Generation of Hydrogen Cyanide from the Reaction of Oxyma

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Generation of Hydrogen Cyanide from the Reaction of Oxyma (Ethylcyano(hydroxyimino) acetate)and DIC(Diisopropylcarbodiimide) Adam Dues McFarland, Jonas Y Buser, Matthew C Embry, Charles Brad Held, and Stanley P. Kolis Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00344 • Publication Date (Web): 04 Sep 2019 Downloaded from pubs.acs.org on September 4, 2019

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Organic Process Research & Development

Generation of Hydrogen Cyanide from the Reaction of Oxyma (Ethyl cyano(hydroxyimino) acetate) and DIC (Diisopropylcarbodiimide) McFarland, Adam D.; Buser, Jonas Y.; Embry, Matthew C.; Held, Charles B.; Kolis, Stanley P.* [email protected] Small Molecule Design & Development, Eli Lilly and Company, Lilly Corporate Center, Indianapolis IN 46285 U.S.A.to

Abstract Evolution of hydrogen cyanide (HCN) during amino acid activation using the reagent combination ethyl cyano(hydroxyimino) acetate/diisopropylcarbodiimide (Oxyma/DIC) is observed under ambient conditions (20 °C) in DMF. Concentration vs time profiles obtained by 1H NMR in the presence and absence of amino acids indicate that HCN is formed upon addition of DIC to the reaction mixture, and that HCN evolution continues to occur even after amino acid activation is complete when Oxyma and DIC are used in excess amounts (compared to the amino acid). A mechanism for the reaction between Oxyma and DIC is proposed and evidence for its validity was gathered by NMR.

Keywords:

oxyma, diisopropylcarbodiimide, hydrogen cyanide, amino acid activation, NMR

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Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Amino acid activation plays a central role In the synthesis of both small molecules and synthetic peptides, and the catalog of reagents available for accomplishing these transformations is expansive.1 The reagent combination of ethyl cyanohydroxyiminoacetate (Oxyma, 2) and diisopropylcarbodiimide (DIC, 3) has seen an increase in use due to many attractive attributes, including prevention of racemization2 and greater thermal stability when compared to the common hydroxybenzotriazole (HOBt) based reagents.3 Due to these benefits, low cost and organic solubility of the diisopropylurea by-product this reagent combination is now the most common system in solid phase peptide synthesis (SPPS).4 During a study of amino acid activation kinetics using Oxyma/DIC, hydrogen cyanide (HCN) generation was observed throughout the course of the reaction. Due to the hazards of exposure to small amounts of HCN gas5, this disclosure will discuss the methods used to conclusively identify the gas, as well as some qualitative understandings of the kinetics and a proposed mechanism for HCN generation. As part of an effort focused on exploring opportunities to optimize cycle time associated with certain amide bond-forming reactions, the activation of several Fmoc-protected amino acids using Oxyma/DIC was studied using 1H NMR. Figure 1 shows a series of 1H NMR spectra collected at different times during an Fmoc-L-Ala-OH (1) activation reaction conducted in DMF-d7 at 20 °C. In addition to the resonances assigned to the reactants and known products of the activation reaction, a singlet at 6.20-6.25 ppm was consistently observed to increase in intensity over the course of the experiment. In this reaction, the integral value of the signal at 6.25-ppm reached greater than 5% of that of the nearby diisopropylurea (5) resonance at 5.6 ppm by the end of the study. Because the resonance was not readily assigned to any of the known reaction species and had been observed at various levels during similar activations of other amino acids, efforts were focused on determining the identity of the compound that engendered this signal. Early assumptions that this was related to the loss of the Fmoc protecting group resulting in the generation of dibenzofulvene were quickly dismissed. Heteronuclear correlation experiments revealed direct connectivity of the 6.25-ppm proton to a carbon resonance at 113.5-ppm, as well as the presence of a 257-ppm nitrogen (referenced to liquid NH3). Direct 13C measurement with and without 1H decoupling unambiguously demonstrated that only one proton was bound to the 113.5-ppm carbon.6 These data, along with the lack of any additional coincident heteronuclear correlations, led logically to the conclusion that the compound under investigation was hydrogen cyanide. This conclusion was ultimately confirmed by spiking a small amount of HCN (prepared by acidifying KCN in DMF with trifluoroacetic acid vapors) into the reaction solution and observing an increase in the 6.25-ppm 1H NMR resonance.


