Remarkably Stereospecific Utilization of ATP α,β-Halomethylene Analogues by Protein Kinases

Abstract

ATP analogues containing a CXY group in place of the α,β-bridging oxygen atom are powerful chemical probes for studying ATP-dependent enzymes. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a novel method based on derivatization of a bisphosphonate precursor with a D-phenylglycine chiral auxiliary that enables preparation of the individual diastereomers of α,β-CHF-ATP and α,β-CHCl-ATP, which differ only in the configuration at the CHX carbon. When tested on a dozen divergent protein kinases, these individual diastereomers exhibit remarkable diastereospecificity (up to over 1000-fold) in utilization by the enzymes. This high selectivity can be exploited in an enzymatic approach to obtain the otherwise inaccessible diastereomers of α,β-CHBr-ATP. The crystal structure of a tyrosine kinase Src bound to α,β-CHX-ADP establishes the absolute configuration of the CHX carbon and helps clarify the origin of the remarkable diastereospecificity observed. We further synthesized the individual diastereomers of α,β-CHF-γ-thiol-ATP and demonstrated their utility in labeling a wide spectrum of kinase substrates. The novel ATP substrate analogues afforded by these two complementary strategies should have broad application in the study of the structure and function of ATP-dependent enzymes.

ATP is an essential nucleotide cofactor for numerous enzymes critical for life processes, ranging from protein kinases and ATPases to nitrogenases.^1,2 Chemical modification of ATP has been a widely employed approach to probe the active-site structures, enzymatic mechanisms and cellular functions of ATP-dependent enzymes.^3,4 Replacement of the α, β-bridging oxygen with a methylene linker (CH₂) facilitates protein crystallization⁵ and, in general, nucleotides with appropriately substituted bridging methylenes (CXY) are useful mechanistic probes^6–9 or for other purposes such as in vivo expansion of the genetic alphabet.¹⁰ A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y).^11–13 Recently, the individual stereoisomers of β, γ-CXY-dGTP (X/Y = hydrogen or halogen) were successfully prepared¹⁴ and exhibited 5–10-fold stereo-specificity in DNA polymerase β catalysis.¹⁵ Among other CXY nucleotide analogues, the α, β-CHN₃ dATP and β, γ-CHF ATP diastereomers, which are inhibitors for pol β and Src kinase, respectively, have been obtained as separate compounds.^6,16 However, the synthesis of individual stereoisomers of α, β-CHF-ATP and its halo homologues, which are substrate mimics of ATP for kinases, has remained an unmet challenge.⁸

We report here a synthetic strategy to prepare the first examples of individual α, β-CHX-ATP diastereomers. The method involves three steps (Scheme 1): (i) conversion of the precursor α, β-CHX-ADP diastereomeric mixture 1 to a D-phenylglycine phosphoramidate derivative 2; (ii) separation of the two diastereomers of 2 using preparative nonchiral reverse phase (RP) HPLC; and (iii) treatment of the isolated individual diastereomers of 2 with 85% H₃PO₄ to afford both individual triphosphate (3) and diphosphate (4) nucleotide analogues in a single reaction. In this synthesis, D-phenylglycine serves a dual role as both a chiral auxiliary ligand and to activate the bisphosphonate moiety for subsequent phosphorylation. H₃PO₄ also plays a dual role, providing an acidic medium to remove the protecting isopropylidene group and serving as the phosphate donor. The use of these reagents in dual roles enabled a concise and efficient chemical route to the α, β-CHX-ATP diastereomers that is 5 steps shorter than the route previously developed for the β, γ-CHX-dGTP stereoisomers.¹⁴ The two isomers of 3 synthesized by the present method are designated as 3x-1 and 3x-2 based on the elution order of their corresponding phosphoramidate precursors 2x-1/2x-2 (x = a or b) under RP-HPLC (Figures S1 and S2). Their α-phosphorusnuclei show detectably different ³¹P NMR chemical shifts (Δδ ∼ 0.13 and 0.19 ppm for 3a-1/2 and 3b-1/2, respectively) (Figures S3 and S4).¹⁷

Scheme 1.

