Synthetic and mechanistic insight into nosylation of glycine

residues

Nicolai Stuhr-Hansen,*a Theis Ivan Sølling,b Kristian Strømgaarda

aDepartment of Drug Design and Pharmacology, University of Copenhagen,

Universitetsparken 2, DK-2100 Copenhagen, Denmark; e-mail: nsh@sund.ku.dk.

bDepartment of Chemistry, University of Copenhagen, Universitetsparken 5,

DK-2100 Copenhagen, Denmark.

Supporting information

Cartesian coordinates of all the calculated structures S1

Nosylation of H-Gly-OC2H4CO-2ClTrt-resin S2

Selected spectra S3

1

S1. Cartesian coordinates of all the calculated structures.

Table S1. PCM-G3MP2 energies

Molecule E(PCM-G3MP2) G(PCM-G3MP2)

-1247.16267 -1247.22544

-1246.71067 -1246.77228

-1286.40156 -1286.46689

-1285.94813 -1286.01201

-1286.39734 -1286.46430

-1285,93721 -1286.00340

-1306.24657 -1306.31173

-1305.78855 -1305.85451

2

-1286.38424 -1286.45082

-1285.92576 -1285.98987

0,1N,-2.6103340952,0.9995115645,-0.3447541059S,-3.3812702437,0.7082108136,1.0626034464O,-3.3324928263,1.182485835,-2.9977124788C,-2.9277849529,2.1844544875,-2.4804668362C,-2.6004419734,2.2864101517,-0.9946945178C,-4.5904124157,-0.5452719044,0.6290338672C,-4.9149334248,-0.7343795168,-0.7019608093C,-5.8055957788,-1.7343954114,-1.0769738326C,-6.3568763144,-2.5697682128,-0.130578309C,-6.0275916928,-2.4046873868,1.207090657C,-5.1684999841,-1.394947438,1.5754096177N,-4.9476073643,-1.2393480204,3.0119044652O,-4.902530417,-0.1306142409,3.4373542712O,-4.8799036601,-2.2378438836,3.6615673151N,-2.7174335424,3.3291672378,-3.16403113O,-4.0492327846,1.9193629509,1.4098145743O,-2.4454519814,0.0713848845,1.9310632965H,-1.8310039464,0.3951790888,-0.4987789701H,-3.3369830671,2.9180117474,-0.5187951202H,-1.6361196381,2.7697658353,-0.8720265727H,-4.4572132871,-0.12634244,-1.4563058519H,-6.0453496774,-1.8561462572,-2.1169459062H,-7.0366489303,-3.3502415036,-0.4177364103H,-6.4360351142,-3.0444760341,1.9633233339H,-2.9873772031,3.363340964,-4.12150583H,-2.4577206842,4.1791526889,-2.7193401638

-1,1N,-3.2065601987,1.0338544987,-1.1068898431S,-3.3943662098,0.6900501086,0.3824577725O,-3.0461482873,3.9729465571,-3.1446521662C,-2.7224120668,3.0475002159,-2.4368120313

3

C,-3.7511505213,2.2545314354,-1.6399609819C,-4.7012529133,-0.5823269289,0.4369491686C,-5.5576804091,-0.6992623331,-0.649250091C,-6.5449435115,-1.6684030837,-0.6975043685C,-6.6779282259,-2.5790228412,0.3354819579C,-5.817204569,-2.5070577919,1.4139220528C,-4.8629990085,-1.5070330813,1.4596749877N,-4.0529715383,-1.4973263221,2.6811776538O,-4.0190985139,-0.4999087047,3.3203714656O,-3.5469850736,-2.5376148146,2.997356142N,-1.4487771319,2.6569032049,-2.2692193701O,-3.9121301089,1.7764928884,1.1864574255O,-2.2612192881,-0.038326442,0.9036647147H,-4.5690197451,2.0764212684,-2.3384444023H,-4.1320985596,2.9330284976,-0.882173007H,-5.4164357306,-0.0316067515,-1.475200485H,-7.1956837331,-1.7217415391,-1.5525297367H,-7.4316204852,-3.345200757,0.3006877649H,-5.8771875094,-3.2129524394,2.2195171664H,-0.7352761577,3.1021577148,-2.7970966192H,-1.2745745036,1.8208504411,-1.7555251702

