Letter pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Oxime Ligation via in situ Oxidation of N‑Phenylglycinyl Peptides Quibria A. E. Guthrie and Caroline Proulx* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, United States S Supporting Information *
ABSTRACT: Mild conditions for oxime ligations via in situ generation of α-imino amide intermediates are reported. The evaluation of a variety of N-terminal N-phenylglycine residues revealed that a metal-free, chemoselective oxidation was possible using oxygen as the only oxidant in buffer at pH 7.0. Moreover, selective unmasking of an inert residue by addition of potassium ferricyanide is demonstrated. These simple and mild conditions, which can be fine-tuned by the electronic properties of the Nphenylglycine residue, offer unique advantages over conventional approaches for oxime ligations.
C
cleavage from the resin and purification, such as periodate oxidation of serine residues, are required to access glyoxylylsubstituted peptides. These conditions have been shown to degrade Met, Cys, Tyr, and Trp residues.9 To circumvent these issues, we report the mild and chemoselective oxidation of an N-terminal N-phenylglycine residue, generating the highly reactive α-imino amide intermediates in situ (Scheme 1b). Chemoselective oxidation of N-phenylglycine residues has been exploited in the past for the synthesis of unnatural amino acids and peptide derivatives.10 However, these transformations often require redox-active catalysts and strong chemical oxidants in stoichiometric amounts.10 Similarly, electron-rich N-terminal N-arylglycines were shown to oxidize to the α-oxo aldehyde in the presence of AgClO4.11 Recently, auto-oxidative coupling reactions with N-phenylglycine esters was reported under aerobic conditions with trace amounts of acid in a MeCN/DCE solvent mixture.12 Herein, we report that oxidation of N-arylglycines and coupling with aminooxy functional groups to give oxime linkages can occur in water, with the electronics of the N-arylglycines dictating optimal conditions. N-(p-MeO-Ph)G-LYRAG13 was synthesized as a test peptide sequence. The installation of the N-arylglycine was accomplished by bromoacetylation of the resin-bound peptide followed by displacement with p-methoxyaniline using standard submononer peptoid synthesis conditions.14,11 To probe the oxidation conditions for in situ generation of the α-imino amide intermediate and ligation, N-(p-MeO-Ph)G-LYRAG (1 mM) and O-benzylhydroxylamine (1 mM) were combined in 100 mM ammonium acetate or phosphate buffer (Figure 1). Ambient atmosphere was used as a mild oxidant by leaving the reaction vessels open to air at different pH. The progress of the reactions was monitored by LCMS at 214 nm, following
hemoselective ligation reactions are utilized in applications ranging from total chemical protein synthesis to siteselective bio-orthogonal protein modifications and beyond.1 For example, α-oxo aldehydes have been used in ligation reactions with aminooxy and hydrazine functional groups to give oximes and hydrazones, respectively.2 α-Effects in aminoxy groups lead to the equilibrium favoring the oxime,3 which has been found to be stable for up to 65 h at pH 8.4 Although the optimal pH for the ligation was found to be near 5,5 addition of p-methoxyaniline catalyzes this reaction at pH 7 by up to 40fold via formation of a protonated aniline Schiff base intermediate (Scheme 1a).6 However, high concentrations of aniline can be toxic to cells7 and may competitively react with endogenous aldehydes and ketones, rendering oxime ligations biorestricted.8 Moreover, additional synthetic steps following Scheme 1. (a) Nucleophilic Catalysis6 and (b) in situ Oxidation of N-Arylglycines for Oxime Ligations (This Work)
Received: March 2, 2018
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.8b00713 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 1. Oxidation and coupling of N-(p-MeO-Ph)G-LYRAG (1) to O-benzylhydroxylamine to give oxime 3 using 1 mM reactant concentration. The extent of the reaction was monitored by LCMS at 214 nm. Each experiment was performed in triplicate.