Synthesis of Individual α, β-CHX (X = F, Cl) ATP Diastereomers

The pure diastereomers of α, β-CHX-ATP (3a-1, 3a-2, 3b-1 and 3b-2) were first evaluated in comparison to ATP, α, β-CH₂-ATP and α, β-CF₂-ATP for their utilization by a prototypical tyrosine kinase Src, which is frequently overexpressed in cancers.¹⁸ The kinetic parameters (Table 1), obtained using the Omnia kinase assay, show that α, β-CF₂-ATP was utilized by Src with similar efficiency to ATP whereas α, β-CH₂-ATP was an extremely poor substrate. An end-point assay based on ¹H NMR revealed the same trend (Figure S5). The specificity constants (k_cat/K_M) of these two nucleotides differ by >1000-fold suggesting that, for Src recognition, the CF₂ group is a much better mimic of the oxygen atom in ATP than CH₂. 3a-2 has a comparable K_M to ATP whereas its k_cat is approximately 2-fold smaller. In contrast, 3a-1’s K_M is over 30-fold greater than that of the active diastereomer whereas its k_cat is over 50-fold smaller. Interestingly, the two α, β-CHF-ATP diastereomers appear to mimic α, β-CH₂-ATP and α, β-CF₂-ATP rather than each other in terms of utilization by Src. Given that CHF phosphonates have long been considered to be isoacidic mimics of biological phosphates,¹⁹ our observation that the specificity constant of the inert diastereomer 3a-1 is at least 1000-fold lower than 3a-2, in agreement with the high selectivity observed from NMR (³¹P of ATP and ADP analogues, Figure S6) and Western-blot analysis (phosphotyrosine, Figure S7). The α, β-CHCl-ATP isomers (3b-1 and 3b-2) were utilized by the Src kinase with even higher diastereoselectivity than that of α, β-CHF-ATP, though their k_cat values are substantially smaller than those of α, β-CHF-ATP (Table 1 and Figure S8). The lower K_M value of 3b-2 than that of 3a-2 may reflect a higher binding affinity of the chlorine analog to the Src kinase than the fluorine one as the same trend was observed with K_i values of 4b-2 and 4a-2 (Table S1). This striking selectivity greatly exceeds that observed with DNA polymerase β¹⁵ in utilizing substrates modified at β, γ-CXY bridge.

Table 1.

Kinetic Parameters of ATP Analogues as Src Substrates

ATP analogue		KM (mM)	kcat (min−1)	Stereospecificity (kcat/KM)S/(kcat/KM)R
ATP		0.085 ± 0.010	167 ± 56	n.a.
α, β-CH2-ATP		1.6 ± 0.47	0.17 ± 0.025
α, β-CF2-ATP		0.034 ± 0.004	84 ± 1.3
3a-1	(R)-CHF	1.6 ± 0.3	1.5 ± 0.6	1.5 × 103
3a-2	(S)-CHF	0.042 ± 0.0053	59 ± 21
3b-1	(R)-CHCl	5.1 ± 0.52	0.22 ± 0.0018	1.8 × 103
3b-2	(S)-CHCl	0.0087 ± 0.0020	0.69 ± 0.10

To assign absolute stereoconfiguration at the CHF carbon as a basis to understand the diastereoselective utilization of α, β-CHF-ATP by Src, we cocrystallized Src with 3a-2, 3b-2, 4a-2 and 4b-2, the enzymatic substrates and products of the better substrate isomers (solved to 2.0–2.4 Å resolutions, Table S2). Given the low observable electron density for the γ-phosphoryl group in the 3b-2/Src structure and the virtually identical complex structures for 3b-2 and 4b-2, we suspect that 3b-2 underwent hydrolysis and lost its γ-phosphoryl group during the course of crystallization. Regardless, the well-defined electron density map allowed us to determine unambiguously the stereoconfiguration of the CHX carbon in 4a-2 and 4b-2 as R (Figure 1A,B). Therefore, the corresponding triphosphate 3a-2 and 3b-2 has the S configuration at the CHX carbon. Interestingly, the conformations of the kinase glycine-rich loop (P loop) and the nucleotide cofactor in the 4a-2/Src structure resemble the previously described active state of Src whereas those in the 4b-2/Src structure are close to the inactive state of Src (Figure 1C).²⁰ This may account for the lower k_cat for 3b-1 than that for 3a-1. Moreover, the fluorine atom in 4a-2 is only 3.4 Å away from the ε-nitrogen of the catalytic lysine (K295) in Src, suggesting the presence of a favorable interaction through an electrostatic interaction with the polar F atom.¹² In contrast, the chlorine atom in the 4b-2 occupies a small hydrophobic pocket formed between the β1 and β2 strands, which may account for the lower K_M of 3b-2 than that of 3a-2. When the opposite diastereomer (4a-1 or 4b-1) is modeled into the Src active site with the same conformation as 4a-2 or 4b-2, the resulting complexes not only lose the halogen-mediated interactions but also have an unfavorable steric overlap between the halogen atom and the main chain amide of Q275/G276 within the glycine-rich loop (P loop) (Figure S9), highlighting the importance of the intimate interaction between ATP and the P loop in kinase catalysis. With its larger Cl atom, 4b-1 is predicted to have a greater steric clash than the corresponding fluorine analog, thus accounting at least in part for the higher diastereoselectivity for 3b-1/2.

Figure 1.

Co-crystal structures of Src and ATP analogs. (A) Co-crystal structure of Src/4a-2 establishes the absolute configuration of the nucleotide and reveals its interaction with the K295 of Src. Stick model of α, β-CHF-ADP overlaid with |2F_o − F_c| electron density (blue mesh, contoured at 1σ). (B) Co-crystal structure of Src/4b-2. (C) Structural overlay reveals that the bisphosphonate moiety of (R)-α, β-CHF-ADP (green) assumes a similar conformation to that of γ-thiol-ATP (cyan) bound to Src (3DQW). (D) Overlay of the α, β-CHX nucleotide bound Src structures with the previously solved active (3DQW) and inactive (2SRC) Src structures reveal different conformations of the glycine-rich loop.

