifts reported for their c-di-GMP monothiophosphates, with about a 1.5 ppm difference between the two diastereomers. In further agreement, we also found that the diastereomer with the larger 31P NMR chemical shift has the shorter retention time on RP HPLC. X-ray structures of c-di-GMP15,16 have shown the 2′-exo/3′-endo conformation typically found for RNA, and also found in cAMP.14,17 The singlets we see in the 1H NMR for the H1′ in c-di-GMP, and for all of the thioates, confirm that this is also the sugar conformation in solution.17 The 1.5 ppm difference in 31P NMR chemical shifts for the diastereomers is, however, well within the envelope of chemical shifts we have observed previously for the various complexes of c-di-GMP,8,9 and the thioate derivatives reported here. Further, the relationship between phosphate and nucleoside in the twelve-member ring of c-di-GMP is strikingly different from that in the six-member ring of c-AMP, as the x-ray structures demonstrate.15,16 In cAMP the phosphate is in a rigid chair conformation directly opposite C4′,14,17 while in the larger twelve-member ring of c-di-GMP the phosphates are moved well away from C4′. Given the small chemical shift differences between the diastereomers relative to the larger chemical shift changes we have seen for different c-di-GMP complexes, and the lack of similarity of the phosphate environment between c-di-GMP and c-AMP, we chose to probe the configuration of the thiophosphate diastereomers using the now well established specificities of venom phosphodiesterase and nuclease P1.1825 These enzymatic studies (described below) gave assignments of c-di-GMP thiophosphate configuration that are opposite to assignments based on c-AMPS.10 We have used our enzymatic assignments for all of the compounds reported below.

The specificity of a variety of enzymes for differential cleavage of the diastereomers of linear 3′,5′-dinucleoside thiophosphates has been well known at least since Eckstein showed, by x-ray crystallography, that ribonuclease A gave exclusively the RP diastereomer of uridine-3′-O-methyl ester from endo-uridine 2′,3′-cyclothiophosphate.21 Our approach to the configuration assignment of the c-di-GMP monothiophosphate diastereomers was to relate their configurations to those of the corresponding linear 3′,5′-diguanosine thiophosphates, where the configuration could be established by enzymatic cleavage by venom phosphodiesterase and by nuclease P1. Venom phosphodiesterase preferentially cleaves the RP diastereomer, while P1 preferentially cleaves the SP diastereomer.18–25 We related results for the linear dimers to the cyclic dimers in two ways. First, as shown in Scheme 2, the linear dimers of 3b, before detritylation, (i) were separated by silica chromatography. A portion of each diastereomer (i) was dephosphitylated26 and deprotected to give ii, which was characterized by cleavage with venom phosphodiesterase. The major portion of each separated diastereomer (i) was used for cyclization, followed by deprotection to give iii, which was characterized by 31P NMR and HPLC retention time. Thus a single linear dimer diastereomer i gave a single deprotected linear dimer ii and a single c-di-GMP monothiophosphate iii. The second approach was to work back from the mixture of the diastereomers of iii (prepared without prior separation of the linear diastereomers 3b) that were then separated by RP HPLC and characterized by 31P NMR and HPLC retention time. Because nuclease P1 cleaves phosphate diesters much faster than either of the thiophosphate diastereomers, we could then use brief treatment of iii with P1 to generate the corresponding linear dimer iv. As shown in Scheme 2, the linear dimers iv obtained from opening of iii have a 5′ phosphate. The presence of this phosphate should have no effect on the enzymatic cleavage of the thiophosphate. Nevertheless, we converted a portion of iv to ii by treatment of iv with bacterial alkaline phosphatase (BAP). The configuration of the linear dimers, ii and iv, obtained from iii, then were determined by treatment with venom phosphodiesterase as well as by treatment with nuclease P1.27 The results of the enzymatic cleavage experiments were identical for both pathways, regardless of the source of the dimer: the c-di-GMP monothiophosphate diastereomer having the longer HPLC retention time and the smaller thiophosphate 31P NMR chemical shift corresponded to the linear dimer that proved the better substrate for venom phosphodiesterase and the poorer substrate for nuclease P1, which must be the RP diastereomer; conversely, the c-di-GMP monothiophosphate diastereomer having the shorter HPLC retention time and the larger thiophosphate 31P NMR chemical shift corresponded to the linear dimer that proved the poorer substrate for venom phosphodiesterase and the better substrate for nuclease P1, which must be the SP diastereomer. The assignments of the di- and trithioate diastereomers were then made based on their retention times and chemical shifts, as shown in Table 1, with the smaller chemical shift/longer retention time assigned the RP configuration.

Table 1
Retention Times on RP HPLC (min) and 31P NMR Chemical Shifts (ppm) at 55 °C for Dilute (0.5 mM) Samples of the Na+ Salts of 57

NMR Characterization

We used 1D 1H and 31P NMR at several temperatures to assess the polymorphism of each of the seven thioate analogs, for both the Na+ and K+ salts. We also used 2D NOESY experiments to assign the syn or anti conformation of the guanine ring relative to the ribose, based on the well-known observation that at low mixing times, syn arrangements give pronounced crosspeaks between the guanine H8 and the ribose H1′, while anti arrangements do not.2830 Further, we have used 2D DOSY (diffusion-ordered spectroscopy)3133 experiments to qualitatively evaluate relative size by means of molecular diffusion coefficients. Our previous work9 with unmodified c-di-GMP in 0.1 M LiCl demonstrated an equilibrium among five different complexes in similar amounts (Fig 2A), four of which contain guanine quartets3436 (Fig 2B): a bimolecular structure, a pair of tetramolecular complexes made of two parallel guanine quartets (one all syn and one all anti), and a pair of octamolecular intercalated complexes made of four stacked quartets (one all syn and one all anti). In 0.1 M KCl, the two octamolecular complexes dominated the equilibrium. We had previously shown8 that c-di-GMP in 0.1 M NaCl appeared to form similar complexes with stabilities somewhat like those in LiCl, but also showed signs of additional aggregation not seen with the other salts.