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Figure 1. Observation of HCN generation during activation of Fmoc-L-Ala-OH by 1H NMR. Reaction progresses from bottom to top. The targeted stoichiometry for this reaction was 1.0:3.0:3.0 (Amino acid:Oxyma:DIC).

Despite confidence in the identification by NMR, further confirmation of the identification of HCN in the solution phase of the reaction mixture was pursued by orthogonal means. Colorimetric methods7 were deemed unreliable due to incompatibility of the reaction matrix with the test mechanism. Instead, headspace gas chromatography with flame ionization detection was used to confirm the presence of HCN in solution. The retention time of HCN was determined by analysis of a DMF reaction mixture containing potassium cyanide acidified with trifluoroacetic acid. The gas chromatograms of an activated Fmoc-L-AlaOH reaction mixture, the Oxyma/DIC reaction mixture studied for mechanistic insight described further below, and control samples are shown in Figure 2. These data further confirm that activation and Oxyma/DIC reaction mixtures contain HCN.

Figure 2. Gas chromatograms confirming the presence of HCN in solutions containing Oxyma and DIC.

A qualitative examination of the concentration vs. time graph measured by NMR (Figure 3) shows trends consistent with expectations for this reaction. Conversion of the limiting reagent, Fmoc-L-Ala-OH (1), to the activated ester (4) is ~90% complete at 4 hours. Based on the balanced chemical equation, one would



expect the molar ratio of the activated ester (4) and by-product diisopropylurea (5) to be approximately equal. However, one can clearly see the ratio departing from unity even at very early time points, and the ratio of diisopropylurea to activated ester steadily increases over the time period monitored.

Figure 3. Concentration vs Time profile for Activation of Fmoc-L-Ala-OH with Oxyma/DIC in DMF at 20 °C. The targeted stoichiometry for this reaction was 1:1.5:1.5 (Amino acid:Oxyma Pure:DIC).

An examination of the secondary axis in Figure 3 illustrates the behavior of the hydrogen cyanide in solution. Clearly, the HCN formation under these conditions is not the dominant process (note the magnitude of the concentrations on the main axis vs the secondary axis). Regarding the HCN behavior, however, two items are noteworthy: (1) HCN evolution starts at the inception of the reaction (when the DIC is added to the reaction mixture) and (2) HCN generation continues even after the main reaction has effectively ceased. This observation led us to hypothesize that Oxyma was reacting directly with DIC to generate HCN during the reaction. A proposed mechanism for the observed HCN formation is shown in Figure 4. Evidence for the mechanism was gathered via NMR studies of Oxyma/DIC solutions in DMF-d7 in the absence of an amino acid or external base. The first step of the proposed mechanism is analogous to the generally accepted mechanism for amino acid activation by DIC.8-9 The transient nature of the open chain intermediate 7 1012 prevented complete structural elucidation of this species at 20 °C. Conducting the reaction at -30 °C, however, sufficiently slowed the reaction kinetics to allow for thorough characterization of 7 in situ (Figure 5), supporting its role in the proposed mechanism. Upon warming this reaction solution to 20 °C, the resonances corresponding to 7 disappeared and were replaced by the resonances characteristic of cyclic product 9 and HCN. After originally characterizing cyclic product 9 in situ, subsequent effort was applied toward isolating a purified sample of the compound for further confirmation of the structure. Oxyma (911 mg, 6.4 mmol) and DIC (1.0 mL, 7.8 mmol, 1.2 equiv) were combined in DMF (10-mL) and held at ambient temperature. After NMR analysis revealed that the reaction had achieved ~33% conversion to 9, 5-mL of the reaction solution was diluted with ethyl acetate (5-mL) and washed with water (3x10-mL). The organic phase was concentrated on a rotary evaporator yielding a thick, red liquid. A Teledyne CombiFlash massguided isolation system was then used to isolate a purified sample of 9 from this mixture, which was


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analyzed by NMR and LC-HRMS. All the characterization data (Figure 6) supports the structure proposed for 9. Confirmation of the presence of 9 itself provides indirect support of the generation of HCN, as formation of 9 from 7 entails the liberation of one equivalent of HCN. i

N NC

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i

N

3

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PrN

PrN C NiPr

O

i

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N i H Pr 8

7

N O NiPr

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6

2

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Proton

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+

N

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Figure 4. Proposed mechanism for HCN generation from reaction of Oxyma with DIC

Figure 5. NMR chemical shift assignments and key correlations for 7 (open-chain intermediate) in DMF-d7 at -30°C.