0,1N,-3.0204974273,0.6679720813,-1.3142502029S,-2.8637596966,0.0051154974,0.1455206249O,-2.0478493187,3.3896374012,-2.8556855711C,-2.3631427214,2.9870229958,-1.7635048529C,-3.5408625068,2.0160991763,-1.5758184745C,-4.4085028626,-0.840742618,0.4536864806C,-5.1771636102,-1.2483887781,-0.6193820156C,-6.4077738512,-1.8632506373,-0.4163834973C,-6.889113518,-2.0509651559,0.8615609089C,-6.1328589168,-1.6421430034,1.9523331387C,-4.9025722246,-1.0660896388,1.7395298301N,-4.1214832742,-0.7649808308,2.9357414134O,-2.987263197,-1.1324389977,2.9578743955O,-4.6791487308,-0.2150270515,3.8348213898N,-1.7342892886,3.3482498427,-0.6366848834O,-2.7888720155,1.0614389516,1.1166257844O,-1.8134623752,-0.9617934647,0.0447886681C,-4.4622611128,2.0049420856,-2.7864995784H,-2.4891615462,0.2093726735,-2.027288651H,-4.0955748394,2.3182216343,-0.6998277306H,-4.8319769676,-1.0755513898,-1.6184738621

4

H,-6.9845211887,-2.1768528847,-1.2654490963H,-7.8437256658,-2.5138268335,1.0231478941H,-6.4852075113,-1.7885863229,2.9539944248H,-0.8945324899,3.8794398949,-0.7119735516H,-1.9299939084,2.9018844898,0.2325172671H,-4.8361831436,3.0034100838,-2.9756633895H,-5.3038296924,1.3496298095,-2.6016605103H,-3.9387063986,1.6678789895,-3.6718743526

-1,1N,-3.3277306277,0.9047151536,-1.2768107684S,-3.1467189291,0.3314123932,0.1408005577O,-2.8012339803,4.4634650303,-1.1462238053C,-2.5955948827,3.2887303211,-1.3698203595C,-3.7502120815,2.2853114805,-1.4204299434C,-4.6312982784,-0.6162849358,0.5576825118C,-5.8178823115,-0.3408089109,-0.1009847107C,-6.9725795227,-1.0655747557,0.1574504839C,-6.9518507176,-2.1094558194,1.0628174455C,-5.7710353997,-2.4239781009,1.715226784C,-4.645153218,-1.66570608,1.4697620427N,-3.4619933576,-2.0431693178,2.2462301701O,-3.0003297393,-1.2386010137,2.992981123O,-3.0685801292,-3.1653255754,2.1293195825N,-1.3801566204,2.806315129,-1.6573739114O,-3.0404909335,1.3458855418,1.1799154789O,-2.1136378006,-0.6908956761,0.1569863705C,-4.4541981748,2.4552071564,-2.7715469334H,-4.4273767291,2.6150756259,-0.6394057378H,-5.8320584602,0.4334236228,-0.8409298327H,-7.878881913,-0.821076924,-0.3646101128H,-7.8375673265,-2.6840789388,1.257778901H,-5.725742897,-3.2382428824,2.4124563918H,-0.5884298988,3.4065898682,-1.596805735H,-1.2450469052,1.8234578741,-1.7265107998H,-4.7511395617,3.4862088488,-2.9273592279H,-5.3393935934,1.8309728634,-2.8106032007H,-3.7966160102,2.1557050216,-3.5809037644