Figure 2. Percent oxidation of substituted N-phenylglycine-LYRAG analogues 1a−g for pH 4.5−7 after 24 h. Each experiment was performed in triplicate.
quenching of a small aliquot with NaOH. Although < 2% ligation product was observed at pH 7 after 24 h, decreasing the pH to 4.5 afforded oxime 3 in 77% conversion, with no trace of the α-oxo aldehyde (Figure 1). The optimal pH was found to be about one unit below the pKa of p-methoxyaniline (pKa 5.3), suggesting that protonation of the N-arylglycine may facilitate its oxidation to 2. Pleased to bypass NaIO4 in the synthesis of α-oxo aldehydes, we explored oxidation of other N-arylglycines to achieve faster kinetics at neutral pH. Seven N-aryl-G-LYRAG analogues (1a− g) were synthesized, and their conversion to the oxime product was quantified after 24 h at different pH (Figure 2). Inspired by aniline catalysis in oxime ligation of α-oxo aldehydes15 and having noticed a relationship between aniline pKa and oxidation rate, electron-rich N-arylglycines were targeted. Moreover, because anthranilic acid derivatives were previously shown to be superior catalysts in ligations with α-oxo aldehydes, we included analogue 1g, which was envisioned to protonate the N-phenylglycine via an intramolecular proton transfer.15a−c 4(NMe2)aniline is known to be unstable in air-saturated water;15a however, analogue 1f was anticipated to oxidize to the desired α-imino amide intermediate. Most analogues were either unreactive under the entire pH range tested (1d,e,g)16 or exhibited similar reactivity trends as analogue 1b (1c), where a pH of ∼4.5 was required to achieve good conversions after 24 h. In contrast, we were pleased to find that N-(p-Me2N-Ph)GLYRAG (1f) was quantitatively oxidized to a mixture of oxime 3 (major) and α-oxo aldehyde (minor) at pH 6.5 and 7.0. This analogue displayed a reverse reactivity trend where lower pH values decreased the oxidation rate, presumably due to protonation of the 4-dimethylamino group, rendering it electron poor. This trend (1) allows reactivity at physiological
pH conditions, which is essential for biomolecule stability, and (2) prevents acid-promoted decomposition during resin cleavage and purification. Moreover, it greatly simplifies the synthesis of the required precursors for oxime ligation, precluding the incorporation and periodate oxidation of Nterminal serine residues. Upon closer examination, we found that oxidation of 1f was complete after 8 h. However, a 70:30 mixture of the oxime and α-oxo aldehyde was observed under these conditions (Table 1, entry 1). Although the aldehyde would convert to the desired oxime after 24 h, to circumvent the competing hydrolysis of the α-imino amide intermediate, the ratio of O-benzylhydroxylamine/N-phenylG-LYRAG peptide was increased to 5:1. We noted inconsistencies in our results under these conditions along with reduced rates of oxidation, which may be due to excess O-benzylhydroxylamine sequestering oxygen and inhibiting N-phenylglycine oxidation. To address these issues, the reaction was performed under an O2 atmosphere, providing oxime 3 in 91% conversion after only 8 h and decreasing the population of the α-oxo aldehyde to ∼8% (Table 1, entry 2). Of note, current aniline-catalyzed oxime ligation procedures require 24 h to go to completion at neutral pH. To more closely mimic typical concentrations of biomolecules, the tandem oxidation/ligation reaction sequence was attempted under dilute conditions, namely 0.1 mM (1f) and 0.5 mM (Obenzylhydroxylamine). While this 10-fold dilution led to an expected increase in aldehyde byproduct (40%), the desired oxime remained the major product (60%) under these low concentrations (Table 1, entry 3). This convenient aerobic one-pot oxidation/oxime ligation reaction sequence should find broad applicability in bioconjugations, in particular, where exposure to periodate B
DOI: 10.1021/acs.orglett.8b00713 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Table 1. Oxidation Conditions for N-Phenylglycine-Terminated Peptides 1f and 1b and Conversions (%) as Determined by LCMS entry
substrate
oxidation conditions at pH 7
time
initial aminooxy/peptide ratioa
% oxidation
aldehyde/oxime ratio
% oxime product
1 2 3 4 5 6 7 8
1f 1f 1f 1f 1b 1b 1b 1b
air O2b air 1 mM K3[Fe(CN)6]d air 1 mM K3[Fe(CN)6] 10 mM K3[Fe(CN)6] 10 mM K3[Fe(CN)6]
8h 8h 8h 5 min 24 h 24 h 24 h 4h
1:1 5:1 5:1c 5:1 1:1 5:1 5:1 5:1
>99 >99 >99c 84 99 96
30:70 8:92 40:60c 14:86 N/A 7:93 6:94 12:87
70 91 60c 70e 1 mM K3[Fe(CN)6] is used (see the Supporting Information). fAppearance of an over-oxidation byproduct (M + 16) is observed after 24 h.
