Aldehyde-mediated N-nitrosation of an amino acid

The N-nitroso Amadori compound is prepared by a multi-step synthetic strategy, using glyceraldehyde and glycine methyl ester hydrochloride as starting materials. Meanwhile, that the N-nitroso Amadori compound can be formed under the simulated gastric conditions in the presence of sodium nitrite is confirmed


Introduction
The N-nitroso compounds, including nitrosamines, nitrosoamides and related compounds, have received considerable attention in recent years because many of them are potent carcinogens.N-nitrosodimethylamine (NDMA), produced as by-product of several industrial processes， was found to be carcinogenic to rats in toxicology studies. 1,2The carcinogenicity of N-nitroso compounds is due to their ability to produce DNA alkylating agents directly 3 or by bioactivation. 4 Nitrosamines are relatively stable so as to require metabolic activation by enzymes to produce the alkylating agents.For example, NDMA 1 is transformed to α-hydroxynitrosamine 2 by the specific enzyme cytochrome P-450. 5The α-hydroxynitrosamine 2 is highly unstable and quickly undergoes intramolecular rearrangement to form the diazohydroxides 3. Then loss of the hydroxyl group leads to the formation of a methyldiazonium ion 4. Alkyl diazonium ions are well known as powerful alkylating agents and are believed to be responsible species in DNA alkylation (Scheme 1).

Scheme 1. Metabolic activation of NDMA.
It has been reported that N-nitroso compounds can be formed endogenously. 6There are two major sources of nitrosating agents (Scheme 2): (1) Sodium nitrite from food.It has been a common practice in food industry to add sodium nitrite to processed meats to inhibit the growth of the bacteria responsible for botulism poisoning.(2) The bioreduction of nitrate both digested and biosynthesized leads to nitrite.Scheme 2. Formation of the active nitrosating agent.N-Nitrosation involves the biomolecular reaction of a substrate having at least one nitrogen atom with a pair of electrons with a nitrosating reagent.Human food contains amino acids and sugars.Amino acids contain a primary amine group, while the sugar has a carbonyl group.It was hypothesized that amino acids can react with sugars to form imines under gastric conditions.When the imines of amino acids and sugars encounter nitrous acid endogenously generated, N-nitroso compound can be formed.The goals of this subject are to synthesize specific N-nitroso Amadori compound and to determine whether the compound can be formed under the endogenous conditions in the simulated gastric conditions.
Glyceraldehyde 5 was chosen as representative of dietary aldehydes because it has fewest hydroxyl groups, which causes fewer problems in synthesis.Basically, our synthetic target is the N-nitroso Amadori compound N-(3-hydroxy-2-oxopropyl)-N-nitrosoglycine (10).The possible route to the formation of compound 10 is shown in Scheme 3.With this standard, we will be able to determine whether this compound can be formed under simulated gastric conditions.The results will be important for the evaluation of the toxicological significance of endogenous nitrosation.Scheme 3. Possible route to the formation of N-nitroso Amadori compound.

Results and Discussion
The synthesis of N-nitroso Amadori compound 10 proved to be very difficult.According to Schieberle's method, 7 a mixture of glyceraldehyde 5 and glycine 6 was refluxed in a mixture of anhydrous methanol and DMF.The Amadori compound may be formed in very low yield in this process.Unfortunately, none of the expected Amadori compounds could be isolated since the high polarity of the expected product could make it very hard to be separated from the complex product mixture.Other conditions 8,9 for Amadori rearrangement were also applied and none of them gave the desired product in a reasonable yield.An alternative synthetic target is methyl N-(3-hydroxy-2-oxopropyl)-N-nitrosoglycinate (11), the methyl ester of compound 10 (Scheme 4).The nitrosation product mixture produced under simulated gastric conditions should be readily methylated by CH2N2 in situ without affecting other functional groups.So if compound 11 can be found in the mixture of methylation products, it will be enough to conclude that compound 10 can be formed in that process.Additionally, esterifying the corresponding carboxylic function group considerably reduces the polarity.

