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Effect of Triazine Impurity in NMI Capping Reagent on Oligonucleotides
Sandy Lorenz & Venkatraman Mohan
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Background
• Automated DNA/RNA synthesis cycle • Impurity profile of desired n-mer oligos
– Failure mode: +85 oligo adduct • Origin of potential impurities
– Amidite monomers, reagents and solvents, reaction conditions • Capping reagents:
– 20/30/50 acetic anhydride/2,6-lutidine/acetonitrile – 20/80 N-Methylimidazole/acetonitrile
• Triazine impurity in NMI • LC/MS analysis of oligos • Summary
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DNA/RNA synthesis cycle O
RO
DMTO B2
PO
NCN
O
RO
HO B1
O
O
RO
B1
O
O
RO
DMTO B2
PO
O
NC
O
RO
B1
O
O
RO
DMTO B2
PO
O
CN
O
RO
B1
O
O
RO
DMTO B2
PO
O
CN
Y
O
RO
B1
O
XO
O
RO
DMTO B1
ONH
O
S
O
OHO
HO B21
OP
YO
O
H/OHOH
O B1
PY
OO5' 3'
Oxidation
Deblock
Couple
Cap
Deblock D
epro
tect
, Pu
rify
X = Acetyl
O
O
O
Capping reagents
20/30/50 acetic anhydride/lutidine/ACN
20/80 N-Methylimidazole/ACN
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Triazine impurity in N-methylimidazole
O
O O H2N
N
N+ + + NH4OH
glyoxal formaldehyde methyl amine amm.hydroxide N-Methylimidazole
O + H2N3 3 NN
N
A. A. Gridnev & I.M. Mihaltseva, Synthetic comm., 24, 1547,1994
1,3,5- trimethyl-hexahydrotriazine
Triazine by-product formation via side reaction
N-methylenemethanamine
Schiff-Base equilibrium
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GCMS of NMI with Triazine Impurity
NM
I N
N
N-m
ethy
lene
met
hana
min
e
Data collected with Shimadzu GCMS 2010
NN
NN
1,3,
5- tr
imet
hyl-h
exah
ydro
tria
zine
Comparison of (A) observed MS & (B) NIST Library match
(A)
(B)
Triazine (ppm) Counts (TIC)
31.4 429,557
87.4 1,479,213
172.4 2,966,143
200.9 3,553,701
291.9 4,930,354
461.2 7,988,014
807.6 13,629,935
R2 = 0.9995
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Experimental Design
N
OO
O+ Oligo
Oligo + 85
Oligo 1: A5C
6G
3T
6 5’- ATA CCG ATT CCG CGA ACT TT-3’
Oligo 2: A
10C
10 5’- ACA CAC ACA CAC ACA CAC AC-3’
Oligo 3: G
10T
10 5’- GTG TGT GTG TGT GTG TGT GT-3’
Oligo #
0 ppm 0.1% 2%
1 2 3
Triazine Levels
Hypothesis
Due to the inherent reactivity of secondary amine groups on G and T bases, the adduct predominantly adds to these. To test the hypothesis, oligos 2 and 3 have been designed.
_ _ _
N-methylenemethanamine Acetic anhydride
Oligos synthesized
NH
NH
O
O
N
NHN
NH
O
NH2
G T
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Oligo Synthesis & Analysis Parameters
Honeywell Burdick & Jackson BioSyn® reagents
Synthesis Parameters Synthesizer: AKTATM OligopilotTM plus 100 Primer Support: GE Healthcare PS200 T80s (polystyrene beads, loading = 80µmole T/gram) (polystyrene beads, loading = 200µmole C/gram) Synthesis Scale: 100µmole, 240µmole Coupling Time: 3 minutes DNA Amidites: A, T, G, C (Prepared at 0.1M in acetonitrile), 1.6 eq.