Partial 1H NMR spectra that display the H8 region are shown in Fig 3 for both the K+ and Na+ salts of 5–7 at 30 °C. Because the H8 region is the most informative, only that section is described in this report. Spectra at lower temperatures (5 and 15 °C, not shown) display increased resonances for the less stable higher order complexes (tetra- and octamolecular) relative to the bimolecular structure, but reduced resonances for them at higher temperature (55 °C, not shown). 31P NMR spectra were also acquired at multiple temperatures, shown in Fig 4 for 30 °C. Those resonances were assigned based on their temperature dependence, and relationship with the 1H NMR spectra using correlations determined from our previous work with unmodified c-di-GMP.9 Except for 6c and 7b (which displayed unusually poor signal to noise ratio due to extensive aggregation), the 31P NMR resonances for the different forms were integrated, and the relative amounts are summarized in Table 2. 2D NOESY plots for the eight samples that display strong crosspeaks characteristic of syn conformations are shown in Fig. 5. The spectra for samples that did not show these characteristic syn crosspeaks are not shown. 2D DOSY plots for all 14 samples at 30 °C are shown in Fig. 6.

Fig 3
1H NMR spectra (H8 region) for 5, 6, and 7 at 30 °C (a = anti, s = syn, B = bimolecular, T = tetramolecular, O = octamolecular, * = residual resonances associated with extreme aggregation)
Fig 4
31P NMR spectra for 5, 6, and 7 at 30 °C (a = anti, s = syn, B = bimolecular, T = tetramolecular, O = octamolecular, *= residual resonances associated with extreme aggregation)
Fig 5
2D NOESY plots for the H8/H1′ region for 5a K+, 5b K+, 5a Na+, 6a K+, 6b K+, 6a Na+ and 7a K+, all at 30 °C, and 7b K+ at 45 °C, all showing the H8/H1′ crosspeaks of syn conformations. Not shown: plots for 5b Na+, 6b Na ...
Fig 6
2D DOSY spectra at 30 °C for all samples, with diffusion coefficients (D) displayed on the vertical axes in m2/sec × 1010
Table 2
Distribution of Complexes at 30 °C Based on Integration of 31P NMR Spectra

For all 14 samples at 30 °C, the H8 regions of the 1H NMR spectra and the 31P NMR spectra are described below, along with syn or anti determinations from the NOESY plots and, where possible, complex size based on diffusion coefficients in m2/sec × 10−10 (D) from the DOSY plots. In our work with unmodified c-di-GMP,9 diffusion coefficients at 30 °C for the bimolecular structure were found to be 3.3 – 3.7, those for the tetramolecular complexes were in the range of 2.0 – 2.1, and those for the octamolecular complexes were 1.8 – 1.9. (Although the mass of the octamolecular complex is twice that of the tetramolecular, its structure is more compact and therefore their diffusion coefficients are more similar than the mass difference would imply.)

Monothioates, K+ salts

The 1H NMR spectrum of the [RP] diastereomer 5a, K+ salt (Fig 3A) displays two separate groups of octamolecular anti H8 resonances (D = 1.5 –1.9); two separate groups of octamolecular syn H8 resonances (D = 1.6 – 1.9); and no resonances with D near 3.5 for the bimolecular structure. The 31P NMR spectrum (Fig 4A) displays one small resonance near −1 ppm for the phosphate of the bimolecular structure; a larger set of nearby downfield octamolecular syn resonances; and a larger set of nearby upfield octamolecular anti resonances. It has a similar pattern for the thiophosphate resonances around 55 ppm.

The 1H NMR spectrum of the [SP] diastereomer 5b, K+ salt (Fig 3B) displays two separate complex groups of octamolecular anti H8 resonances (D = 1.5 – 1.7); one large and one small octamolecular syn resonance (D = 1.6 – 1.7); and a small resonance (D = 3.4) for the bimolecular structure. The 31P NMR spectrum (Fig 4B) displays one small resonance near −1 ppm for the phosphate of the bimolecular structure; a larger set of nearby downfield octamolecular syn resonances; and a larger set of nearby upfield octamolecular anti resonances. However, the pattern for the thiophosphate resonances is different, with both sets of octamolecular syn and anti resonances appearing upfield of the small resonance near 56 ppm for the bimolecular structure.

Monothioates, Na+ salts

The 1H NMR spectrum of the [RP] diastereomer 5a, Na+ salt (Fig 3C) displays a group of multiple tetramolecular anti H8 resonances (D = 2.0 – 2.2); a group of several tetramolecular syn H8 resonances (D = 1.7 – 2.2); and one large broad H8 resonance for the bimolecular structure (D = 3.8). The 31P NMR spectrum (Fig 4C) displays one large resonance near −1 ppm for the phosphate of the bimolecular structure; a smaller set of nearby downfield tetramolecular syn resonances; and a smaller set of nearby upfield tetramolecular anti resonances. It has a similar pattern for the thiophosphate resonances near 55 ppm.

The 1H NMR spectrum of the [SP] diastereomer 5b, Na+ salt (Fig 3D) displays two separate groups of tetramolecular anti H8 resonances (D = 2.1 – 2.4); no syn H8 resonances (no strong H8-H1′ NOESY crosspeaks); and two separate sharp resonances for the bimolecular structure (D = 3.7 – 3.9). The 31P NMR spectrum (Fig 4D) displays one small resonance near −1 ppm for the phosphate of the bimolecular structure; and a larger set of nearby upfield tetramolecular anti resonances (but no downfield syn resonances). It has a similar pattern for the thiophosphate resonances near 56 ppm.