0,1N,-2.1874484307,0.0542100304,-0.8002813939

5

S,-3.0856057518,1.0395516648,0.1243324831N,-2.1423883761,1.0944587161,-5.5755922855C,-2.2412928218,0.489092075,-3.2388436377C,-1.4275182783,0.5715318964,-1.9553751293C,-4.6039980564,0.1236440767,0.35863513C,-4.9405136473,-0.8815628698,-0.5272309706C,-6.0869592378,-1.6450310243,-0.3319511697C,-6.8908671102,-1.4263485441,0.7653918793C,-6.5625996615,-0.4280028084,1.6739439375C,-5.4446719927,0.3407784441,1.4532251076N,-5.2244007674,1.4318172985,2.398598961O,-5.0023272821,2.5135160341,1.948838931O,-5.3283268555,1.1837955698,3.5604664564C,-1.4388315996,0.9426614628,-4.4432544353O,-3.3415935992,2.2199546957,-0.6474100333O,-2.4965699184,1.163694649,1.4282058602O,-0.2480200109,1.145561817,-4.3863742478H,-1.7042132478,-0.6265928522,-0.2485758522H,-1.666159178,1.3377834654,-6.4157343401H,-3.1149673323,0.8937684561,-5.6287960914H,-3.1396479918,1.0896528801,-3.1592250723H,-2.5562284357,-0.5356926789,-3.411399826H,-1.0948839325,1.582246818,-1.7757977204H,-0.5505421861,-0.0514421378,-2.0300491305H,-4.3002327468,-1.0897853306,-1.358927098H,-6.3299644268,-2.41617481,-1.0379185005H,-7.7702824896,-2.0200963467,0.9259960664H,-7.1780736345,-0.2381776471,2.5307171219

-1,1N,-1.9431948105,0.2551988316,-0.59073939S,-2.9391798738,1.1365258625,0.1722857402N,-2.3034825488,1.2504184914,-5.267834548C,-2.2174557846,0.196483931,-3.0797287828C,-1.4155424389,0.6984741596,-1.8686192265C,-4.4699172445,0.1818653093,0.3343378325C,-4.7023618551,-0.8860332828,-0.5151383622C,-5.8412266901,-1.671375517,-0.3974363533C,-6.7602959571,-1.4200047279,0.6024495276C,-6.5373710091,-0.3765521622,1.4868012943C,-5.4160190939,0.4102469436,1.3291762447N,-5.2971090388,1.5120202927,2.2873619911O,-5.3120138174,2.6261834774,1.8674863357O,-5.2603043457,1.2243938961,3.4466491433C,-1.5504905499,0.57458779,-4.3857815349

6

O,-3.3068132307,2.367417463,-0.5171364963O,-2.5718628887,1.3210989423,1.5713566344O,-0.3984285611,0.2801083946,-4.6203152374H,-1.9066148239,1.5235112196,-6.1396060354H,-3.24396439,1.5092027929,-5.0786672266H,-3.2318255347,0.5752763091,-3.0399200761H,-2.2666832981,-0.8876698685,-3.050438589H,-1.3364280456,1.7793114422,-1.9317367843H,-0.409573097,0.306516986,-1.9536029703H,-3.9666948225,-1.1182915692,-1.2570031246H,-5.9936119518,-2.4878085847,-1.0787983805H,-7.637407023,-2.0301418068,0.7085906218H,-7.2280742745,-0.1707310156,2.2817607525