by several orders of magnitude (5 min vs 8 h). To the best of our knowledge, these conditions represent one of the fastest reported oxime ligation reactions. Furthermore, oxidation of a methionine side chain was not observed under these conditions (1 mM K3[Fe(CN)6], 5 min) using N-(p-Me2N-Ph)G-LYRAM as the peptide substrate. In addition to identifying substituted N-phenylglycines that could undergo mild in situ oxidation at pH 7, we were also interested in pursuing them as masked electrophiles, where residues that are inert at pH 7 could be selectively unmasked in the presence of chemical oxidants. In principle, formation of αimino amide intermediates may be confined to environments under oxidative stress, such as cancer cells, leading to environmentally triggered ligations. Peptide 1b, which exhibited very low reactivity at pH 7 under aerobic conditions, was treated with 1−10 mM potassium ferricyanide (Table 1, entries 6−8). Gratifyingly, with 1 mM K3[Fe(CN)6], oxime 3 was obtained in 66% conversion after 24 h, with the major byproduct being unreacted peptide 1b (Table 1, entry 6). Increasing K3[Fe(CN)6] concentration by 10-fold led to >99% oxidation after 24 h; however, appearance of a byproduct with M + 16 relative to the oxime was observed over time, likely a result of tyrosine oxidation (Table 1, entry 7). Quenching this reaction after 4 h led to 84% oxime formation with no detectable overoxidation byproducts (Table 1, entry 8). The selective unmasking of N-(p-MeO-Ph)glycines with potassium ferricyanide is a unique feature of this chemistry, rendering it orthogonal to aldehydes at pH 7. To confirm this, glucose was selectively coupled to O-benzylhydroxylamine in the presence of peptide 1b and aniline catalyst (100 mM) at pH 7 to give oxime 6 (Scheme 3a).18 Conversely, peptide 1b was treated with 1 mM K3[Fe(CN)6] in the presence of O-benzylhydroxylamine (1 mM) and glucose (1 mM) with no aniline catalyst (Scheme 3b). Oxime 3 was obtained as the only detectable product under these conditions; however, reactivity with glucose is observed to give 6 as a minor product when excess O-benzylhydroxylamine and glucose are used under prolonged reaction times, likely facilitated by the release of 1 equiv of p-anisidine upon formation of 3. In summary, mild aqueous conditions (O2, pH 7) were shown to provide oxime ligation products in >90% yield after 8 h using N-arylglycine-terminated-peptides in a one-pot oxidation/ligation sequence. Under these mild conditions, a peptide containing a C-terminal methionine residue gave the desired oxime ligation product in 90% conversion with no sign of side-chain oxidation to the sulfoxide or sulfone after 8 h. The
reagents is problematic (e.g., in the presence of Met, Ser, Trp) and/or where competing aldehyde functional group may exist. To demonstrate this, N-(p-Me2N-Ph)G-LYRAM, possessing a C-terminal methionine, was subjected to our mild ligation conditions (O2, pH 7) with O-benzylhydroxylamine (5 mM) for 6 h, giving the oxime product in 90% conversion with no sign of methionine oxidation over that time (see the Supporting Information). In the oxime ligation of two peptides, namely 1f and aminooxyacetyl-GRGDSGG (4), >99% oxidation of 1f was observed after only 4 h, albeit affording an oxime/α-oxo aldehyde ratio of ∼1:1 (Scheme 2). As a point of comparison, Scheme 2. Ligation of 1f with Aminooxyacetyl-GRGDSGG (4) to Give 5 under O2 Atmosphere at pH 7