Summary for monothioates

Because both diastereomers of these monothioates are unsymmetrical molecules, each has two kinds of H8s, and there are different possible orientations of the monomers in any complex. This situation presumably accounts in part for the multiple H8 resonances. In spite of the resulting complexity of the spectra, several conclusions can be noted. 1) For the K+ salts, the higher order complexes are present in much larger amounts than for the Na+ salts (Table 2). 2) The [SP] diastereomer in the Na+ salt shows a larger fraction of higher order complexes than does the [RP] diastereomer (Table 2). 3) The higher order K+ thioates primarily have diffusion coefficients consistent with octamolecular complexes (1.5 – 1.9), while those of the Na+ thioates primarily are consistent with tetramolecular complexes (1.8 – 2.4). 4) While both syn and anti higher order complexes are present in the K+ thioates (with more anti than syn), in the Na+ thioates, the [SP] diastereomer has only anti complexes, while the [RP] diastereomer has both syn and anti.

Dithioates, K+ salts

The 1H NMR spectrum of the [RP,RP] diastereomer 6a, K+ salt (Fig 3E) displays two close octamolecular anti H8 resonances (D = 1.8) and a third upfield octamolecular anti H8 resonance (D = 1.6); two syn octamolecular H8 resonances (D = 1.6 – 1.7); and no resonances with D near 3.5 for the bimolecular structure. The 31P NMR spectrum (Fig 4E) displays a small monothiophosphate resonance near 55 ppm for the bimolecular structure; a larger set of nearby downfield octamolecular syn resonances; and a larger set of nearby upfield octamolecular anti resonances.

The 1H NMR spectrum of the [SP,RP] diastereomer 6b, K+ salt (Fig 3F) displays several broad complex octamolecular anti H8 resonances (D = 1.7 – 2.5); a large octamolecular syn H8 resonance (D = 1.7); and several small resonances for the bimolecular structure (D = 3.2 – 3.4). The 31P NMR spectrum (Fig 4F) displays two small resonances for the monothiophosphates of the bimolecular structure ([RP] near 55 ppm and [SP] near 56 ppm); a larger set of nearby downfield octamolecular syn resonances; and a larger set of nearby upfield octamolecular anti resonances.

The 1H and 31P NMR spectra of the [SP,SP] diastereomer 6c, K+ salt both exhibit a poor signal to noise ratio with few resonances. This behavior is also seen (see below) for the Na+ salt of 6c, as well as both salt forms of 7b, all of which have two sulfurs in the [Sp] configuration. The spectra are significantly worse at temperatures below 30°, where there are often no resonances, while heating the sample to 55° gives spectral quality comparable to the other samples at this temperature (not shown). A possible explanation is that the presence two [Sp] sulfurs promotes extensive aggregation such that only a small amount of non-aggregated material is visible at 30° C. The 1H NMR spectrum (Fig 3G) displays several residual H8 resonances that are difficult to assign (D = 1.4 – 1.6); and no resonances with D near 3.5 for the bimolecular structure. Even though the NOESY was acquired at 45° C for this sample, the spectrum was not good enough to assign syn or anti conformations. The 31P NMR spectrum (Fig 4G), displays a small monothiophosphate resonance near 57 ppm for the bimolecular structure; and a larger set of nearby upfield resonances (but no downfield resonances).

Dithioates, Na+ salt

The 1H NMR spectrum of the [RP,RP] diastereomer 6a, Na+ salt (Fig 3H) displays two complex groups of multiple tetramolecular anti H8 resonances (D = 2.0 – 2.5); two tetramolecular syn H8 resonances (D = 2.1 – 2.2); and two H8 resonances for the bimolecular structure (D = 3.2 – 3.3) that overlap with the upfield tetramolecular anti resonances but are resolved in the DOSY spectrum (Fig 6H). The 31P NMR spectrum (Fig 4H) displays one large monothiophosphate resonance near 55 ppm for the bimolecular structure; a smaller set of nearby downfield tetramolecular syn resonances; and a smaller set of nearby upfield tetramolecular anti resonances.

The 1H NMR spectrum of the [SP,RP] diastereomer 6b, Na+ salt (Fig 3I) displays two broad tetramolecular anti H8 resonances (D = 1.9 – 2.1); no syn H8 resonances; and two sharp H8 resonances for the bimolecular structure (D = 3.2 – 3.4). The 31P NMR spectrum (Fig 4I) displays a set of two monothiophosphate resonances for the bimolecular structure ([RP] near 55 ppm and [SP] near 56 ppm); and one set of nearby upfield tetramolecular anti resonances (but no downfield syn resonances).

The 1H NMR spectrum of the [SP,SP] diastereomer 6c, Na+ salt (Fig 3J) displays primarily one residual H8 resonance that is consistent with the bimolecular structure (D = 3.5). The 31P NMR spectrum (Fig 4J) displays one monothiophosphate resonance near 57 ppm for the bimolecular structure; and a set of broad nearby upfield resonances that are difficult to assign.

Summary for dithioates

The [RP,RP] and [SP,SP] dithioates (6a and 6c) are symmetrical molecules, while the [SP,RP] dithioate is not. The presence of multiple H8 resonances for 6a and 6c therefore indicates additional complexity that we are unable to define at this time. None-the-less, all four conclusions noted above for the monothioates hold true for these dithioates as well. In particular, diastereomers with one or two sulfurs in the [SP] configuration (5b, 6b, and 6c) tend not to form syn complexes for the Na+ salts, but do for the K+ salts; and Na+ thioates form primarily tetramolecular complexes while K+ thioates form primarily octamolecular complexes. In addition, the presence of two sulfurs in the [SP] configuration (6c) results in spectra with a poor signal to noise ratio that may be the result of extensive aggregation.

