Enzymatic Fluoromethylation as a Tool for ATP‐Independent Ligation

Abstract S‐adenosylmethionine‐dependent methyltransferases are involved in countless biological processes, including signal transduction, epigenetics, natural product biosynthesis, and detoxification. Only a handful of carboxylate methyltransferases have evolved to participate in amide bond formation. In this report we show that enzyme‐catalyzed F‐methylation of carboxylate substrates produces F‐methyl esters that readily react with N‐ or S‐nucleophiles under physiological conditions. We demonstrate the applicability of this approach to the synthesis of small amides, hydroxamates, and thioesters, as well as to site‐specific protein modification and native chemical ligation.

Amide bond formation is one of the most important reactions in biology and also in synthetic chemistry, since amides occur in many therapeutics, agrochemicals, food additives, plastics, and dyes.Consequently, a large body of chemical literature documents methodologies, reagents and processes to connect amines with carboxylates, continuously expanding the scope, efficiency, selectivity, and economic viability of condensation reactions. [1]Biocatalysis plays an increasing role in this development, empowered by the growing list of known amide bond forming enzymes and increasing ability to engineer enzymes with tailormade amide bond forming activities. [2]Under physiological conditions, the central challenge to condensation reactions between amines and carboxylates is that the reaction equilibrium strongly favors the educts.The same problem applies to the formation of other anhydrides including esters, thioesters, and hydroxamates, with notable exceptions such as disulfides and thioethers.
2d] ATP-dependent ligases conjugate their carboxylate substrates with either phosphate or adenosine-5'-monophosphat (A, Figure 1).2d,3] The most promising examples include an ATP-recycling cascade that uses polyphosphates instead of ATP as the stoichiometric dehydration agent. [4]here are surprisingly few documented enzymes that activate carboxylates without ATP or other NTPs.A rare example participates in the biosynthesis of the nucleoside antibiotic capuramycin (B, Figure 1).In this process a S-adenosyl methionine (SAM)-dependent methyltransferase (CapS) methylates the carboxylate of a biosynthetic intermediate. [5]The resulting methyl ester is conjugated with the amine nucleophile of aminocaprolactam by the acyltransferase CapW.2e,6] This present report is inspired by this idea.We describe an MT-catalyzed process for the formation of hydroxamates, thioesters and amides without the need of ATP or acyltransferases.We have harnessed this methodology for the synthesis of low molecular weight hydroxamates, thioesters and amides, for specific labeling of iso-Aspartate (isoAsp) containing peptides, for C-terminal protein labeling, and protein-protein ligation.
Conjugation of Cys to 1 and 6 likely proceeds via nucleophilic attack of the Cys thiolate onto the F-methyl ester, followed by S-to-N acyl transfer. [9]To test as to whether F-methyl esters would also react with amines directly, we examined the products of the same IAAMT and HMT catalyzed reaction, containing 10 mM 6-aminopenicillanic acid instead of Cys (Figure 2).HPLC analysis showed that this reaction converted 80 % of 6 to the corresponding conjugate 9 ([C 11 H 23 N 2 O 4 ] + calcd: m/z 374.1169, found: 374.1175, Figure S21) within 21 h (Figure S20).20 % of 6 remained unreacted.These results demonstrate that a thioester intermediate is not required for conjugation, and that comparatively bulky nucleophiles are tolerated.

Peptide and protein labeling:
Encouraged by these results with small carboxylates we explored as to whether MTs could also mediate conjugation to peptides or proteins.First, we studied the example of protein-L-isoaspartate O-methyltransferase (PIMT).This enzyme methylates the α-carboxylate of isoAsp moieties in proteins and peptides that underwent spontaneous isomerization of the peptide backbone (10, Figure 3). [10]The resulting isoAsp methyl ester (11) reacts to a succinimide intermediate (12) that then hydrolyzes either to reproduce the starting isoAsp moiety, or an Asp residue with an eupeptide bond (13).10b,11] Hence, multiple rounds of methylation by PIMT, cyclization and hydrolysis may be necessary to restore the activity and stability of damaged protein.At the present time, the physiological function of this activity in animals, plants, fungi and bacteria is not clear. [12,13,14]In order to address this question, a number of reports describe labeling techniques to identify and quantify potential PIMT substrates. [15,16]15a,c,16-17] This Scheme is elegant, but the protocols published so far are not efficient enough to detect isoAspor succinimide-containing peptides in complex samples.Hence, improving the efficiency of isoAsp-conjugation reactions is an important objective.

