Generation of Hydrogen Cyanide from the Reaction of Oxyma (Ethyl Cyano(hydroxyimino)acetate) and DIC (Diisopropylcarbodiimide)

Article abstract of DOI:10.1021/acs.oprd.9b00344

Evolution of hydrogen cyanide (HCN) during amino acid activation using the reagent combination ethyl cyano(hydroxyimino)acetate (Oxyma)/diisopropylcarbodiimide (DIC) is observed under ambient conditions (20 °C) in N,N-dimethylformamide (DMF). Concentration versus time profiles obtained by ¹H NMR spectroscopy 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 relative to the amino acid. A mechanism for the reaction between Oxyma and DIC is proposed, and evidence for its validity was gathered by NMR spectroscopy.

Full text of DOI:10.1021/acs.oprd.9b00344

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Communication
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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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.*
skolis@lilly.com
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Small Molecule Design & Development, Eli Lilly and Company, Lilly Corporate Center, Indianapolis IN
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6285 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
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conditions (20 °C) in DMF. Concentration vs time profiles obtained by H 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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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. The reagent
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combination of ethyl cyanohydroxyiminoacetate (Oxyma, 2) and diisopropylcarbodiimide (DIC, 3) has seen
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an increase in use due to many attractive attributes, including prevention of racemization and greater
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thermal stability when compared to the common hydroxybenzotriazole (HOBt) based reagents. Due to
these benefits, low cost and organic solubility of the diisopropylurea by-product this reagent combination
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is now the most common system in solid phase peptide synthesis (SPPS). During a study of amino acid
activation kinetics using Oxyma/DIC, hydrogen cyanide (HCN) generation was observed throughout the
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course of the reaction. Due to the hazards of exposure to small amounts of HCN gas , 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
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was studied using H NMR. Figure 1 shows a series of H NMR spectra collected at different times during
an Fmoc-L-Ala-OH (1) activation reaction conducted in DMF-d at 20 °C. In addition to the resonances
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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
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of a 257-ppm nitrogen (referenced to liquid NH ). Direct C measurement with and without H decoupling
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unambiguously demonstrated that only one proton was bound to the 113.5-ppm carbon. 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)
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into the reaction solution and observing an increase in the 6.25-ppm H 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
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solution phase of the reaction mixture was pursued by orthogonal means. Colorimetric methods 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-Ala-
OH 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
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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.
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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-d in the absence of an amino acid or
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external base. The first step of the proposed mechanism is analogous to the generally accepted
mechanism for amino acid activation by DIC. The transient nature of the open chain intermediate 7 10-
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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 mass-
guided 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.
iPrN
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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.
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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
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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.
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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
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amounts of these two materials. Independent H 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-L-
Leu-OH:Oxyma:DIC
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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
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ratio of the HCN H 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
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5 °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.
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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-Ala-
OH·H O (44.0 mg, 0.13 mmol) and Oxyma (29.0 mg, 0.20 mmol, 1.5 equiv), in 0.7 mL of DMF-d . A stock
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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 5-
mm 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-d were prepared in a 5-mm NMR tube and immediately
7
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.
1
1
13
Hydrogen cyanide. H NMR (500 MHz, DMF-d ) δ 6.21 (s, J = 260 Hz, 1H). C{1H} NMR (126 MHz, DMF-
7
CH
1
13
15
1
15
d , H- C HSQC) δ 113.5. N NMR (51 MHz, DMF-d , H- N HMBC) δ 257.
7
7
1
Activated FMOC-L-Ala-OH ester (4): H NMR (500 MHz, DMF-d ) δ 8.23 (d, J= 6.8 Hz, 1H), 7.94 (d, J= 7.7
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-d ) δ 168.7, 157.3, 156.6, 144.4, 141.4, 133.4, 128.0, 127.4, 125.5,
7
120.3, 107.7, 66.9, 64.0, 49.3, 47.3, 16.5, 13.6.
1
ethyl-2-cyano-2-(((N,N'-diisopropylcarbamimidoyl)oxy)imino)acetate (7). H NMR (500 MHz, DMF-d -
7
,
3
=
0C) 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
1
3
1
7.1 Hz, 3H), 1.32 (d, J = 5.9 Hz, 6H). C{ H} NMR (126 MHz, DMF-d -30 °C) δ 156.1, 155.1, 134.9,
7
,
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
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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
1
(
by dissolving in DMF-d7) and LC-MS (by dissolving in acetonitrile). H NMR (400 MHz, DMF-d ) δ 1.12 (d,
7
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{ H} NMR (101 MHz, DMF-d ) δ 156.5, 150.6, 149.4, 63.3, 48.0, 46.9,
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0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
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0
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9
0
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0
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24.2, 18.2, 13.4. MS(ESI) calculated for C H N O [M+H]+ 242.1499, found 242.1499.
1
1
20
3
3
1
Activated FMOC-L-Leu-OH ester (11): H NMR (500 MHz, DMF-d ) δ 8.21 (d, J= 7.4 Hz, 1H), 7.98 –
7
7
4
.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,
.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
3
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). C{1H}NMR (126
MHz, DMF-d ) 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,
7
5
2.1, 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
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El-Faham, A.; Albericio, F., Peptide Coupling Reagents, More than a Letter Soup. Chem. Rev. 2011,
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Jad, Y. E.; Khattab, S. N.; de la Torre, B. G.; Govender, T.; Kruger, H. G.; El-Faham, A.; Albericio, F.,
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379-8385.
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reactions that use this reagent combination for amino acid activation.
Cdc.gov CDC - The Emergency Response Safety and Health Database: Systemic Agent:
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Hydrogen Cyanide (AC) - NIOSH.
https://www.cdc.gov/niosh/ershdb/emergencyresponsecard_29750038.html (accessed 11/9/2018).
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.
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.
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the carbon-13 satellites in the proton spectrum
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