Trithioates, K+ salt

The 1H NMR spectrum of the [RP] diastereomer 7a, K+ salt (Fig 3K) displays two complex groups of octamolecular anti H8 resonances (D = 1.6 –1.9); a large and a small octamolecular syn H8 resonance (D = 1.6 – 1.9); and two H8 resonances for the bimolecular structure (D = 3.1 – 3.2). The 31P NMR spectrum (Fig 4K) displays one small monothiophosphate resonance near 56 ppm for the bimolecular structure; a set of larger nearby downfield octamolecular syn resonances; and a set of larger nearby upfield octamolecular anti resonances. It also has one small dithiophosphate resonance near 114 ppm for the bimolecular structure; and two sets of larger nearby upfield octamolecular syn and anti resonances (but no nearby downfield resonances).

The 1H NMR spectra of the [SP] diastereomer 7b, K+ salt (Fig 3L) displays a group of multiple anti H8 resonances that reflect complexes that are at least as large as octamolecular (D = 1.1 – 1.3); one or two small syn H8 resonances (D = 1.6); and no resonances with D near 3.5 for the bimolecular structure. The NOESY was acquired at 45 °C because the signal to noise ratio was somewhat better than that at 30 °C. The 31P NMR spectrum (Fig 4L) shows one small monothiophosphate resonance near 58 ppm for the bimolecular structure; and a set of larger nearby upfield syn and anti resonances (but no nearby downfield resonances). It also has one small dithiophosphate resonance near 116 ppm for the bimolecular structure; and a larger set of nearby upfield syn and anti resonances (but no nearby downfield resonances).

Trithioates, Na+ salt

The 1H NMR spectrum of the [RP] diastereomer 7a, Na+ salt (Fig 3M) displays two small tetramolecular anti H8 resonances (D = 1.8 – 1.9); no syn H8 resonances; and two large H8 resonances for the bimolecular structure (D = 3.1 –3.2). The 31P NMR spectrum (Fig 4M) displays one monothiophosphate resonance near 56 ppm for the bimolecular structure, and a set of nearby upfield tetramolecular anti resonances (but no nearby downfield resonances). It also has a dithiophosphate resonance near 114 ppm for the bimolecular structure, and a set of nearby upfield tetramolecular anti resonances (but no nearby downfield resonances).

The 1H NMR spectrum of the [SP] diastereomer 7b, Na+ salt (Fig 3N) displays two H8 resonances (D = 2.7 – 2.8) that appear to be anti (no strong NOESY crosspeaks), but their size is difficult to assess. The NOESY was acquired at 45 °C because the signal to noise ratio was somewhat better than that at 30 °C. The 31P NMR spectrum (Fig 4N) shows a dithiophosphate resonance near 58 ppm for the bimolecular structure; and a set of nearby upfield anti resonances (but no nearby downfield resonances). It also has one dithiophosphate resonance near 116 ppm for the bimolecular structure; and a set of nearby upfield anti resonances (but no nearby downfield resonances).

Summary for trithioates

The trithioates are both unsymmetrical molecules, each with two kinds of H8s, and so have multiple possible orientations in any complex. The trends noted above also apply to these trithioates. Again, diastereomers with one or two sulfurs in the [SP] configuration (5b, 6b, 6c, 7a, and 7b) tend not to form syn complexes as the Na+ salts, but do as the K+ salts. Further, all the Na+ thioates form primarily tetramolecular complexes while the K+ thioates form primarily octamolecular complexes. In addition, the results for the [SP] trithioate support the rationale that the presence of two sulfurs in the [SP] configuration (6c, 7b) results in extensive aggregation.

DISCUSSION

We have found that the K+ salts of all seven thioates display more extensive higher order complex formation than do the Na+ salts. None-the-less, 1D 31P NMR always shows small amounts of the bimolecular structure in the K+ salts, even when the less sensitive 2D DOSY cannot detect it. Although K+ and Na+ ions are both known to stabilize guanine quartets in nucleic acids, in general the larger and more easily dehydrated K+ does so more effectively.35,36 K+ has frequently been found to be located between the planes of the quartets, whereas Na+ is often within the planes. It is perhaps for this reason that K+ can better stabilize the stacked quartet structures of c-di-GMP and its analogs, thereby promoting octamolecular complexes. Without this additional stabilization, tetramolecular complexes are dominant with Na+.

We have also found that the presence of sulfur in the [SP] configuration (5b, 6b, 6c, 7a, and 7b) specifically stabilizes anti complexes and/or destabilizes syn complexes in the Na+ salts, to the extent that no syn complexes are observed. However, for the K+ salts, both syn and anti complexes are present. Examination of the x-ray structures15,16 of the bimolecular form shows that the [SP] sulfur is directed more toward the interior of the molecule, where the guanine rings are stacked, than is the [RP]. The [SP] sulfur may therefore be better positioned to create stabilizing interactions with key parts of the anti complexes and/or destabilizing clashes with parts of the syn complexes for the Na+ salts.

In addition, we have found that for both the K+ and Na+ salts, the presence of two [SP] sulfurs (6c and 7b) gives poor spectra, perhaps as the result of extensive aggregation. This aggregation may take the form of end-to-end stacking of the tetra- or octamolecular complexes, forming extended guanine quartet structures similar to ones seen in other cases,33,35,36 although other forms of aggregation cannot be excluded. Some of the residual resonances we do observe in the K+ salts of 6c and 7b, which must reflect very low concentrations, given the poor signal to noise ratio, show diffusion coefficients that are unusually small (1.1 – 1.4), and may represent aggregates just small enough to be detected. We also note that our preliminary NMR used thioate samples with 0.1 M salts, just as in our original work on unmodified c-di-GMP.9 However, the aggregation was so extensive that we switched to using just the stoichiometric amount, with no excess salt, for the work presented here.