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To test as to whether F-methylation could help in this regard, we incubated a synthetic isoAsp-containing peptide (10, [C 103 H 165 N 25 O 32 ] 2 + calcd: m/z 1132.604,found: 1132.595, Figure 3 and S22) with 50 μM HMT and 2.8 μM human PIMT (PDB: 1I1N), 5 mM FMeI or MeI and SAH (50 μM) in 0.1 M phosphate buffer at pH 7 in the presence of 1 M methoxyamine.The progress of this reaction was monitored by liquid chromatography-coupled MS (Quadrupole Time of Flight Mass Spectrometer) and the signal intensities for different peptide species were used to estimate their relative concentration (Figure 3).The FMeI containing reaction afforded almost complete conversion of substrate peptide to the succinimide-containing derivative within the first hr (  [10b,11] the accumulating product is likely a mixture of two regioisomers (Figure 3).The MeI-containing reaction converted all peptide to the methyl ester in the first hr (  S22), but formation of the conjugate (15) occurred much more slowly.Fitting the time dependent concentrations of the different peptide species to a sequential two-step reaction (see Supporting Information for details) shows that the rate of intermolecular conjugation with methoxyamine is similar in both reactions (k 2 , Figure 3), consistent with the idea that methoxyamine reacts with the same electrophilic succinimide intermediate (12).Formation of this obligatory intermediate, on the other hand, occurs 10-fold faster with FMeI (k 1 ) than with MeI (k 1 ') as the conjugating agent.
F-methylation was also effective in labelling this model peptide with hydroxylamine-derivatized biotin in a one-pot reaction (16, [C 121 H 198 N 29 O 37 S] 3 + calcd: m/z 894.141, found: 894.134, Figure S23).PIMT-mediated conjugation to biotin has been proposed as a strategy for enriching and identifying low-abundance isoAsp-containing peptides and proteins in proteome samples.15a] A second type of SAM-dependent MTs with potential applications in protein ligation may be the carboxylate MT LahS B from Lachnospiraceae bacterium (PDB: 6UAK). [18]his enzyme methylates the C-terminal Val, Ile or Met residues of several translated precursors of peptide natural products (LahA2-A5).Unlike other protein carboxylate

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MTs, such as the eukaryotic leucine carboxyl MT or the lanthipeptide maturing enzyme OlvSA which require their substrates to adopt a stable three-dimensional fold for recognition, [19] LahS B appears to recognize unmodified substrates by just the last 15, or possibly fewer residues.Based on this report, we designed an expression plasmid coding for a green fluorescent protein (GFP, PDB: 2B3P) [20] with a 15-residue C-terminal tag mimicking the terminus of the precursor peptide LahA2 (sequence: DGDEVDYSL-FAATAM, GFP-tag-1).Incubation at pH 8.0 in the presence of 1 mM SAM, 10 μM LahS B for 4 h at 25°C converted GFP-tag-1 (20 μM) completely to methylated GFP-tag-1 (17) as inferred by HR-ESI-MS (17, calcd: m/z 30509.3,found: 30509, Figure S24).A reaction containing 1 mM FMeI, 20 μM SAH, 10 μM LahS B , 10 μM HMT and 2 mM cysteine converted more than 90 % of GFP-tag-1 (20 μM) to the cysteine adduct 19 as inferred by relative signal intensities of the corresponding HR-ESI-MS spectrum (calcd: m/z 30598.8,found: 30598, A, Figure 4 and S25).Increasing or decreasing the reaction pH (6.5-8.5)reduced this yield significantly, presumably due to an increased rate of ester hydrolysis or reduced concentration of anionic Cys (Figures S26-S29).