oxime 5 was previously reported to proceed to 78% conversion, requiring 22 h reaction time, 100 mM p-methoxyaniline catalyst, and synthesis and isolation of glyoxylyl-LYRAG. To shorten reaction times, we evaluated mild oxidants such as potassium ferricyanide, which was previously shown to be compatible with biomolecules possessing free sulfhydryl groups and glycoproteins in other oxidative coupling reactions.17 Substrate 1f (1 mM) was treated with K3[Fe(CN)6] (1 mM) in the presence of O-benzylhydroxylamine (5 mM), and the reaction was monitored by LCMS following quenching of a small aliquot with tris(2-carboxyethyl)phosphine (TCEP). Remarkably, after 5 min, peptide 1f showed 70% conversion to oxime 3 (Table 1, entry 4). The reaction proved sensitive to oxidant concentration, with appearance of byproducts when treated with >1 mM potassium ferricyanide; however, careful control over the reaction conditions reduced the reaction time C
DOI: 10.1021/acs.orglett.8b00713 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
(4) (a) Rose, K.; Zeng, W. G.; Regamey, P. O.; Chernushevich, I. V.; Standing, K. G.; Gaertner, H. F. Bioconjugate Chem. 1996, 7, 552−556. (b) Kalia, J.; Raines, R. T. Angew. Chem., Int. Ed. 2008, 47, 7523−7526. (5) Shao, J.; Tam, J. P. J. Am. Chem. Soc. 1995, 117, 3893−3899. (6) Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Angew. Chem., Int. Ed. 2006, 45, 7581−7584. (7) Tuley, A.; Lee, Y. J.; Wu, B.; Wang, Z. U.; Liu, W. R. Chem. Commun. 2014, 50, 7424−7426. (8) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13−21. (9) (a) Geoghegan, K. F.; Stroh, J. G. Bioconjugate Chem. 1992, 3, 138−146. (b) Clamp, J. R.; Hough, L. Biochem. J. 1965, 94, 17−24. (c) Haney, C. M.; Horne, W. S. Chem. - Eur. J. 2013, 19, 11342− 11351. (10) For select examples, see: (a) Zhao, L.; Li, C. Angew. Chem., Int. Ed. 2008, 47, 7075−7078. (b) Zhao, L.; Basle, O.; Li, C. J. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 4106−4111. (c) Zhang, G.; Zhang, Y.; Wang, R. Angew. Chem., Int. Ed. 2011, 50, 10429−10432. (d) Zhu, S. Q.; Rueping, M. Chem. Commun. 2012, 48, 11960−11962. (e) Huo, C. D.; Wang, C.; Wu, M. X.; Jia, X. D.; Xie, H. S.; Yuan, Y. Adv. Synth. Catal. 2014, 356, 411−415. (f) San Segundo, M.; Guerrero, I.; Correa, A. Org. Lett. 2017, 19, 5288−5291. (11) Proulx, C.; Yoo, S.; Connolly, M. D.; Zuckermann, R. N. J. Org. Chem. 2015, 80, 10490−10497. (12) Huo, C.; Yuan, Y.; Wu, M.; Jia, X.; Wang, X.; Chen, F.; Tang, J. Angew. Chem., Int. Ed. 2014, 53, 13544−13547. (13) The LYRAG peptide sequence was chosen for comparison to aniline-catalyzed oxime ligations reported in ref 6. (14) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am. Chem. Soc. 1992, 114, 10646−10647. (15) (a) Crisalli, P.; Kool, E. T. J. Org. Chem. 2013, 78, 1184−1189. (b) Larsen, D.; Pittelkow, M.; Karmakar, S.; Kool, E. T. Org. Lett. 2015, 17, 274−277. (c) Crisalli, P.; Kool, E. T. Org. Lett. 2013, 15, 