CONCLUSION

Seven thiophosphate analogs of c-di-GMP were synthesized by new routes, and 1D 1H and 31P NMR, along with 2D NOESY and DOSY, were used to assess the impact of the thioate substitution(s) on the polymorphism of both the Na+ and K+ salts. The results demonstrate the dramatic effects of [SP] thiophosphates, as well as major differences between the Na+ and K+ salts. We find that in all cases, the K+ salts display more extensive higher order complex formation than the Na+ salts, resulting primarily in octamolecular complexes for the K+ salts, but tetramolecular complexes for the Na+ salts. Further, the presence of just one [SP] sulfur is sufficient to shift the population of the complexes entirely to the anti form, with no detectable syn complexes. The presence of two [SP] sulfurs promotes extensive aggregation. Phosphorothioate derivatives of c-di-GMP are likely to prove useful for further exploration of the mechanisms of c-di-GMP function, where an understanding of the very different behavior of these diastereomers may be valuable.

EXPERIMENTAL

General methods

Pivaloyl chloride was freshly distilled before each use. The amidite coupling reactions were carried out in anhydrous acetonitrile that had been dried over 3Å molecular sieves. The starting materials for all reactions in dry pyridine were dried by concentration from pyridine three times.

Preparative silica gel chromatography was carried out on pre-packed silica gel flash columns from AnaLogix using gradients of methanol in CH2Cl2 containing 0.5% pyridine. Analytical reverse phase HPLC was carried out on a Waters 2960 system, with an Atlantis C18 column, 100Å, 4.6 mm × 50 mm, 3.0 μm. Gradients of acetonitrile and 0.1 M triethylammonium acetate (TEAA) buffer (pH 6.8) were used with a flow rate of 1.0 mL/min. ESI-MS was acquired using a Waters Micromass single quadropole LCZ system. Semi-preparative reverse phase HPLC purification was performed on a Waters Novapak C18 19 × 300 mm column using gradients of CH3CN in 0.1 M TEAA (pH 6.8) or a Beckman ultrapore RPSC C3 10 × 250 mm column using gradients of CH3CN in 0.1 M diisopropylethyl ammonium acetate (pH 6.8). Desalting of pure samples was performed on a Waters Novapak C18 19 × 300 mm column using gradients of CH3CN in 0.1 M ammonium bicarbonate. Sodium and potassium salts of 57 were obtained by ion exchange using 10 mL of AG 50W-X2 sulfonic acid resin, which had previously been converted to the Na+ or K+ forms, respectively.

NMR

The 1H and 31P NMR spectra were acquired on a Varian Inova 500 MHz spectrometer, with the latter referenced to neat phosphoric acid. The samples were 31 mM cyclic dimer in 0.30 mL H2O containing 10% D2O, pH 6.8 (adjusted with HCl or either KOH or NaOH, as appropriate). The thioates were quantified by OD measurement at 260 nm using ε = 26,100 OD M−1 cm−1.8

The NOESY and DOSY spectra were acquired at 30 °C (unless otherwise noted) on a Varian Inova 500 MHz spectrometer. NOESY data were collected by 4096 (t2) times 512 (t1) complex data points with spectral widths of 8000 Hz in both dimensions. The mixing time for NOESY spectra was 150 ms, the number of scans per each t1 increment was 16, and the relaxation delay for each scan was 2 s. The DOSY spectra were collected over a spectral width of 8000 Hz using 16 scans for each of the 256 increments, with a relaxation delay of 2.5 sec and a diffusion delay of 0.1 sec. The gradient pulse strength was arrayed from 400 to 20,000. Both NOESY and DOSY acquisitions used 1 s 1H presaturation during the relaxation delay to suppress water.

Syntheses of 2a and 2b

2-N-Isobutyryl-2′-O-tert-butyldimethylsilyl-3′-O-(H-phosphonate)-guanosine, sodium salt (2a)

To 2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-guanosine37,38 (3.85 g, 5.0 mmol) dissolved in 15 mL of dry pyridine was added diphenyl phosphite (1.9 mL, 10 mmol, 2 eq). After 15 min at room temperature the reaction mixture was poured into saturated NaHCO3 and extracted with CH2Cl2 (3 × 50 mL). The organic layers were concentrated, and the residue was purified by silica gel chromatography. Product fractions were combined, concentrated to dryness, and the residue detritylated by shaking a CH2Cl2 solution with NaHSO4/SiO2 (1.36 g, 3.0 mmol) for 30 min. After filtration, the filtrate was evaporated with frequent addition of toluene (50 mL × 3), the last time with about 100 mL remaining. The bulk of the product was deposited on the wall of the flask, leaving the 4,4′-dimethoxytritanol in solution. Hexane (15 mL) was added to the solution, which was allowed to stand for 30 min. The liquid was decanted and the residue washed with 20 mL 15% hexane in toluene. The residue was then dissolved in CH2Cl2, made into a foam, and dried in a desiccator over P2O5 to give 2a (2.25 g, 3.7 mmol, 74%). The product was confirmed by LC-MS in negative mode, with m/z (M-1) 530.4 (calcd for C20H33N5O8PSi: 530.2).

2-N-Isobutyryl-2′-O-tert-butyldimethylsilyl-3′-O-(H-thiophosphonate)-guanosine, sodium salt (2b)

To 2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-guanosine37,38 (3.21 g, 4.1 mmol) dissolved in 15 mL of dry pyridine was added diphenylphosphite (1.6 mL, 8.2 mmol, 2 eq). After 15 min lithium sulfide (Li2S, 0.94 g, 20.5 mmol, 5 eq) was added. After 40 min, the mixture was poured into saturated NaHCO3 and extracted with CH2Cl2 (3 × 50 mL). The organic layers were concentrated and the residue was purified by silica gel chromatography. Product fractions were combined, concentrated to dryness and the residue dissolved in CH2Cl2. Detritylation occurred on standing overnight and the product was separated from 4,4′-dimethoxytritanol as described for 2a to give the H-thiophosphonate 2b (1.81 g, 2.9 mmol, 71%). The product was confirmed by LC-MS in negative mode, with m/z (M-1) 546.4 (calcd for C20H33N5O7PSSi: 546.2).