BMI activator reagent cat. # BR731 (0.3M 5-benzylthio-1H-tetrazole, 0,5% NMI, 99.5% ACN) Deblock reagent cat. # SR674 (3% dichloroacetic acid in toluene (v/v)) Capping reagent, Cap A cat. # BR644 (20% acetic anhydride , 30% 2,6-lutidine, 50% acetonitrile (v/v)) Capping reagent, Cap B cat. # BR654 (20% N-methylimidazole, 80% acetonitrile (v/v)) Oxidation reagent cat. # BR664 (0.05M iodine, 10% water, 90% pyridine (v/v)) Acetonitrile cat. # BB017
LC/MS Parameters Agilent 6120 TOF Column: Agilent Eclipse plus C18, 3.5µm, 2.1 X 150 mm ESI Negative Mode Buffer A: HFIP and Diisopropylamine in water Buffer B: Methanol
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LCMS Analysis of Oligo A5C6G3T6
0 ppm triazine in capping reagent
0.1% triazine in capping reagent
2% triazine in capping reagent
UV (260nm) Absorbance Chromatograms
Higher amounts of Triazine result in multiple additions of the + 85 adduct
n+85
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LCMS Analysis of Oligo A5C6G3T6
Extracted Ion Chromatogram (M-3H)3-
UV at 260nm
n n +
85
n +
(2 x
85)
n +
(3 x
85)
n +
(4 x
85)
RT (min) % Area
MW (Daltons) from MS
Deconvolution
Expected MW (Daltons) ID
13.74 18.55 6052.18 6052.07 n
14.10 23.68 6137.22 6137.12 n + 85
14.44 14.05 6222.28 6222.17 n + (2 x 85)
14.71 5.97 6307.34 6307.23 n + (3 x 85)
15.16 3.95 6392.53 6392.28 n + (4 x 85)
Peak ID (M-3H)3- Ions Extracted (m/z)
1 n 2015.9 - 2016.1 2 n + 85 2044.6 - 2044.8 3 n + (2 x 85) 2072.9 - 2073.1 4 n + (3 x 85) 2100.9 - 2101.1
5 n + (4 x 85) 2129.6 - 2129.8
Analysis shows adducts overwhelm the desired oligo product
1 2 3
4 5
2% triazine in capping reagent
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LCMS Analysis of Oligo G10T10
Total Ion Chromatogram Base Peak Chromatogram
Extracted Ion Chromatogram (M-3H)3-
1 2
Peak ID (M-3H)3- Ions Extracted (m/z)
1 n 2089.5 - 2089.7 2 n + 85 2117.9 - 2118.1 3 n + (2 x 85) 2146.3 - 2146.5 4 n + (3 x 85) 2174.6 - 2174.8 5 n + (4 x 85) 2202.9 - 2203.1 6 n + (5 x 85) 2259.7 - 2259.9 7 n + (6 x 85) 2288.0 - 2288.2 8 n + (7 x 85) 2316.4 - 2316.6 9 n + (8 x 85) 2344.7 - 2344.9
3 4 5 6 7 8 9
2% triazine in capping reagent
+ 85 adducts are distributed over G and T bases
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LCMS Analysis of Oligo A10C10
Total Ion Chromatogram
UV at 260nm
RT (min) MW (Daltons)
from MS Deconvolution
ID Ion Abundance %
13.28 - 13.40 5728.14 n - 234 100
5611.16 n - 351 65.5
5845.22 n - 117 27.1
13.55-13.74 5845.17 n - 117 100
5728.19 n - 234 55.9
5962.19 n 50
14.01-14.19 5845.21 n - 117 100
5962.21 n 98.3
5930.22 n-32 71
No + 85 adduct observed on oligo sequence without G or T bases
2% triazine in capping reagent
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Summary
• LCMS analysis of the failure mode (oligos with n+85 adduct)
• +85 adduct impurity affects oligo API quality
• GCMS analysis of NMI: identification and validation of triazine impurity
• Triazine and acetic anhydride adds to oligo, resulting in +85 adduct
• Higher spiked amounts of triazine in capping reagent correspond to higher Oligo-adducts
• Evidence for adduct formation limited to sequences containing either G or T
• No +85 adduct formed in sequence containing only A and C
• Additional experiments to investigate mechanistic details are in progress