0,1N,-2.3949988139,0.8580910834,-0.2436366101S,-3.2107714643,0.5946966183,1.1158732015O,-2.6369117081,0.8193193777,-2.9265133878C,-2.4615536339,1.9169680197,-2.4491576485C,-2.331312248,2.1228699558,-0.9454104148C,-4.5341720005,-0.52175143,0.6565006845C,-4.8683935656,-0.6512673202,-0.6773739455C,-5.8564670443,-1.54688962,-1.0765590377C,-6.4951740577,-2.3406637086,-0.1497938223C,-6.1547766036,-2.2417822942,1.1933183538C,-5.1995217985,-1.3322724845,1.5813715632N,-4.9561101423,-1.2385212996,3.0174328037O,-4.8761992759,-0.1497151939,3.4966161529O,-4.8932283521,-2.2586115607,3.633277266N,-2.3629580571,3.020259635,-3.1998161555O,-3.7668994665,1.8510863103,1.5207297131O,-2.3704836309,-0.1445428282,2.0120673178C,-2.1238279431,4.3808334866,-2.7493213063H,-1.7451213989,0.142283588,-0.4956482279H,-3.1284503767,2.758873177,-0.5928210935H,-1.3959967308,2.6250852691,-0.7298622535H,-4.3418558454,-0.0830058255,-1.4170432158H,-6.1036818317,-1.6233189554,-2.1184187192H,-7.25076176,-3.0392545386,-0.4545465695H,-6.6369966771,-2.8522746042,1.9302822162H,-2.4287080906,2.8596979624,-4.1811999611H,-2.1812690522,5.0269998178,-3.6119251043H,-1.1421387507,4.4933056056,-2.3047728632H,-2.8734346796,4.7038197568,-2.0380769364

7

-1,1N,-3.2182420981,1.0358054594,-1.4352216411S,-3.2547803892,0.6606370713,0.0529953771O,-3.485445431,2.8730728112,-4.1543237174C,-3.0697472898,2.8629015688,-3.0131852352C,-3.8873834649,2.2365296496,-1.8926912857C,-4.6057825824,-0.5189042393,0.3068278036C,-5.6369922359,-0.5704522728,-0.6164617821C,-6.6725522966,-1.485943101,-0.4929028271C,-6.6765979963,-2.3975795568,0.5456741018C,-5.6408725664,-2.3886350722,1.4655245522C,-4.6424599798,-1.4446521459,1.3436252775N,-3.6204910892,-1.4823322934,2.3914417757O,-3.4855404188,-0.5191401852,3.0784375023O,-3.0231900204,-2.5074354127,2.5349976907N,-1.8845127069,3.4206390543,-2.7103603734O,-3.550245891,1.7705827336,0.9477003301O,-2.0887712932,-0.1311106934,0.4094294741C,-1.1947398516,3.4412947882,-1.4361948736H,-4.8560225555,2.0287868465,-2.3358109835H,-4.0391225741,2.9696420421,-1.106721006H,-5.6186198793,0.0971978931,-1.453627366H,-7.4622620379,-1.4930085373,-1.2209723635H,-7.4671583978,-3.1178429076,0.6403356938H,-5.6127510998,-3.0959711183,2.2717790535H,-1.3767358984,3.7437914272,-3.5049625654H,-0.5360605664,4.2995114346,-1.4229471033H,-0.6065527926,2.5457659118,-1.2761603803H,-1.8942745966,3.5404098439,-0.6210951284

0,1N,-2.3949988139,0.8580910834,-0.2436366101S,-3.2107714643,0.5946966183,1.1158732015O,-2.6369117081,0.8193193777,-2.9265133878C,-2.4615536339,1.9169680197,-2.4491576485C,-2.331312248,2.1228699558,-0.9454104148C,-4.5341720005,-0.52175143,0.6565006845C,-4.8683935656,-0.6512673202,-0.6773739455C,-5.8564670443,-1.54688962,-1.0765590377C,-6.4951740577,-2.3406637086,-0.1497938223C,-6.1547766036,-2.2417822942,1.1933183538C,-5.1995217985,-1.3322724845,1.5813715632N,-4.9561101423,-1.2385212996,3.0174328037O,-4.8761992759,-0.1497151939,3.4966161529