Synthesis of the [RP] monothiophosphate (c-GpGps) (5a)

2-N-Isobutyryl-2′-O-tert-butyldimethylsilyl-guanosinylyl (3′→5′)-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-3′-O-(H-phosphonate)-guanosine, sodium salt (3a)

To a mixture of phosphoramidite 1 (1.45 g, 1.5 mmol, 1.5 eq) and H-phosphonate 2a (0.61g, 1.0 mmol) dissolved in 20 mL CH3CN at room temperature was added pyridinium trifluroacetate (0.58 g, 3 mmol, 2 eq to 1). The mixture was stirred for 30 min, followed by addition of anhydrous tert-butylhydroperoxide (0.87 mL, 6.25 mmol) in decane. After 30 min, the mixture was poured into aqueous NaHCO3 and extracted with ethyl acetate. The organic layers were then concentrated and purified to >80% purity by silica gel chromatography. The product fractions were concentrated to dryness, the residue dissolved in CH2Cl2 and treated with NaHSO4/SiO2 (0.91 g), and the product isolated as described above for 2a to give 0.64 g crude 3a as a mixture of two unresolved diastereomers. The 3a mixture was confirmed by LC-MS in negative mode, with m/z (M−1) 1113.0 (calcd for C43H68N11O16P2Si2: 1112.4).

Cyclo-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-guanosinylyl-(3′→5′)-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-[RP]-P-thioguanosinylyl-(3′→5′), sodium salt (4a)

To a solution of a portion of crude 3a from above (0.28 g) in 15 mL dry pyridine was added pivaloyl chloride (Pv-Cl, 0.12 mL, 1.0 mmol). After 3 min, elemental sulfur (40 mg, 1.25 mmol) was added, and the reaction was stirred for 40 min. The mixture was then concentrated, evaporated with toluene three times and CH3CN three times, filtered to remove excess sulfur, and concentrated to dryness to give crude 4a (0.25 g). The residue was not further purified before deprotection, which is described below. 4a in the residue was confirmed by LC-MS in negative mode, with m/z (M-1) 1127.1 (calcd for C43H66N11O15P2SSi2: 1126.4).

Cyclo-P-guanosinylyl (3′→5′)-[RP]-P-thioguanosinylyl-(3′→5′), ammonium salt (c-GpGps [RP], 5a)

To the crude 4a from above (0.25 g) was added 4 mL of methylamine in water (40%). After 40 min the mixture was concentrated and evaporated with pyridine three times to remove water. To the residue was added TEA•3HF (2.2 mL, 13.3 mmol) and an additional 1.5 mL TEA. After being heated at 50 °C for 4 hrs, the reaction was quenched with 7 mL isopropyl trimethylsilyl ether.39 Ethyl ether (10 mL) was added to complete precipitation of 5a. The mixture was allowed to stand overnight, the supernatant was decanted, and the product purified by semi-preparative RP HPLC on a Waters Novapak C18 column using a gradient of 2–20% CH3CN in 0.1 M TEAA over 90 min. The pure fractions were pooled, lyophilized, and desalted by C18 RP-HPLC using a gradient of CH3CN in 0.1 M ammonium bicarbonate to give pure 5a (12 μmol, 3% from 2a). 5a was confirmed by LC-MS in negative mode, with m/z (M-1) 705.3 (calcd for C20H23N10O13P2S: 705.1).

Synthesis of the [RP] (5a) and [SP] (5b) monothiophosphates (c-GpsGp)

2-N-Isobutyryl-2′-O-tert-butyldimethylsilyl-P-thioguanosinylyl (3′→5′)-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-3′-O-(H-phosphonate)-guanosine, sodium salt (3b)

To a mixture of phosphoramidite 1 (1.45 g, 1.5 mmol, 1.7 eq) and H-phosphonate 2a (0.54 g, 0.90 mmol) dissolved in 20 mL CH3CN at room temperature was added pyridinium trifluroacetate (0.58 g, 3.0 mmol, 2 eq to 1). After 30 min, elemental sulfur (0.16 g, 5.0 mmol) dissolved in 30 mL 1:1 pyridine:CH2Cl2 was added, and the mixture was stirred for 40 min. The mixture was then concentrated, evaporated with toluene three times and CH3CN three times, filtered to remove excess solid sulfur, and concentrated to dryness. The residue was purified to >80% purity by silica gel chromatography. The product fractions were concentrated to dryness, the residue dissolved in CH2Cl2 and treated with NaHSO4/SiO2 (0.91 g) as described above for 2a to give 0.72 g of crude 3b as a mixture of two partially resolved diastereomers. The 3b mixture was confirmed by LC-MS in negative mode, with m/z (M−1) 1128.9 (calcd for C43H68N11O15P2SSi2:1128.4).

Cyclo-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-thioguanosinylyl (3′→5′)-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-guanosinylyl-(3′→5′), sodium salt (4b)

To a solution of a portion of crude 3b from above (0.41 g) in 15 mL dry pyridine was added Pv-Cl (0.17 mL, 1.4 mmol). After 3 min, water (0.10 mL, 5.6 mmol, 4 eq to Pv-Cl) was added, followed by iodine (0.432 g, 1.7 mmol). After 5 min the mixture was poured into aqueous NaHCO3 containing Na2SO3 (0.34 g) and extracted using CH2Cl2. The organic layers were concentrated with addition of toluene three times and purified to >80% purity by silica gel chromatography to give crude 4b (0.14 g) as a mixture of two partially resolved diastereomers. The 4b mixture was confirmed by LC-MS in negative mode, with m/z (M-1) 1126.7 (calcd for C43H66N11O15P2SSi2: 1126.4).