Figure 6. Structural characterization data for 9 (cyclic product). (A) NMR chemical shift assignments and key correlations in DMF-d7 at 25°C and (B) HRMS characterization supporting a M+H formula of C11H20N3O3+, with major fragment identities proposed.

With good evidence for the proposed reaction mechanism in hand, we embarked on a deeper examination of the time course of the reaction. The concentration vs time graphs for the reaction of Oxyma with DIC in DMF at 20 °C and 50 °C are shown in Figure 7. Qualitatively, a few items are of note. Oxyma and DIC both appear to react via first order mechanisms, with DIC disappearing more rapidly than Oxyma (vide infra). As discussed above, open chain intermediate 7 does not accumulate to an appreciable extent at ambient temperature, but instead rapidly cyclizes to form cyclic product 9 and HCN. The proposed reaction mechanism and conservation of mass should imply that HCN and 9 are formed in a 1:1 ratio, but examination of the concentration vs time data in Figure 7 shows an ~20 mM difference in concentration ([9] > [HCN]. This discrepancy is explained by the reasonable assumption that the HCN gas is escaping to the vapor phase over the course of the reaction, and as such is not observable by NMR.


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Figure 7B illustrates that at 50 °C and 250mM Oxyma/DIC concentration, the HCN yield is ~20% after just 5 hours of reaction time. The rate and yield of the HCN-forming reaction under specific conditions should be carefully considered, especially in the context of potential unintended operations that could occur at manufacturing scale.

Figure 7. Concentration vs. Time Plots for reaction of DIC with Oxyma in the absence of amino acid at 20 °C (A) and 50 °C (B).

Diisopropylurea (5) also forms during the reaction. While this is expected in the case of the reaction where an amino acid is present based on the commonly accepted reaction mechanism for activation, its presence in this reaction is likely the result of reaction of residual water from reagents or the reaction solvent. The presence of diisopropylurea likely explains the exaggerated discrepancy between the concentration of Oxyma and DIC in the reaction solution. The most obvious pathway for DIC consumption is from direct reaction with water from the reagents or solvents used. In addition, hydrolysis of 7 (Figure 4) would regenerate Oxyma but consume DIC, which is also a reasonable explanation for the discrepancy in molar amounts of these two materials. Independent 1H NMR experiments examining the course of the direct reaction between DIC and water at both 20 °C and 50 °C indicate that the reaction is very slow, therefore hydrolysis of 7 is likely the primary mechanism for forming urea in this system.

Figure 8. Summary of Activated Amino Acid Concentration (A) and HCN concentration (B) for various stoichiometries of Fmoc-LLeu-OH:Oxyma:DIC