Scheme 4. Methylation of compound 10.
Our synthesis of the N-nitroso Amadori compound 11 is summarized in Scheme 5.The first step is the reductive amination reaction to form methyl ((2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl) glycinate ( 14).Later, it was found that it was not necessary to separate the product 14; the crude product mixture directly underwent the nitrosation reaction with nitrous acid to afford methyl N-((2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl)-Nnitrosoglycinate (15), which was easily deprotected to give methyl N-(2, 3-dihydroxypropyl)-N-nitrosoglycinate (16) under acidic conditions.Using Et3N as the base, the primary hydroxyl group of diol 16 was selectively protected by the TBDMS group to afford methyl N- (3-((tert-butyldimethylsilyl)oxy)-2-hydroxypropyl)-Nnitrosoglycinate (17).CrO3 10 and KMnO4 11 have been used to oxidize β-hydroxynitrosamine.But the reaction was slow to give N- (3-((tert-butyldimethylsilyl)oxy)-2-oxopropyl)-N-nitrosoglycinate (18) with less than 10% yield after 24 hours along with some decomposition products.Then we found that Swern-type oxidation can transform the β-hydroxynitrosamine 17 to β-ketonitrosamine 18 at a much faster reaction rate and also in good yield.At last, the TBDMS group was successfully removed under acidic conditions using TFA/THF/H2O.It is noteworthy to mention that using HF or TBAF, common reagents for desilylation, caused the total decomposition of the starting material.A possible reason is that the fluoride ion can react with the nitroso group as a nucleophile to cause the decomposition.

Scheme 5. Synthesis of N-nitroso Amadori compound 11.
With the standard compound 11 in hand, we have tried to determine whether it can be produced under the simulated gastric conditions.At last, we demonstrated that N-nitroso Amadori compound 11 can be produced under the simulated gastric conditions.Our preliminary experiments used glycine methyl ester and glyceraldehyde.The mixture of glycine methyl ester and glyceraldehyde in pH 4 buffer was stirred for 40 hours at 37 ºC.Then sodium nitrite was added to the solution.The reaction continued for another hour.Then the extract was analyzed by HPLC and one peak had the same retention time as the standard 11.The compound was isolated by column chromatography and the 1 H NMR and 13 C NMR are identical with the standard 11 previously synthesized.
The result supports our hypothesis that imines of glyceraldehyde and glycine can undergo Amadori rearrangement in vivo and then following nitrosation can give N-nitroso Amadori compound as one of the products.

Conclusions
N-Nitroso Amadori methyl ester 11 was successfully synthesized by a multi-step synthetic strategy or under the simulated gastric conditions.The results indicated that the regular consumption of amino acids and sugar aldehydes in the diet in the presence of sodium nitrite may produce N-nitroso Amadori compounds which could be potentially toxic substances.Further investigation will be reported on the chemical and biological properties of these compounds in due course.

Experimental Section
General.All air-and moisture-sensitive reactions were carried out in flame-dried glassware under a nitrogen atmosphere.Reactive liquid compounds were measured and transferred by gas-tight syringes and added to the reaction flask through rubber septa.Moisture-sensitive and hygroscopic solid compounds were transferred under a nitrogen atmosphere in a glove bag.The reaction mixture was concentrated by using a rotary evaporator attached to a water aspirator.Residual solvents were usually removed under reduced pressure using a vacuum pump.Analytical thin-layer chromatography (TLC) was performed on glass-backed silica gel plates.Compounds were visualized under a UV lamp or by developing in iodine, vanillin, phosphomolybdic acid solution or with the potassium permanganate solution followed by heating on a hotplate to ~350 °C.Flash chromatography was performed on 230-400mesh silica gel with technical grade solvents which were distilled prior to use.1H NMR spectra were recorded on a Bruker (Billerica, MA) AMX-250, Bruker AMX-300, Bruker AMX-500 at 250, 300, or 500 MHz, respectively, as CDCl3 solutions with tetramethylsilane (δ = 0 ppm) as the internal standard. 13C spectra were obtained on the same instruments at 62.5, 75, or 125MHz, respectively, with CDCl3 (δ= 77 ppm) as the internal reference.Chemical shifts are reported in parts per million (ppm).Multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet), etc. Infrared spectra were recorded on a Thermo Nicolet NEXUX 670 FT-IR spectrometer as neat liquids with NaCl cells.Optical rotations were measured on a Jasco DIP-370 digital polarimeter.