Cyclo-[RP]-P-thioguanosinylyl (3′→5′)-P-guanosinylyl-(3′→5′), ammonium salt (c-GpsGp [RP], 5a) and cyclo-[SP]-P-thioguanosinylyl (3′→5′)-P-guanosinylyl-(3′→5′), ammonium salt (c-GpsGp [SP], 5b)

The crude mixture of two diastereomers of 4b from above (0.14 g) was treated in the same way as the single diastereomer 4a. C18 RP-HPLC using 0.1 M TEAA buffer and CH3CN gave two major fractions, each containing 5a or 5b as the major component. Fractions from each peak were combined, further purified by C18 RP-HPLC using a different gradient, and then desalted to give pure 5a (18 μmol, 3% from 2a) and 5b (10 μmol, 2% from 2a). 5a had the same LC-MS profile as above, and 5b (with a different retention time, Table 1) was confirmed by LC-MS in negative mode, with m/z (M-1) 705.3 (calcd for C20H23N10O13P2S: 705.1).

Synthesis of the [RP,RP] (6a) and [SP,RP] (6b) dithiophosphates (c-GpsGps)

Cyclo-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-thioguanosinylyl (3′→5′)-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-thioguanosinylyl-(3′→5′), sodium salt (4c)

To a solution of a portion of crude 3b from above (0.29 g) dissolved in 15 mL dry pyridine was added Pv-Cl (0.10 mL, 0.8 mmol). After 3 min, elemental sulfur (25.6 mg, 0.8 mmol) was added, and the reaction was stirred for 40 min. The mixture was then concentrated, evaporated with toluene three times and CH3CN three times, filtered to remove excess sulfur, and concentrated to dryness to give crude 4c (0.22 g) as a mixture of two partially resolved diastereomers. The residue was not further purified before deprotection, which is described below. The 4c mixture was confirmed by LC-MS in negative mode, with m/z (M-1) 1142.9 (calcd for C43H66N11O14P2S2Si2: 1142.3).

Cyclo-[RP]-P-thioguanosinylyl (3′→5′)-[RP]-P-thioguanosinylyl-(3′→5′), ammonium salt (c-GpsGps [RP,RP], 6a) and cyclo-[SP]-P-thioguanosinylyl (3′→5′)-[SP]-P-thioguanosinylyl-(3′→5′), ammonium salt (c GpsGps [SP,RP], 6b)

The crude mixture of two diastereomers of 4c from above (0.22 g) was deprotected as described for 4a. C18 RP-HPLC using 0.1 M TEAA buffer and CH3CN resolved the mixture into two major fractions, each containing 6a or 6b as the major component. The fractions for each diastereomer were pooled, lyophilized and individually further purified by C18 RP-HPLC, and then desalted to give pure 6a (29 μmol, 3% from 2a) and 6b (16 μmol, 2% from 2a). These were characterized by LC-MS with different retention times (Table 1) and m/z (M-1) of 721.6 for 6a and 721.5 for 6b (calcd for C20H23N10O12P2S2: 721.0).

Synthesis of the [RP,RP] (6a), [SP,RP] (6b), and [SP,SP] (6c) dithiophosphates (c-GpsGps)

2-N-Isobutyryl-2′-O-tert-butyldimethylsilyl-P-thioguanosinylyl (3′→5′)-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-3′-O-(H-thiophosphonate)-guanosine, sodium salt 3c

To a mixture of phosphoramidite 1 (5.81 g, 6.0 mmol, 1.5 eq) and H-thiophosphonate 2b (2.52 g, 4.0 mmol) dissolved in 40 mL CH3CN was added pyridinium trifluroacetate (2.31 g, 12 mmol, 2 eq to 1). The mixture was stirred for 30 min, followed by addition of elemental sulfur (0.384 g, 12 mmol) dissolved in 30 mL 1:1 pyridine:CH2Cl2. After 40 min, the mixture was concentrated and evaporated with toluene three times and CH3CN three times, filtered to remove excess sulfur, and concentrated to dryness. The residue was purified to >80% purity by silica gel chromatography. The product fractions were concentrated to dryness, the residue dissolved in CH2Cl2 and treated with NaHSO4/SiO2 (2.73 g) as described above for 2a to give 2.33 g of crude 3c as a partially resolved mixture of diastereomers. The 3c mixture was confirmed by LC-MS in negative mode, with m/z (M-1) of 1144.8 (calcd for C43H68N11O14P2S2Si2: 1144.3).

Cyclo-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-thioguanosinylyl (3′→5′)-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-thioguanosinylyl-(3′→5′), sodium salt 4d)

To a solution of a portion of crude 3c from above (0.41g) in 20 mL of dry pyridine was added diphenyl chlorophosphate (DPP-Cl, 0.1 mL, 0.5 mmol) dissolved in 3 mL pyridine. After 20 min, H2O (12 μL, 0.67 mmol) was added, followed by I2 (0.42 g, 1.65 mmol). After 5 min the mixture was poured into aqueous NaHCO3 containing Na2SO3 (0.34 g) and extracted with CH2Cl2. The organic layers were concentrated with addition of toluene three times and then purified to >80% purity by silica gel chromatography to give crude 4d (0.21 g) as a partially resolved mixture of three diastereomers. The 4d mixture was confirmed by LC-MS in negative mode, with m/z (M-1) of 1142.3 (calcd for C43H66N11O14P2S2Si2: 1142.3).

Cyclo-[RP]-P-thioguanosinylyl (3′→5′)-[RP]-P-thioguanosinylyl-(3′→5′), ammonium salt (c-GpsGps [RP,RP], 6a), cyclo-[SP]-P-thioguanosinylyl (3′→5′)-[RP]-P-thioguanosinylyl-(3′→5′), ammonium salt (c GpsGps [SP,RP], 6b), and cyclo-[SP]-P-thioguanosinylyl (3′→5′)-[SP]-P-thioguanosinylyl-(3′→5′), ammonium salt (c-GpsGps [SP,SP], 6c)

The crude mixture of three diastereomers of 4d from above (0.21 g) was deprotected as described for 4a. C18 RP-HPLC using 0.1 M TEAA buffer and CH3CN gave three major fractions, each containing 6a, 6b, or 6c as the major component. The fractions containing 6a and 6b were further purified by C18 RP-HPLC, and the fractions containing 6c were further purified on a Beckman C3 column. Desalting gave pure 6a (25μmol, 4% from 2b), 6b (12 μmol, 2% from 2b), and 6c (4μmol, 1% from 2b). 6a and 6b had the same LC-MS profiles as above, and 6c (with a different retention time, Table 1) was confirmed by LC-MS in negative mode, with m/z (M-1) 721.4 (calcd for C20H23N10O12P2S2: 721.0).