8

O,-4.8932283521,-2.2586115607,3.633277266N,-2.3629580571,3.020259635,-3.1998161555O,-3.7668994665,1.8510863103,1.5207297131O,-2.3704836309,-0.1445428282,2.0120673178C,-2.1238279431,4.3808334866,-2.7493213063H,-1.7451213989,0.142283588,-0.4956482279H,-3.1284503767,2.758873177,-0.5928210935H,-1.3959967308,2.6250852691,-0.7298622535H,-4.3418558454,-0.0830058255,-1.4170432158H,-6.1036818317,-1.6233189554,-2.1184187192H,-7.25076176,-3.0392545386,-0.4545465695H,-6.6369966771,-2.8522746042,1.9302822162H,-2.4287080906,2.8596979624,-4.1811999611H,-2.1812690522,5.0269998178,-3.6119251043H,-1.1421387507,4.4933056056,-2.3047728632H,-2.8734346796,4.7038197568,-2.0380769364

0,1N,-2.6266073006,1.0379271206,-0.3171602531S,-3.4003004341,0.7174087738,1.0612156301O,-3.2508900086,1.1358784297,-2.9912969466C,-2.8898271027,2.1702426215,-2.4871918098C,-2.6457002638,2.3196317926,-0.9898415417C,-4.6018845012,-0.5369846019,0.6297129939C,-4.9508743137,-0.7036881493,-0.6961819191C,-5.8460185878,-1.7022383259,-1.0703542705C,-6.3730429024,-2.5577337295,-0.1283553091C,-6.0155421886,-2.4179435208,1.2068646073C,-5.1557653824,-1.4092077113,1.5715685034N,-4.8966785197,-1.2794892049,3.0023228161O,-4.9434346635,-0.1866355664,3.476972082O,-4.6962277563,-2.2809557765,3.6190969323N,-2.675480018,3.2911027574,-3.1863314303O,-4.08250626,1.9139232041,1.4541701431O,-2.4769803672,0.086360943,1.9589532645H,-1.859700744,0.4245274435,-0.5037830136H,-3.4373879262,2.9234510552,-0.5726969213H,-1.7133083015,2.8453144851,-0.8249344686H,-4.5082226427,-0.0795365373,-1.4463721854H,-6.1091211785,-1.8076485991,-2.1058147968H,-7.05571596,-3.334712313,-0.415005231H,-6.4137486007,-3.0731417549,1.9556421327H,-2.8640103292,3.2971395792,-4.1643656704H,-2.3938687465,4.144983585,-2.7602753382

9

-1,1N,-3.109956108,0.8958967771,-1.2072910823S,-3.3503773421,0.5912290688,0.2796768586O,-3.1131650775,4.0500639301,-2.9515974664C,-2.7237777803,3.0740962213,-2.3425967127C,-3.7224811084,2.0547365857,-1.8107930352C,-4.7029763671,-0.6051247839,0.3925385156C,-5.6066794351,-0.7004538802,-0.6527074824C,-6.6379502856,-1.6286273114,-0.6328502053C,-6.7636275717,-2.5080468952,0.4258847528C,-5.8560827362,-2.4527996928,1.4709589787C,-4.8621535546,-1.4964355058,1.4475807825N,-3.9858387095,-1.4806869933,2.6206716163O,-3.9498007468,-0.4905833575,3.2812845064O,-3.4038856553,-2.4915432443,2.880673669N,-1.436118448,2.8295936142,-2.0930637169O,-3.7941354349,1.7336346851,1.0643393219O,-2.230856051,-0.1512625283,0.8332707946H,-4.3231779698,1.7719612761,-2.6718685719H,-4.3764074532,2.6103537653,-1.1463466682H,-5.4894538437,-0.0574281764,-1.5017601015H,-7.3277812636,-1.6709538814,-1.4551027112H,-7.550402,-3.2384290721,0.4415170689H,-5.9243312184,-3.1332249289,2.2976816996H,-0.7370928383,3.4456954005,-2.4424057072H,-1.177215001,1.9852919277,-1.635235104

S2. Nosylation of H-Gly-OC2H4CO-2ClTrt-resin (Table 1, entry 11).

Fmoc-Gly-OC2H4CO2Bu-t was and loaded on resin after liberation of the

corresponding free acid, followed by nosylation as described above:

Fmoc-Gly-OC2H4CO2Bu-t.

