Kinetic resolution

Selected Cell

Cell:

Value:


Kinetic resolution

LS_ATA WT	0,3 μg/mL
LS_ATA V37A	0,3 μg/mL

Amine donors:
(±) phenylethylamine (±)-PEA, (±) phenylpropylamine (±)-PPA	8 mM
R-phenylbutylamine (R)-PΒA	4 mM
diluted in DMSO	5% final concetration
Sodium pyruvate	16 mM	(8 mM when R-PBA is used as amine donor)
pyridoxal 5'-phosphate (PLP)	0,1 mM
CHES buffer 50 mM pH= 9
total reaction volume	2 mL

Reaction Conditions	750 rpm, 35 C

Sample preparation

sample 200 μL
quenched with 15 μL NaOH 5M
extraction with 400 μL ΕtΟΑc
Να2SO4 was used to dry the organic phase

GC Analytics
Column: cyclodex-B
Carrier: Ηe constant flow 1,4 mL/min
Injection : 1 μL split 20
Detector: FID, 250 C
Oven:
initial temperature 80 oC
linear gradient to 120 oC with a slope of 1,75 oC/min
linear gradient to 220 oC with a slope of 20 oC/min, kept for 3 min.
	RT
(R)-PEA	13.8 min
(S)-PEA	14.0 min
Acetophenone	15.4 min
(R)-PPA	18,8 min
(S)-PPA	18,9 min
Propiophenone	21,0 min
(R)-PBA	23,6 min
Butyrophenone	24,6 min


Stock solutions
(±) phenylethylamine/ DMSO 160 mM, 1 mL
(±) phenylpropylamine/ DMSO 160 mM, 1 mL
R-phenylbutylamine/DMSO 80 mM, 1mL
Pyruvate/CHES 32 mM 6,5 mL

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									Peak Area
								mM	R-PEA	acetophenone	R-PPA	propiophenone	R-PBA	butyrophenone
								8	641888	958500	721279	1067384	523294	1293958
								calib.factor:	80236	119812.5	90159.875	133423	65411.75	161744.75