Figure 8 shows a summary of the behavior of the HCN generation at 20 °C for various reaction stoichiometries (see legend in Figure) during the activation of Fmoc-L-Leu-OH. The trend for the amino acid activation is shown in subfigure A, while the trend for HCN generation (displayed as the HCN concentration as a percentage of the initial amino acid concentration) is shown in subfigure B. Analysis of the figure indicates that while the HCN generation is exacerbated under conditions where Oxyma and DIC are used in excess, measurable amounts of HCN are formed when the amino acid:Oxyma:DIC ratios are 1:1:1 - as well as when the Oxyma and DIC are used in a sub stoichiometric amounts (1:0.5:0.5) compared to the starting amino acid. In this latter case, ~0.07% HCN is present at 5 hours (signal-to-noise ratio of the HCN 1H NMR resonance is 20:1 at this data point). This is consistent with generation of HCN by a parallel reaction pathway to the desired, kinetically favored activation. Without the use of NMR, it is likely that this phenomenon would continue to exist undiscovered. Despite many years of using Oxyma in combination with various carbodiimides for amino acid activation, to our knowledge this is the first reported observation of the generation of this toxic gas. While it is unlikely that one would consider NMR the most preferable technique for the identification and concentration monitoring of a small molecule like HCN, this study represents yet another example of the versatility that modern NMR spectroscopy provides for comprehensive reaction and process characterization.13-14 In the interest of appraising the broader synthetic organic chemistry community to this finding, there are many studies to be completed with regard to the reaction characterization, and these will be reported in a future, more comprehensive study. In conclusion, the data demonstrate the generation of HCN when using the combination of Oxyma and DIC for amino acid activation. The generation is exacerbated when the reagents are used in excess with respect to the amino acid component and does occur to a measurable extent even when sub stoichiometric amounts of Oxyma/DIC (vs amino acid) are used. A mechanism is proposed to account for the formation of HCN that involves direct reaction between Oxyma and DIC. As we continue to use this chemistry in both a development and manufacturing environment, we will characterize the chemistry to raise awareness of and understand the extent of HCN generation and keep researchers and operators safe. A subsequent publication will explore a variety of different characteristics of this reaction, including the effect of amino acid structure on the rate of HCN generation, as well as the effect of using different carbodiimide activators (EDCI, DCC) on the course of the HCN formation reaction.

Experimental Section All NMR data were collected on a 500 MHz Bruker UltraShield system equipped with an Avance III console, a 5-mm BBFO probe, and a BCU-II unit for precise temperature control. Spectral data were processed using MestReNova software. Gas chromatography was performed on an Agilent GC 7890A equipped with a FID detector and an Agilent G1888 Headspace auto sampler using a DB-624 capillary column (30 m length x 0.32 mm id x 1.8µm film thickness, Agilent Co., USA). Data acquisition and processing were accomplished using Empower® 3 software. Chromatographic conditions for the separation and quantitation of HCN were performed using an oven programming at initial temperature of 45 °C for 5 min


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followed by a ramp rate of 10 °C/min to a final temperature of 175 °C with a hold of 1 min and a total run time of 19 min. The temperature of the injector was set to 140 °C with a split ratio of 10:1 using helium carrier gas with a constant flow rate of 1.8 mL/min. The headspace sampler oven temperature was set at 85 °C, the sample transfer loop temperature set at 95 °C, and the transfer line temperature set at 110 °C. The sample equilibration time was set to 10 min on low agitation. The vial pressurization was set to ~10 psig and the pressurization time to 0.5 min. The sample loop fill, loop equilibration, and injection times were set to 0.2, 0.1 min and 1.0 min, respectively. General Procedure for activation of Fmoc-Protected Amino Acids with DIC/Oxyma. Fmoc-L-Ala-OH activation conditions are described as an example. A solution was prepared by dissolving Fmoc-L-AlaOH·H2O (44.0 mg, 0.13 mmol) and Oxyma (29.0 mg, 0.20 mmol, 1.5 equiv), in 0.7 mL of DMF-d7. A stock solution of DIC in DMF-d7 was prepared at a concentration of approximately 250 mg DIC/mL. 100 µL of this stock solution (25.0 mg DIC, 0.20 mmol, 1.5 equiv) was combined with the Ala/Oxyma solution in a 5mm NMR tube, vigorously mixed, and immediately transferred to the spectrometer for monitoring at 20 °C. The reported concentrations of all species measured by NMR are referenced to the theoretical concentration of the amino acid. Procedure for reaction of Oxyma and DIC. Solutions containing Oxyma (29.0 mg, 0.2 mmol) and DIC (25.0 mg, 0.2 mmol) in approximately 0.8 mL of DMF-d7 were prepared in a 5-mm NMR tube and immediately transferred to the spectrometer for monitoring at 20 and 50 °C. The reported concentrations of all species measured by NMR are referenced to the theoretical concentration of the Oxyma.