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methylation protein has the capacity to slowly undergo intramolecular condensation.The same species (GFP-tag-1-H 2 O) also forms in some of the conjugation reactions described above as a minor side product.
The same methodology was used to conjugate small molecules to a nanobody (PDB: 3OGO) [23] with a C-terminal tag-1 (24-28, Figure 4 and S37-S42).These results suggest that MT-mediated C-terminal protein labeling may be an efficient and easy to implement alternative to transpeptidase-mediated ligation technologies. [24]One potential advantage of the present approach over these established methodologies is that conjugation to the F-methyl esters does not require catalysis and therefore tolerate nucleophiles that are not accepted by transpeptidases.
Finally, we as to whether the F-methylated Cterminus of GFP-tag-1 could also react with the N-terminal Cys residue of another protein (Figure 5).To this end we incubated 0.3 mM GFP-tag-1 with 0.6 mM of a nanobody variant that features an N-terminal Cys residue (nanobody-C16) together with HMT (20 μM), LahS B (20 μM), SAH (40 μM), a total of 8 mM FMeI in 50 mM phosphate buffer at pH 8.0 at 15 °C.After 48 h the reaction mixture was analyzed by SDS-PAGE and HR-ESI-MS (Figure 5  Chemoselective conjugation of a protein with an activated C-terminus to a protein with an N-terminal Cys to form an eupeptide bond has been termed native chemical ligation. [25]One of the most important discoveries in chemical biology, this methodology has enabled the assembly of chemically defined large proteins from synthetic peptides and recombinant protein fragments. [26]Although there are established protocols for the production of peptides with activated C-termini, [27] we anticipate that MTmediated in situ activation of recombinant proteins can open up new options for protein synthesis, not the least because this approach is simple to implement and requires only off-the-shelf chemical reagents.Combining protein Fmethylation with ligation auxiliaries and selenium-assisted ligation, [28] provide promising options to further improve the observed conjugation efficiency and possibly suppress unwanted side products. In conclusion, MT-mediated F-methylation of carboxylic acids produces reactive F-methyl esters that readily condense with N-and S-nucleophiles under physiological conditions.We have illustrated possible applications of this reaction in the synthesis of small thioesters, hydroxamates, and amides, as a tool for isoAsp-specific and C-terminal protein conjugation, and for protein synthesis by native chemical ligation.The simplicity and broad substrate scope of MT-mediated ligation makes this approach amenable to further improvement and adaptation.As an exciting example we note the recent discovery that (formal) substitution of the sulfur atom in F-SAM to tellurium generates a more stable reagent for enzyme-catalyzed F-methylation. [29]

Figure 1 .
Figure 1.A: ATP-dependent ligases activate carboxylate substrates by producing phosphate or adenosine-5'-monophosphat (AMP) esters.B: Specific carboxylate MTs (CapS) combined with matching acetyltransferases can mediate amide bond formation by a SAM-dependent mechanism.C: Carboxylate MT-catalyzed F-methylation provides a more broadly applicable mechanism for ATP-independent anhydride-formation.D: HMT-catalyzed production of F-SAM as a reagent for carboxylate F-methylation.

Figure 2 .
Figure 2. MT-catalyzed F-methylation activates low molecular weight carboxylates for uncatalyzed formation of hydroxamates, amides and thioethers.The methyl esters of the same carboxylates react much less efficiently with nucleophiles.

Figure 3 .
Figure 3. PIMT-mediated peptide labeling.Top: PIMT-catalyzed methylation or F-methylation of isoAsp moieties in a model peptide (10) produces the corresponding methyl ester (11) or F-methyl ester (14).Both esters collapse to a succinimide intermediate (12) that can react with an external nucleophile to form an O-methyl hydroxamic acid (15).Bottom: Reaction progress of nucleophilic trapping of an isoAsp-containing peptide upon PIMT-dependent F-methylation (left) and methylation (right).The reactions are fit to a 2-step sequential kinetic model as described in the SI.The ranges correspond to the standard error of the fit.The constants k 1 and k 1 'describe the rate at which intermediate 12 is formed; k 2 describes the rate at which intermediate 12 is conjugated to methoxyamine.