(±)-Phenylethylamine
	WT_rac-PEA			V37A_rac-PEA		WT_rac-PEA				(area/ calibration factor)*dilution factor			product/all	(S-R)/(S+R)
0a	13818	281103*	0a	13675	282496		Peak Area (μVxsec)			Concetration (mM)
	14663	12935		13799	311735	Time (h)	(R)-PEA	(S)-amine	Acetophenone	(R)-PEA	(S)-amine	Acetophenone	Conversion	eeS	Conversion (av.)	STDEV	eeS (av.)	Sample-blank
0b	13675	298804		14656	9128	0			12935			0.2159207094418362			0.011081847295718792		0.04679965994044166	0
	13803	328145	0b	13669	290433		298804	328145	10491	7.448128022334115	8.179495488309486	0.17512363067292644	0.011081847295718792	0.04679965994044166
	14659	10491		13796	319346	0.5	82524	211847	220760	2.0570317563188594	5.280597238147465	3.68509128847157	0.33431777219056674	0.4393197699501649	0.2847493909465689	0.07010027702014204	0.4205151115719509	0.2736675436508501		WT_PEA	stden	V37A_PEA	stdev
30a	13730	82524		14655	9147		141033	330421	216479	3.5154544094919986	8.236228127025276	3.6136296296296297	0.23518100970257114	0.4017104531937368					0	0		0	1.6729672036897944E-4
	13831	211847	30a	13709	83625	1	50055	247527	307887	1.2476943018096616	6.169973577945062	5.1394804381846635	0.40928722891982583	0.6635885234994051	0.37435956209926247	0.049395180119689464	0.6554108924675982	0.36327771480354365	0.5	0.2736675436508501	0.07010027702014204	0.2873519299230639	0.004922559944271144
	14644	220760		13803	297708		66974	312733	291351	1.6694251956727653	7.795328780098709	4.863449139280125	0.3394318952786991	0.6472332614357913					1	0.36327771480354365	0.049395180119689464	0.3693660712423863	0.005457761397861714
30b	13702	141033		14634	245083	2	7177	258808	398080	0.17889725310334514	6.451169051298669	6.6450495565988525	0.5005643360236447	0.9460345508205349	0.4732452689391101	0.038634995182329164	0.9384918439167436	0.4621634216433913	2	0.4621634216433913	0.038634995182329164	0.45280249595759947	0.005274176074302656
	13804	330421	30b	13703	87183		11606	324552	403991	0.28929657510344486	8.089934692656662	6.743720396452791	0.4459262018545756	0.9309491370129522					3	0.4690464003956384	0.029081519544050416	0.44125232206819387	0.004000282269271721
	14640	216479		13797	307429	3	0	259400	388423	0	6.465925519716835	6.4838476786645804	0.5006919873681643	1	0.4801282476913572	0.029081519544050416	1	0.4690464003956384	5	0.466594056150971	0.025223763996256175	0.4470601830771638	0.004924932920517936
1a	13736	50055		14633	245307		0	313465	398038	0	7.813574953886036	6.644348461137193	0.45956450801455	1
	13819	247527	1a	13725	31482	5	0	281121	412315	0	7.007353307742161	6.8826708398539385	0.49551179801549156	1	0.47767590344668975	0.025223763996256175	1	0.46659405615097094
	14635	307887		13804	286648		0	330674	420356	0	8.242534523156687	7.016897235263432	0.4598400088778879	1						PPA
1b	13715	66974		14630	295245															WT_PPA		V37A_PPA
	13803	312733	1b	13719	33606														0	0	0	0	0
	14630	291351		13797	295711														0.5	0	0	0.1264470701604698	5.471440518089584E-4
2a	13753	7177		14627	295771	V37A_rac-PEA				(area/ calibration factor)*dilution factor			product/all	(S-R)/(S+R)					1	0.04851364281814551	9.083929512377831E-4	0.16675134473937653	8.976455323364297E-4
	13822	258808	2a	13804	267044		Peak Area (μVxsec)			Concetration (mM)									2	0.08397959060975262	3.650178781516578E-4	0.22198760364278664	0.0010378843435749933
	14628	398080		14626	348819	Time (h)	(R)-PEA	(S)-amine	Acetophenone	(R)-PEA	(S)-amine	Acetophenone	Conversion	eeS	Conversion (av.)	STDEV	eeS (av.)	Sample-blank	3	0.10188206653312079	9.372757437770114E-4	0.25084450056469787	0.00113372546481943
2b	13734	11606	2b	13798	276444	blank	282496	311735	9128	7.041627199760706	7.770452166109975	0.15237141366718832	0.010182225589965411	0.04920477053536418	0.010063928944522236	1.6729672036897944E-4	0.04831015480385831	0	5	0.1271718524657365	0.0017918784630496054	0.2901883999386844	7.824684667235417E-4
	13802	324552		14623	350425		290433	319346	9147	7.23946856772521	7.9601675058577195	0.1526885758998435	0.009945632299079061	0.04741553907235244
	14625	403991	3a	13799	278755	0.5	83625	297708	245083	2.084475796400618	7.420808614586968	4.0911090245174755	0.30089663438497755	0.5614069592718175	0.29741585886758615	0.004922559944271144	0.5597700057425654	0.2873519299230639
3a	13822	259400		14622	346316		87183	307429	245307	2.1731641657111522	7.663118799541353	4.094848200312989	0.29393508335019475	0.5581330522133133
	14633	388423	3b	13798	277678	1	31482	286648	295245	0.784735031656613	7.145121890423251	4.9284507042253525	0.38328922028143475	0.8020809103196807	0.37943000018690853	0.005457761397861714	0.7989928839746296	0.3693660712423863		WT_R-PBA		V37A_R-PBA
3b	13804	313465		14623	337186		33606	295711	295771	0.8376788474001695	7.371030460142579	4.937231090245175	0.3755707800923824	0.7959048576295787					0	0		0	7.360192323641805E-4
	14627	398038	5a	13803	281044	2	0	267044	348819	0	6.656463432873025	5.822748043818466	0.46659583056943293	1	0.4628664249021217	0.005274176074302656	1	0.45280249595759947	0.5	0.0011139030385069776		0.04345104024103226	0.003247172492746368
5a	13813	281121		14625	358370		0	276444	350425	0	6.890772221945261	5.849556598852374	0.45913701923481043	1					1	0.005705668111923702		0.08058099446943627	0.005572326334906005
	14629	412315	5b	13796	295021	3	0	278755	346316	0	6.94837728700334	5.780966092853417	0.4541448777319785	1	0.4513162510127161	0.004000282269271721	1	0.44125232206819387	2	0.012918795338813606		0.14906632696849326	0.00947283665047408
5b	13801	330674		14622	365781		0	277678	337186	0	6.921531482127723	5.628561293688055	0.4484876242934538	1					3	0.016451358452715142		0.19504926365888614	0.013248807241302106
	14625	420356				5	0	281044	358370	0	7.005433969789122	5.982180490349505	0.4606065654866731	1	0.457124112021686	0.004924932920517936	1	0.4470601830771638	5	0.024409415120107088		0.28774630237254556	0.019477306006023775
							0	295021	365781	0	7.3538311979660005	6.105890453834116	0.4536416585566989	1