Hydrogen cyanide. 1H NMR (500 MHz, DMF-d7) δ 6.21 (s, 1JCH = 260 Hz, 1H). 13C{1H} NMR (126 MHz, DMFd7, 1H-13C HSQC) δ 113.5. 15N NMR (51 MHz, DMF-d7, 1H-15N HMBC) δ 257. Activated FMOC-L-Ala-OH ester (4): 1H NMR (500 MHz, DMF-d7) δ 8.23 (d, J= 6.8 Hz, 1H), 7.94 (d, J= 7.7 Hz, 2H), 7.76 (t, J= 7.6 Hz, 2H), 7.46 (t, J= 7.5 Hz, 2H), 7.36 (td, J= 7.5, 1.3 Hz, 2H), 4.62 (p, J= 7.2 Hz, 1H), 4.45 (q, J= 7.1 Hz, 2H), 4.38 (dd, J= 14.9, 7.5 Hz, 3H), 4.30 (t, J= 7.2 Hz, 1H), 1.60 (d, J= 7.3 Hz, 3H), 1.36 (t, J= 7.1 Hz, 3H). 13C NMR (126 MHz, DMF-d7) δ 168.7, 157.3, 156.6, 144.4, 141.4, 133.4, 128.0, 127.4, 125.5, 120.3, 107.7, 66.9, 64.0, 49.3, 47.3, 16.5, 13.6. ethyl-2-cyano-2-(((N,N'-diisopropylcarbamimidoyl)oxy)imino)acetate (7). 1H NMR (500 MHz, DMF-d7, 30C) δ 10.49 (b, 1H), 4.45 (q, J = 7.1 Hz, 2H), 4.18 (m, 1H), 4.12 (m, 1H), 1.36 (d, J = 6.4 Hz, 6H), 1.33 (t, J = 7.1 Hz, 3H), 1.32 (d, J = 5.9 Hz, 6H). 13C{1H} NMR (126 MHz, DMF-d7, -30 °C) δ 156.1, 155.1, 134.9, 107.6, 64.3, 46.6, 46.1, 21.9, 21.1, 13.5. ethyl-4-isopropyl-5-(isopropylimino)-4,5-dihydro-1,2,4-oxadiazole-3-carboxylate (9). Oxyma (911 mg, 6.4 mmol) was dissolved in DMF (10-mL), and DIC (1.0 mL, 7.8 mmol, 1.2 equiv) was added to the solution. After the reaction had achieved ~33% conversion to 9 in situ (2.1 mmol, 511 mg), 5-mL of the reaction solution was diluted with ethyl acetate (5-mL), and the organic phase was washed with water (3x10-mL). The organic phase was concentrated on a rotary evaporator and the resulting thick, red liquid mixture was transferred to a 2.5-g loading cartridge for purification using a Teledyne CombiFlash mass-guided isolation



system equipped with a 12-g silica column. Elution was carried out with a gradient system (100:0 hexane:ethyl acetate to 80:20 hexane:ethyl acetate over 25 minutes. The elution solvent was removed from a fraction representing a peak with M+H=242. The colorless oil that resulted was analyzed by NMR (by dissolving in DMF-d7) and LC-MS (by dissolving in acetonitrile). 1H NMR (400 MHz, DMF-d7) δ 1.12 (d, J = 6.3 Hz, 6H), 1.38 (t, J = 7.1 Hz, 3H), 1.48 (d, J = 6.9 Hz, 6H), 3.75 (hept, J = 6.3 Hz, 1H), 4.47 (q, J = 7.1 Hz, 2H), 4.71 (hept, J = 6.9 Hz, 1H). 13C{1H} NMR (101 MHz, DMF-d7) δ 156.5, 150.6, 149.4, 63.3, 48.0, 46.9, 24.2, 18.2, 13.4. MS(ESI) calculated for C11H20N3O3 [M+H]+ 242.1499, found 242.1499. Activated FMOC-L-Leu-OH ester (11): 1H NMR (500 MHz, DMF-d7) δ 8.21 (d, J= 7.4 Hz, 1H), 7.98 – 7.89 (m, 2H), 7.81 –7.73 (m, 2H), 7.48 –7.44 (m, 2H), 7.36 (dd, J= 7.4, 1.1 Hz, 2H), 4.59 (ddd, J= 9.8, 7.5, 4.8 Hz, 1H), 4.45 (q, J= 7.1 Hz, 2H), 4.41 –4.35 (m, 2H), 4.30 (t, J= 7.4 Hz, 1H), 1.96 –1.83 (m, 2H), 1.82 – 1.72 (m, 1H), 1.36 (t, J= 7.1 Hz, 3H), 1.01 (d, J= 6.4 Hz, 3H), 0.99 (d, J= 6.4 Hz, 3H). 13C{1H}NMR (126 MHz, DMF-d7) δ 168.8, 157.3, 156.8, 144.4, 141.4, 133.5, 128.0, 127.4, 125.6, 120.3, 107.7, 66.8, 64.0, 52.1, 47.3, 39.7, 24.9, 22.6, 21.2, 13.6.