1646−1649. (d) Rashidian, M.; Mahmoodi, M. M.; Shah, R.; Dozier, J. K.; Wagner, C. R.; Distefano, M. D. Bioconjugate Chem. 2013, 24, 333−342. (e) Wendeler, M.; Grinberg, L.; Wang, X. Y.; Dawson, P. E.; Baca, M. Bioconjugate Chem. 2014, 25, 93−101. (f) Yuen, L. H.; Saxena, N. S.; Park, H. S.; Weinberg, K.; Kool, E. T. ACS Chem. Biol. 2016, 11, 2312−2319. (16) In aniline-catalyzed hydrazone ligations, a >50-fold decrease in reactivity at pH 7 was observed when 3,5-dimethoxyaniline was compared with 2,4-dimethoxyaniline (see ref 15f). Given the pKa of 3,5-dimethoxyaniline (3.8), it is possible that 1d and other unreactive analogues would undergo the tandem oxidation/oxime ligation reaction at lower pH values (< 4.5). The pKa of 3,5-dimethoxyaniline can be found in: Bryson, A. J. Am. Chem. Soc. 1960, 82, 4858−4862. (17) (a) Obermeyer, A. C.; Jarman, J. B.; Netirojjanakul, C.; El Muslemany, K.; Francis, M. B. Angew. Chem., Int. Ed. 2014, 53, 1057− 1061. (b) Yamagishi, Y.; Ashigai, H.; Goto, Y.; Murakami, H.; Suga, H. ChemBioChem 2009, 10, 1469−1472. (18) (a) Thygesen, M. B.; Munch, H.; Sauer, J.; Clo, E.; Jorgensen, M. R.; Hindsgaul, O.; Jensen, K. J. J. Org. Chem. 2010, 75, 1752−1755. (b) Carrasco, M. R.; Brown, R. T. J. Org. Chem. 2003, 68, 8853−8858.
Scheme 3. Selective Ligation at pH 7 of (a) Glucose in the Presence of 1b under Aniline Catalysis and (b) 1b in the Presence of Glucose and Potassium Ferricyanide Oxidant
reaction time can be reduced to 5 min if treated with potassium ferricyanide, which constitutes one of the fastest reaction conditions to yield oximes. We further demonstrated that inert residues can be unmasked by K3[Fe(CN)6] at pH 7. This allows for orthogonal functionalization in the presence of other aldehydes, which is a unique feature of this chemistry. The chemoselective oxidation of N-phenylglycines to generate protonated aniline Schiff base intermediates in situ under mild conditions should minimize problems associated with (1) aniline catalyst toxicity in vivo, (2) competition with endogeneous aldehyde and ketone groups in live cells, and (3) over-oxidations of proteins/peptides upon prolonged exposure to periodate reagents previously required to synthesize α-oxo aldehydes. Further mechanistic studies and applications of this concept are underway and will be reported in due course.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00713. Experimental procedures; LC−MS analytical data for 1a−g before and after oxime ligations (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Caroline Proulx: 0000-0003-3851-793X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS C.P. gratefully acknowledges North Carolina State University for startup support. REFERENCES
(1) (a) Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011, 7, 876−884. (b) Kent, S. B. H. Chem. Soc. Rev. 2009, 38, 338−351. (2) For reviews, see: (a) El-Mahdi, O.; Melnyk, O. Bioconjugate Chem. 2013, 24, 735−765. (b) Kolmel, D. K.; Kool, E. T. Chem. Rev. 2017, 117, 10358−10376. (c) Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P. Chem. - Eur. J. 2014, 20, 34−41. (3) Sander, E. G.; Jencks, W. P. J. Am. Chem. Soc. 1968, 90, 6154− 6162. D
DOI: 10.1021/acs.orglett.8b00713 Org. Lett. XXXX, XXX, XXX−XXX