(±)-Phenylropylamine
WT_PPA			V37A_PPA			WT_rac-PPA				(area/ calibration factor)*dilution factor			product/all	(S-R)/(S+R)
0a	18840	193387	0a	18772	338773		Peak Area (μVxsec)			Concetration (mM)
	18991	214301		18910	440913	Time (h)	(R)-PPA	(S)-amine	Propiophenone	(R)-PPA	(S)-amine	Propiophenone	Conversion	eeS	Conversion (av.)	STDEV	eeS (av.)	Sample-blank
0b	18836	198153	0b	18768	346361	blank	193387	214301	0	4.2898684143029255	4.7537998472158485	0	0	0.05129903259355195	0	0	0.05871060036100353
	18987	226213		18907	451202		198153	226213	0	4.395591719709017	5.018041562280338	0	0	0.0661221681284551
30a	18846	173158	30a	18801	227405	0.5	173158	209890	0	3.841132210975226	4.655951441813778	0	0	0.09589398717654185	0	0	0.09633847013031693
	18991	209890		18926	408483		176289	214069	0	3.9105866107289966	4.748653433692094	0	0	0.09678295308409202
30b	18846	176289		20994	136690	1	162856	206497	28257	3.6126048311402386	4.5806851440288705	0.4235701490747472	0.04915597363394781	0.11815526068557715	0.04851364281814551	9.083929512377831E-4	0.1251533376024464
	18988	214069	30b	18789	230538		165162	215462	28320	3.6637584069410036	4.779554097651533	0.42451451398934215	0.047871312002343215	0.13215141451931564
1a	18851	162856		18919	413062	2	149826	215152	49683	3.323562726767312	4.7726774244085854	0.7447441595527008	0.08423769722664798	0.17898613067088973	0.08397959060975262	3.650178781516578E-4	0.18057650685556764
	18992	206497		20986	137382		152449	220363	50410	3.3817482555294136	4.888272083340843	0.7556418308687408	0.08372148399285725	0.18216688304024553
	21021	28257	1a	18802	198704	3	145386	228728	63259	3.2250710196747723	5.073831346815864	0.9482473036882696	0.10254482056738719	0.22277166852884409	0.10188206653312079	9.372757437770114E-4	0.22172753368988318
1b	18850	165162		18928	428783		148189	232116	63381	3.287249455481166	5.148986730516208	0.9500760738403423	0.10121931249885441	0.22068339885092228
	18989	215462		20980	186680	5	119378	208750	69943	2.6481403174083815	4.6306630305332614	1.0484399241510085	0.125904803053452	0.27236931929003316	0.1271718524657365	0.0017918784630496054	0.2750873393357165
	21019	28320	1b	18800	199084		116373	205903	70282	2.5814809525856153	4.567508550782707	1.0535215067866859	0.128438901878021	0.2778053593813998
2a	18854	149826		18921	434627
	18990	215152		20975	186817
	21014	49683	2a	18827	136879
2b	18849	152449		18940	373627	V37A_rac-PPA				(area/ calibration factor)*dilution factor			product/all	(S-R)/(S+R)
	18982	220363		20972	216473		Peak Area (μVxsec)			Concetration (mM)
	21013	50410	2b	18824	138601	Time (h)	(R)-PPA	(S)-amine	Propiophenone	(R)-PPA	(S)-amine	Propiophenone	Conversion	eeS	Conversion (av.)	STDEV	eeS (av.)	Sample-blank
3a	18853	145386		18941	375061	blank	338773	440913	0	7.514939433977698	9.780692353444367	0	0	0.13100145443165578	0	0	0.1312265695630782
	18985	228728		20974	215968		346361	451202	0	7.6832626487115245	10.00893135665949	0	0	0.13145168469450066
	21014	63259	3a	18829	111014	0.5	227405	408483	136690	5.044483480040317	9.061303600964399	2.048972066285423	0.12683395942978978	0.2847639835945953	0.1264470701604698	5.471440518089584E-4	0.2841812459924499
3b	18850	148189		18935	394314		230538	413062	137382	5.113982245427914	9.162878719607807	2.059345090426688	0.1260601808911498	0.28359850839030454
	18981	232116		20966	251463	1	198704	428783	186680	4.407814451827933	9.511614784292902	2.798318131056864	0.1673860759823934	0.36666735725202276	0.16675134473937653	8.976455323364297E-4	0.3691778568081796
	21008	63381	3b	18828	114385		199084	434627	186817	4.416243922254773	9.641251166330921	2.8003717499981264	0.16611661349635962	0.37168835636433645
5a	18863	119378		18929	397024	2	136879	373627	216473	3.0363617961981424	8.288099334654135	3.2449127961445927	0.22272149870021588	0.46375165032340454	0.22198760364278664	0.0010378843435749933	0.4620466378751208
	18993	208750		20964	252327		138601	375061	215968	3.074560606921871	8.319909494106996	3.237342886908554	0.22125370858535742	0.4603416254268371
	21007	69943	5a	18841	74903	3	111014	394314	251463	2.462603236750273	8.746995268127867	3.769410071726764	0.25164616552887553	0.5606259696672261	0.25084450056469787	0.00113372546481943	0.5566466043045257
5b	18871	116373		18933	402715		114385	397024	252327	2.5373815125631	8.807110701961378	3.7823613619840657	0.25004283560052015	0.5526672389418255
	18999	205903		20960	289735	5	74903	402715	289735	1.6615595352145287	8.933353113011748	4.343104262383547	0.2907416886975693	0.6863476669639753	0.2901883999386844	7.824684667235417E-4	0.6807195704085487
	21010	70282	5b	18838	78764		78764	406074	292539	1.7472073913145953	9.00786519502162	4.385135996042662	0.28963511117979956	0.6750914738531221
				18924	406074
				20956	292539