Acknowledgements The authors wish to thank Drs. Christopher Burcham, Michael Kopach and Scott May for their critical reading of the manuscript and their helpful suggestions. In addition, the authors wish to acknowledge Trent Oman for helpful discussions during the preparation of the manuscript. References 1. El-Faham, A.; Albericio, F., Peptide Coupling Reagents, More than a Letter Soup. Chem. Rev. 2011, 111, 6557-6602. 2. Jad, Y. E.; Khattab, S. N.; de la Torre, B. G.; Govender, T.; Kruger, H. G.; El-Faham, A.; Albericio, F., Oxyma-B, an excellent racemization suppressor for peptide synthesis. Org. Biomol. Chem. 2014, 12, 8379-8385. 3. Subirós-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.; Albericio, F., Oxyma: An Efficient Additive for Peptide Synthesis to Replace the Benzotriazole-Based HOBt and HOAt with a Lower Risk of Explosion[1]. Chem. Eur. J. 2009, 15, 9394-9403. 4. A SciFinder search executed prior to submission of this note returned over 17,000 entries of reactions that use this reagent combination for amino acid activation. 5. Cdc.gov CDC - The Emergency Response Safety and Health Database: Systemic Agent: Hydrogen Cyanide (AC) - NIOSH. https://www.cdc.gov/niosh/ershdb/emergencyresponsecard_29750038.html (accessed 11/9/2018). 6. Phasing of the multiplicity-edited HSQC data originally suggested the resonance was a methylene; however, this experiment was optimized for a proton-carbon coupling constant of 145 Hz. The one-bond H-C coupling for the species of interest was measured as 260 Hz based on the spacing of the carbon-13 satellites in the proton spectrum 7. Udhayakumari, D., Chromogenic and fluorogenic chemosensors for lethal cyanide ion. A comprehensive review of the year 2016. Sens. Actuators, B 2018, 259, 1022-1057. 8. Rebek, J.; Feitler, D., Improved method for the study of reaction intermediates. Mechanism of peptide synthesis mediated by carbodiimides. J. Am. Chem. Soc. 1973, 95, 4052-4053.


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9. Rebek, J.; Feitler, D., Mechanism of the carbodiimide reaction. II. Peptide synthesis on the solid phase. J. Am. Chem. Soc. 1974, 96, 1606-1607. 10. Hickenboth, C. R.; Menta, F.; Bojkova, N. V. Polymerizable compositions containing ethylenically unsaturated monomers having episulfide functional groups and related methods. US20130225777A1, 2013. 11. Nesvadba, P.; Bugnon Folger, L.; Carroy, A.; Faller, M.; Spony, B. O-Iminoisourea compounds as initiators and polymerizable compositions thereof. WO2010128062A1, 2010. 12. Park, Y.-I.; Noh, S.-M.; Song, Y.-K.; Kim, B.-J.; Kim, J.-C.; Nam, J.-H. Blocked-isocyanate doublecurable at low temperature. WO2017160016A1, 2017. 13. Buser, J. Y.; Luciani, C. V., A new method for determination of gas–liquid mass transfer coefficients by direct measurement of gas uptake by flow NMR. Reaction Chemistry & Engineering 2018, 3, 442-446. 14. Buser, J. Y.; McFarland, A. D., Reaction characterization by flow NMR: quantitation and monitoring of dissolved H2 via flow NMR at high pressure. Chem. Commun. 2014, 50, 4234-4237.


Generation of Hydrogen Cyanide from the Reaction of Oxyma

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