10

Fmoc-glycine (1.49 g, 5 mmol) was refluxed in thionyl chloride (15 mL) for 2

hours. Excess of thionyl chloride was removed in a gentle stream of nitrogen a

the acid chloride was taken up in dichloromethane (5 mL) and added to an ice-

cooled solution of tert-butyl 3-hydroxypropionate (0.73 g, 5 mmol) in DIPEA-

dichloromethane (1:7, 25 mL) under vigorous stirring. The resulting yellow solu-

tion was stirred at room temperature for 2 hours, poured into ice (200 g) and

extracted with pentane (50 mL) and thenethyl acetate (2 x 50 mL). The pooled

organic phases were washed with sodium carbonate (sat., 10 mL), dried over

sodium sulphate, filtered and evaporated. Separation on silica (60F) with ethyl

acetate-heptane (1:4) gave Fmoc-Gly-OC2H4CO2Bu-t (1.69 g, 40%) as white crystals.

Mp: 78-79 ˚C. Anal. Calcd for C24H27NO6: C, 67.75; H, 6.40; N, 3.29. Found: C,

67.67; H, 6.43; N, 3.45. 1H NMR (400 MHz, CDCl3): 1.45 (s, 9H), 2.58 (t, J = 6.2

Hz, 2H), 3.99 (t, J = 5.2 Hz, 2H), 4.23 (t, J = 6.9 Hz, 1H), 4.38-4.42 (m, 4H), 5.31

(br-s, 1H), 7.29-7.33 (m, 2H), 7.37-7.41 (m, 2H), 7.59 (d, J = 8.1 Hz, 2H) 7.76 (t,

J = 8.1 Hz, 2H). 13C NMR (100 MHz, CDCl3): 28.07, 34.90, 42.70, 47.13, 61.11,

67.24, 81.30, 120.00, 125.09, 127.09, 127.73, 141.31, 143.82, 156.24, 169.58,

169.81.

Attachment to solid phase

The tert-butyl ester (0.43 g, 1.0 mmol) was added in one portion to a cooled

mixture of triflouroacetic acid (5 mL) and dichloromethane (2 mL), and stirring

was maintained under nitrogen for 15 minutes. Evaporation followed by co-

evaporation with dichloromethane. 2-chlorotrityl chloride polystyrene resin (1-

2% DVB cross linking, 100-200 mesh, ready for loading of 0.25 mmol carboxylic

11

acid) was used as a solid support, and after swelling the resin in dry DCM for

15-30 min, and the Fmoc-protected amino acid was loaded to the resin using

diisopropylethylamine (DIPEA) (resin/amino acid/DIPEA in 1:4:8) in DCM for 1.5

h, followed by capping with methanol (DCM/MeOH/DIPEA 17:2:1). Fmoc

deprotection was performed with 20% piperidine in DMF (1×5 and 1×15 min;

Wash step in between) followed by washing of the resin with DCM and drying.

The resin was swelled in DIPEA (6 equiv) in THF (2.5 mL) for ca. 30 min. 2-

Nitrobenzenesulfonyl (Ns) chloride (4 equiv) in DCM (1 mL) was added slowly

while agitating the solution for 1 h followed by draining and washing with THF,

MeOH, and DCM. An inhomogenous mixture of TFA/triisopropylsilane(TIPS)/H2O

(18:1:1, 4 mL) was added and shaking was maintained for 2 h. The peptide

containing solution was filtered into a flask and combined with two successive

washes with DCM. After gentle evaporation in a stream of nitrogen the crude

peptide which was analyzed by LC-MS.

S3. Selected NMR spectra

Compound 1, Scheme 2 (Table, Entry 1)

Compound 2, Scheme 2 (Table, Entry 1)

Fmoc-Gly-OC2H4CO2Bu-t (for use in Table, Entry 11)

12

13

14

15

16

17

18

19