Synthesis of the [RP] (7a) and [SP] (7b) trithiophosphates (c-GpsGpss)

Cyclo-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-thioguanosinylyl (3′→5′)-2-N-isobutyryl-2′-O-tert-butyldimethylsilyl-P-dithioguanosinylyl-(3′→5′), sodium salt (4e)

To a solution of a portion of crude 3c from above (0.85 g) dissolved in 20 mL dry pyridine was added DPP-Cl (0.22 mL, 1.05 mmol) dissolved in 3 mL pyridine. After 20 min, elemental sulfur (0.068 g, 2.1 mmol) was added, and the mixture was stirred for 40 min. The mixture was concentrated, evaporated with toluene three times and CH3CN three times, filtered to remove excess sulfur, and concentrated to dryness. The residue was purified to >80% purity by silica gel chromatography to give crude 4e (0.85 g) as a mixture of two partially resolved diastereomers. The 4e mixture was confirmed by LC-MS in negative mode, with m/z (M-1) 1158.7 (calcd for C43H66N11O13P2S3Si2: 1158.3).

Cyclo-[RP-P-thioguanosinylyl (3′→5′)-P-dithioguanosinylyl-(3′→5′), ammonium salt (c-GpsGpss [RP], 7a) and cyclo-[SP]-P-thioguanosinylyl (3′→5′)-P-dithioguanosinylyl-(3′→5′), ammonium salt (c-GpsGpss [SP], 7b)

A portion of the crude mixture of two diastereomers of 4e from above (0.52 g) was deprotected as described for 4a. C18 RP-HPLC using 0.1 M TEAA buffer and CH3CN gave two major fractions, each containing 7a or 7b as the major component. The fractions containing 7a were further purified by C18 RP-HPLC, and the fractions containing 7b were further purified on a Beckman C3 column. Desalting gave pure 7a (32μmol, 4% from 2b) and 7b (6 μmol, 1% from 2b). The products had different retention times (Table 1) and were confirmed by LC-MS in negative mode, with m/z (M-1) 737.4 for 7a and 737.1 for 7b (calcd for C20H23N10O11P2S3: 737.0).

Enzymatic Cleavage Experiments

Preparation of ii

To i (0.2 g, 0.13 mmol) dissolved in 9 mL of dry pyridine and ethylene glycol (0.08 g, 1.3 mmol, 10 eq) was added adamantoyl chloride (0.13 g, 0.65 mmol, 5 eq). After 3 hrs, the reaction was quenched with saturated NaHCO3 and extracted three times with CH2Cl2. The organic layers were combined and concentrated. To the residual oil was added 5 mL of aq 40 % methylamine. After 1 hr the mixture was dried by concentration from pyridine three times. To the residue was added TEA·3HF (1.8 mL, 11 mmol, 125 eq to TBS) and 1.4 mL TEA. After 4 hrs at 50 °C, the reaction was quenched with 5.4 mL isopropyl trimethylsilyl ether and the product collected by centrifugation. To the residue was added 7 mL of 0.5 M acetic acid. After 18 h the mixture was concentrated, the residue dissolved in H2O, and the solution washed with ethyl ether three times. The aqueous layers were concentrated, and the residue was purified by semi-preparative RP HPLC.

SVPD cleavage of ii

To 5 OD (0.20 μmol) of ii dissolved in 250 μL buffer (pH 7.0) containing 100 mM Tris-Cl and 2 mM MgCl2 was added 0.003 units of SVPDE. The solution was maintained at 37° C, with 10 μL aliquots analyzed by RP-HPLC over 80 h. The percentage of remaining linear dimer was determined by integration of the corresponding peak in the chromatograms.

P1, BAP, and SVPD cleavage of iii

To 6 OD (0.23 μmol) of iii dissolved in 600 uL TEAA buffer (pH 7.0) was added 0.30 units of nuclease P1. The solution was maintained at 37° C with 15 μL aliquots analyzed by RP-HPLC to monitor the disapperance of cyclic dimer. After the completion of ring-opening, the solution was heated at 95° C for 30 min. After cooling to room temperature, 2.0 units of BAP were added, and the removal of 5′-phosphate was monitored by HPLC by injection of 15 μL aliquots. After completion of dephosphorylation, the mixture was heated at 95° C for 30 min. After cooling to room temperature, 0.004 units of SVPDE were added. The mixture was maintained at 37° C with 15 μL aliquots analyzed by HPLC over 40 h.

P1 and SVPD cleavage of iii

To 6 OD (0.23 μmol) of iii dissolved in 600 uL TEAA buffer (pH 7.0) was added 0.30 units of nuclease P1. The solution was maintained at 37° C with 15 μL aliquots analyzed by RP-HPLC to monitor the disapperance of cyclic dimer. After the completion of ring-opening, the solution was heated at 95° C for 30 min. After cooling to room temperature, the solution was divided into two equal parts. To one was added 0.001 units of SVPDE. The mixture was maintained at 37° C with 15 μL aliquots analyzed by HPLC over 47 h.

P1 cleavage of iii

The pH of the second part above was adjusted to between 5 and 6 using acetic acid and 0.25 units of nuclease P1 was added. The solution was maintained at 37° C with 15 μL aliquots analyzed by HPLC over 50 h.

Acknowledgments

This work was supported by NIH grant GM079760.

Footnotes

In honor of and in celebration of Morris J. Robins’ 70th birthday

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