Methyl N-(3-((tert-butyldimethylsilyl)oxy)-2-hydroxypropyl)-N-nitrosoglycinate (17).
The N-nitroso diol 16 (50 mg, 0.026 mmol), TBDMSCl (80 mg, 0.53 mmol) and catalytic amount of DMAP were dissolved in 5 mL CH2Cl2 under argon.Then the solution was cooled to 0 ºC and 50 μL Et3N was added to the solution.Then the solution was naturally warmed to RT and stirred for 4 hours.TLC was used to monitor the completion of the reaction.Upon completion, the reaction mixture was poured into 10 mL water and extracted by CH2Cl2 (3 × 10 mL).The extracts were combined, washed with 10 mL water, and dried with MgSO4.The solvents were removed under reduced pressure at room temperature.Flash chromatography (ethyl acetate/hexane, 1:1) gave 47 mg (60%) of the desired product.In CDCl3, compound 17 exists as 5:1 mixture of Z and E isomers.Major product, Z-isomer, 1   (18).A solution of trifluoroacetic anhydride (525mg, 2.5 mmol) in 0.5 mL CH 2 Cl 2 was added to a solution of dimethylsulfoxide (180 μL, 2.5 mmol) in 2.5 mL CH2Cl2 at -78 ºC.The mixture was stirred for 10 min, and a solution of alcohol 17(140 mg, 0.5 mmol)in 1.5 mL CH2Cl2 was added.After stirring for 2h at -45 ºC, trimethylamine(560 μL, 4 mmol) was added, and the solution was allowed to warm to room temperature.Then saturated aqueous NaHCO3 solution (5 mL) was added and the mixture was separated.The aqueous layer was extracted with ethyl ether, and the combined organic layer was dried over MgSO4 and concentrated under reduced pressure.Flash chromatography (ethyl acetate/hexane, 1:3) gave 85 mg (61%) desired product.In CDCl3, compound 18 exists as 5:3 mixture of Z and E isomers.Major product, Z-isomer, 1   (11).Mono-TBDMS protected N-nitroso Amadori compound 18 (24 mg, 0.079 mmol) was dissolved in a mixture of 2 mL of THF, 2 mL of water and 2 mL of TFA.

Nitrosation of methyl glycine and glyceraldehyde under simulated gastric conditions
Methyl glycine hydrochloride (1 g, 7.8 mmol) and 1.6g (17.7 mmol) D, L-glyceraldehyde were dissolved in 10 mL pH 4 buffer (0.05 M).The resulting solution was heated to 37 ºC and stirred for 40 hours at 37 ºC.The solution turned to a light yellow color.Then the reaction mixture was cooled to room temperature and sodium nitrite (2 g, 29 mmol) was added.The solution was stirred for another hour.The reaction was monitored by TLC.When TLC showed the completion of the reaction, the solution was extracted with ethyl acetate (3 × 10 mL).The existence of the compound 11 was determined by HPLC.The HPLC separation was performed on a reverse phase HPLC C-18 column (10 mm×25 cm).The program was as follows: mobile phase: water/CH3CN; flow rate: 1 mL/ min; isocratic: 20% CH3CN/80% water; standard 11 RT: 11.8 min.The combined organic layer was washed with 10 mL water, dried over MgSO4 and concentrated under reduced pressure.Flash chromatography (ethyl acetate/hexane, 1:1) separated the product (5 mg) which has the same Rf value as the synthesized standard.The separated product's 1 H NMR and C NMR are identical with those of the previously synthesized standard 11.