(R)-Phenylbutylamine
WT_R-PBA			V37A_R-PBA			WT_R-PBA in kinetic res mode				(area/ calibration factor)*dilution factor			product/all	(S-R)/(S+R)
0a	23593	157278*	0a	23594	163846		Peak Area (μVxsec)			Concetration (mM)
	24675	22169		24675	23145	Time (h)	(R)-PBA	(S)-amine	Butyrophenone	(R)-PBA	(S)-amine	Butyrophenone	Conversion	Sample-blank
30a	23594	161019	30a	23600	138798	blank	157278		22169	4.808860793358991		0.274123271389025	0.0539295948791479	0
	24676	22613		24675	37824	0.5	161019		22613	4.9232439125998		0.279613403217106	0.05374227779936793	0.0011139030385069776
1a	23590	165193	1a	23604	127252	1	165193		24513	5.050866243450145		0.30310721058952456	0.05661350643433331	0.005705668111923702
	24673	24513		24675	50392	2	170320		27681	5.207627070060043		0.3422800430925888	0.06167311202045618	0.012918795338813606
2a	23592	170320	2a	23607	102704	3	165208		29002	5.05132487664678		0.3586144217973072	0.06628806757599769	0.016451358452715142
	24674	27681		24673	67217	5	166662		32480	5.095781721173948		0.4016204544506081	0.07305640766676859	0.024409415120107088
3a	23590	165208	3a	23610	88331
	24674	29002		24672	75921
5a	23593	166662	5a	23618	62835
	24674	32480		24674	85530
			24a	24676	49468	V37A_R-PBA in kinetic res mode				(area/ calibration factor)*dilution factor			product/all	(S-R)/(S+R)
							Peak Area (μVxsec)			Concetration (mM)
						Time (h)	(R)-PBA	(S)-amine	Butyrophenone	(R)-PBA	(S)-amine	Butyrophenone	Conversion	eeS	Conversion (av.)	STDEV	Sample-blank
						blank	163846		23145	5.009680982392307		0.2861916692813832	0.05404051194300076		0.05352006775271233	7.360192323641805E-4	0
			0b	23586	171988	0.5	138798		37824	4.243824695104473		0.46769987897597914	0.09926720568304796		0.09697110799374459	0.003247172492746368	0.04345104024103226
				24673	23801	1	127252		50392	3.890799435881168		0.6231052321636406	0.13804129196054502		0.1341010622221486	0.005572326334906005	0.08058099446943627
			30b	23588	147301	2	102704		67217	3.140230921814506		0.8311490790272945	0.2092847017538283		0.2025863947212056	0.00947283665047408	0.14906632696849326
				24672	38090	3	88331		75921	2.7007685927986946		0.9387754471165216	0.2579376528545566		0.24856933141159845	0.013248807241302106	0.19504926365888614
			1b	23588	137755	5	62835		85530	1.92121446070469		1.0575922866120848	0.3550389052813628		0.3412663701252579	0.019477306006023775	0.28774630237254556
				24669	50971
			2b	23592	117794	0	171988		23801	5.258627081525873		0.29430321540575505	0.0529996235624239
				24671	70956	0.5	147301		38090	4.503808566503724		0.4709890120081177	0.0946750103044412
			3b	23596	99012	1	137755		50971	4.211934400165108		0.6302646608313407	0.13016083248375218
				24669	76976	2	117794		70956	3.601615917629478		0.8773824189038594	0.19588808768858293
			5b	23604	74327	3	99012		76976	3.0273460043493716		0.9518206927891013	0.2392010099686403
				24668	89501	5	74327		89501	2.272588640420108		1.1066943440204395	0.327493834969153





Note:	In the case of phenylethylamine and pheylbutylamine a small amount of the coresponding ketone is present in the sample at time 0.
	This however is not due to the enzyme, since sample 0h was taken before the addtition of the enzyme.
	It has to do with purity of the reagents. Therefore this 0 h conversion was abstracted from later measurements.