We attempted to obtain the individual α, β-CHBr-ATP diastereomers, motivated by the Br atom’s well-known utility in producing phase information for protein cocrystals²¹ and its ability to form stronger halogen bonds than F and Cl.²² Our auxiliary strategy failed to make available individual α, β-CHBr-ATP diastereomers because the CHBr phosphoramidate precursors could not be well resolved by RP-HPLC. However, the observed high stereoselectivity of Src toward 3a and 3b suggested an alternative method to produce individual α, β-CHX-ATP diastereomers. Incubation of a α, β-CHBr-ATP diastereomer mixture¹¹ with the Src kinase, Src-tide, and a tyrosine phosphatase YopH affords a catalytic cycle for phosphorylation of Src-tide (Figure 2). Using this enzymatic system, we succeeded in converting only one diastereomer of α, β-CHBr-ATP into the diphosphate form, which could be readily separated from the remaining triphosphate diastereomer by anion-exchange HPLC (Figure S10). The same method also succeeded in the enzymatic resolution of a mixture of α, β-CHCl-ATP diastereomers (Figure S11).

Figure 2.

Enzymatic approach to resolve a mixture of α, β-CHX-ATP diastereomers.

To test the generality of diastereoselective utilization of α, β-CHX-ATP by kinases, we measured the kinetic parameters of 3a-1 and 3a-2 along with ATP for a panel of 12 divergent kinases using an established microfluidic assay based on mobility shift of peptide substrates upon phosphorylation.²³ The K_M and k_cat values could be calculated for both 3a-1 and 3a-2 for nine out of the twelve kinases, which display stereospecificity constants ranging from 8 to 174 (Table S3). The different stereospecificity determined for Src kinase using the fluorescence-based and mobility shift-based assays may be due to the differences between the two assays and the Src proteins used. The kinetic parameters could be derived for 3a-2 but not 3a-1 (activity below detection limit) for three other kinases, which again show preference for the same diastereomer. These results suggest that high stereospecificity for α, β-CHF-ATP seems to be common to many protein kinases.

To examine further the scope of this selectivity, we tested the ability of α, β-CHF-ATPs to serve as the phosphoryl donor in the whole proteome in HEK293 cell lysates, taking advantage of a chemical-immunological method for detecting thiophosphorylation developed by Shokat.²⁴ The previously unavailable individual diastereomers of α, β-CHF-γ-thiol-ATP were synthesized by treating the separated diastereomers 2a with thiophosphate followed by deprotection with HCl at −20 °C (Scheme 1). (S)-α, β-CHF-γ-S-ATP produced intense labeling of numerous proteins throughout the lane, whereas the R diastereomer caused no visible labeling of any bands (Figure 3), demonstrating that protein kinases generally exhibit high selectivity for (S)-α, β-CHF-γ-S-ATP in phosphorylating a wide spectrum of substrates. Although it produced a labeling pattern that is similar overall to that of γ-S-ATP, (S)-α, β-CHF-γ-S-ATP labeled a prominent band of ∼33 kDa not labeled by γ-S-ATP (Figure 3), suggesting that certain cellular kinases utilize (S)-α, β-CHF-γ-S-ATP more efficiently than γ-S-ATP. Identification of such kinases and their substrates using our probes will provide valuable new insight for understanding biological phosphorylation mechanisms.

Figure 3.

Utilization of γ-S-ATP, (R)-α, β-CHF-γ-S-ATP and (S)-α, β-CHF-γ-S-ATP by various kinases to phosphorylate substrate proteins in cell lysates. All γ-S nucleotides were used at 0.25 mM, and thiolphosphorylation was detected using a monoclonal antibody.

In conclusion, we describe the first preparation of the individual diastereomers of α, β-CHX-ATP where X = F or Cl. Remarkable stereospecificity (>1000-fold) was observed in the utilization of these α, β-CHX-ATP diastereomers by Src kinase. Crystal structure analysis established the absolute configuration of the CHF and CHCl nucleotide analogues and reveals a potential electrostatic interaction between the fluorine atom in (R)-α, β-CHF-ADP and the ε-amino group of the catalytic lysine. Finally, we demonstrate that the observed selectivity is general for a wide range of eukaryotic protein kinases. Our results raise the interesting possibility that an engineered kinase capable of accepting (R)-α, β-CHX-ATP (the inert isomer) with high efficiency can be used for kinase substrate identification as in the bump−hole approach.²⁵ Beyond providing new mechanistic insights on one of the most centrally important enzymes utilizing ATP, individual α, β-CHX-ATP stereoisomers should have wide application in the study of numerous additional ATP-dependent enzymes.